1 Congreso Latinoamericano de Glicobiología

Transcripción

1er Congreso Latinoamericano de Glicobiología
Oaxaca, Oaxaca
4-7 de Agosto de 2011
Evento avalado por El Consejo Mexicano de
Medicina Molecular, A.C.
Comité Organizador
Dr. Iván Martínez Duncker R.
Profesor-Investigador a Tiempo Completo
Jefe del Laboratorio de Glicobiología Humana
Coordinador del Departamento de Bioquímica y Biología Molecular
Facultad de Ciencias-Universidad Autónoma del Estado de Morelos
Investigador Nacional – CONACYT
[email protected]
Dr. Miguel A. Mayoral Chávez
Profesor-Investigador
Jefe del Laboratorio de Bioquímica
Coordinador de la Maestría en Ciencias Médicas y Biológicas
Centro de Investigación en Ciencias Médicas y Biológicas
Facultad de Medicina y Cirugía
Universidad Autónoma Benito Juárez de Oaxaca
[email protected]
Dra. Blanca Ortiz Quintero
Investigador en Ciencias Médicas
Departamento de Bioquímica – Unidad de Investigación
Instituto Nacional de Enfermedades Respiratorias
[email protected]
Bienvenidos,
Nos es muy grato recibirlo en esta Ciudad de Oaxaca como asistente al 1er
Congreso Latinoamericano de Glicobiología. El gran impacto que la
glicobiología tiene en diversas áreas de las ciencias de la vida hace
indispensable la labor de generar un foro regional que permita la difusión de
los avances que estamos realizando en esta disciplina, además de fomentar
la interacción académica que permita acrecentar la consolidación de un
grupo de glicobiólogos en nuestra región.
Esperamos que este paso sea el primero de una fructífera caminata que nos
permita hacer incidir activamente a la glicobiología en la comprensión de la
gran variedad de procesos biológicos que se ven enriquecidos por la
participación de los glicanos. La glicobiología ha mostrado desde sus inicios
una intensa actividad en la generación de conocimientos e innovación
tecnológica transdisciplinaria que ha resuelto y continúa resolviendo los
mecanismos de diversas funciones celulares. Esta expansión de la
glicobiología y la complejidad asociada a un creciente acervo de
conocimientos especializados amerita del apoyo de una comunidad de
investigadores comprometida a organizar, encausar y hacer reconocer a la
Glicobiología en la investigación latinoamericana.
Les agradecemos su asistencia a este evento, esperando se lleven una grata
experiencia personal y académica.
Atentamente.
El Comité Organizador.
PROGRAMA
JUEVES 4 DE AGOSTO
0800-1615
Curso Pre-congreso de Glicobiología
1400-1630
REGISTRO Y COLOCACIÓN DE CARTELES
1630-1715
Inauguración del Evento
CONFERENCIAS PLENARIAS
1715-1800
Dr. Hudson Freeze, Sanford Burnham Institute, EUA
“Dark matter matters: glycobiology today”
1800-1845
Dr. Pedro Antonio Prieto Trejo
“Oligosacáridos de la leche humana: su estudio,
síntesis y experiencias clínicas”
1845-1930
Dra. Anne Harduin Lepers, Universidad de Lille, Francia
“Insights into evolutionary history of animal
sialyltransferases: a phylogenetic approach”
2000-2230
Recepción de Bienvenida
VIERNES 5 DE AGOSTO
INMUNOGLICOBIOLOGÍA
Coordinador: Dra. Blanca Ortiz Quintero
0900-0930
Dra. Patricia Gorocica Rosete, Inst.Nac.Enf.Res., México
“Interacciones de los carbohidratos de la pared celular de
Histoplasma capsulatum con moléculas de la célula
huésped y sus posibles roles en la inmunomodulación de
la respuesta a histoplasmosis.
0930-1000
Dr. Ricardo Lascurain Ledesma, Fac.Med.UNAM, México
“Identificación de los receptores de superficie de
linfocitos T CD4+ reconocidos por la lectina del
Amaranthus Leucocarpus”
1000-1030
Dr. Francisco Urrea, Inst.Nac.Enf.Res., México
“Las vías de la glicosilación de linfocitos T reguladas por
STAT-6, en un modelo de ratón “knock out”
1030-1100
RECESO
1100-1130
Dra. Marta Toscano, Inst. Biol. y Med. Exp, Argentina
“Interacciones de Galectina-1 en la modulación de las
funciones inmunes celulares”
1130-1200
Dr. José Luis Montiel Hernández, Fac. Farmacia-UAEM,
México
“La glicosilación del linfocito y su papel en
enfermedades crónicas inflamatorias”
1200-1300
SESIÓN DE CARTELES
1300-1430
COMIDA
1430-1500
Dr. Julio Reyes Leyva, CIBIOR-IMSS, México
“Glicobiología de las infecciones virales”
las
Abstracts seleccionados para ponencia oral
__________________________________________________________________
1500-1520
Mtra. Maricela Carrasco Yépez, ESM- Instituto Politécnico
Nacional, México
“Naegleria fowleri usa residuos de D-manosa en Nglicanos para adherirse e infectar la mucosa nasal
murina”
1520-1540
Dra. Adriana Obregón Cardenas, Inst.Biot., FCB, UANL,
México
“Caracterización parcial de la glicosilación de la proteína
inmunodominante BPM de Entamoeba histolytica”
__________________________________________________________________
1540-1610
Dr. Ruben T Almaraz, Johns Hopkins University, EUA
“Cell surface glycan engineering through metabolic
labeling: Implications for cell adhesion and cancer cell
metastasis”
1610-1640
Dr. Edgar Zenteno Galindo, Facultad de Medicina – UNAM,
México
“El papel de los glicoconjugados en la inmunidad innata:
relevancia de la adhesina específica para GlcNAc de
Mannheimia haemolytica en la septicemia enzoótica”
SÁBADO 6 DE AGOSTO
GLICANOPATÍAS
Coordinador: Dr. Miguel Ángel Mayoral
0830-0900
Dr. Miguel Ángel Mayoral, Fac. Med. – UABJO, México
“Sialilación en la metástasis cerebral”
0900-0930
Dr. Pedro A. Hernández Cruz, Fac.Med. – UABJO, México
“Papel de los O-glicanos en el cáncer de mama”
0930-1000
Dr. Carlos Martínez Duncker, Academia Mexicana de Cirugía
“F-18-Fluordesoxiglucosa y diagnóstico en oncología:
Quo vadis México?”
__________________________________________________________________
1000-1020
Dra. Carmen González, Universidad Autónoma de San Luis
Potosí, México
“Papel del ácido hialurónico endotelial luminal en la
inhibición mediada por vasoinhibinas sobre los efectos de
la bradiquinina”
1020-1040
Mtro. José Agustín Atzin, Inst.Nac.Enf.Resp, México
“Caracterización de los epítopes sacarídicos presentes en
MUC1 expresada en células tumorales de pacientes con
adenocarcinoma pulmonar”
1040-1100
M.B. Roberta Salinas Marín, Facultad de Ciencias-UAEM,
México
“Inhibición del transporte de CMP-ácido siálico mediada
por morfolinos como estrategia antimetastásica en una
línea de hepatocarcinoma celular”
__________________________________________________________________
1100-1130
RECESO
1130-1200
Dra. Carla Asteggiano, CEMECO, Argentina
“Los proteoglicanos de heparán sulfato y los desórdenes
congénitos de la O-glicosilación: bases moleculares y
bioquímica de la exostosis múltiple hereditaria”
1200-1230
Dr. François Foulquier, Universidad de Lille, Francia
“The COG complex and Golgi glycosylation”
1230-1300
Dra. Rosella Mollicone, INSERM, Francia
“Un nuevo perfil de CDG tipo IIx”
1300-1330
Dr. Hudson Freeze, Sanford Burnham Institute, EUA
“Tools for identifying new human glycosylation disorders”
1330-1500
COMIDA
1500-1600
SESIÓN DE CARTELES
GLICOTECNOLOGÍA
Coordinador: Dr. Octavio T. Ramírez Reivich
1600-1630
Dr. Octavio T. Ramírez Reivich, Instituto de Biotecnologia
UNAM, México
“Respuesta transcripcional de genes selectos asociados a
la N-glicosilación y estrés por frío durante la producción
de anticuerpos monoclonales por células CHO en
hipotermia”
1630-1700
Dr. Héctor Mora Montes, Universidad de Guanajuato, México
“Glicobiología de la pared celular de Candida albicans”
__________________________________________________________________
1700-1720
QFB Diana Fabiola Díaz Jiménez, Universidad de Guanajuato,
México
“La
caracterización
bioquímica
de
las
manosiltransferasas recombinantes Mnt1 y Mnt2 de
Candida albicans revela nuevas funciones en la
biosíntesis de O-mananos”
1720-1740
Dr. Enrique García Hernández, Instituto de Química-UNAM,
México
“Plegamiento y homodimerización de la aglutinina de
gérmen de trigo”
1740-1800
Mtro. Ramsés Gamboa Suasnavart, Inst.Inv.Biom. -UNAM,
México
“Efecto de las condiciones de cultivo en la Omanosilación
de
APA
45/47
recombinante
de
Mycobacterium tuberculosis en Streptomyces lividans”
__________________________________________________________________
1800-1830
Dr. Arturo Flores Carreón, Universidad de Guanajuato, México
“Glicosilación de proteínas en el hongo patógeno de
humanos, Sporothrix schenckii”
1830-1900
Dr. Vicente Vérez Bencomo, Centro de Química Biomolecular,
Cuba
“Glicoconjugados sintéticos: Herramientas para explotar
el reconocimiento molecular en medicina”
2000
CENA DE CLAUSURA
DOMINGO 7 DE AGOSTO
0900-1400
Paseo turístico
PRESENTACIONES ORALES
(en orden de aparición)
1
Dark matter matters: glycobiology today
Hudson Freeze
Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
[email protected]
Astronomers were unaware of dark matter, but its discovery revised our concept of the
Universe. Micro RNA was dark matter too, and its discovery spawned both basic science
and medical applications along with a Nobel Prize. What of Glycobiology? Is it dark matter
or merely standing in the shadows? Genomics and proteomics are well-established,
glycomics much less so. Why? Technical barriers, lack of common language and
databases, and the absence of template-driven biological functions all contribute to this
situation. However, like off stage actors awaiting the cue, the field is poised to join the
ensemble as a full partner. In the United States, massive, long-term Glycobiology program
grants are being funded, the National Academy of Sciences will report on the field’s impact
and importance, the American Societies for Glycobiology and Matrix Biology will hold joint
meetings and high profile papers are now appearing more frequently than ever before.
Traditional boundaries to communication are fading. An opportunity for advancing the field
is to identify and foster grass roots alliances with scores of patient and family advocacy
groups on the social networking landscape. Laboratory work does not exist in isolation.
Outreach can bring all stakeholders, scientists, physicians, and affected families into
greater communication. The basic science roots of biology, medicine, and pathology
predate the creation of traditional disciplines. Weakness in any area of science, weakens
the whole. Glycobiology can merge artificial silos and isolated towers in stressing its
universality and broad application. Glycobiology will become part of greater field of biology
and has the chance to take a leading role on the emerging world stage by partnering with
all the stakeholders.
Supported by The Rocket Fund and a Sanford Professorship in Glycobiology.
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2
Oligosacáridos De La Leche Humana: Su Estudio, Síntesis y
Experiencias Clínicas
Dr. José Antonio Prieto Trejo
Investigador Independiente
El interés por el estudio de los oligosacáridos de la leche humana ha ido variando tanto
por sus motivos como por su importancia potencial para la salud. Inicialmente la leche
humana se consideraba objeto de estudio de la bioquímica de hidratos de carbono
debido a la variedad y cantidad de estructuras solubles que no se encontraban en otros
fluidos corporales o tejidos. Posteriormente estas estructuras se pusieron en el contexto
de la naciente ciencia de la glicobiología que descubría las mismos oligosacáridos, o
algunos muy similares, formando parte de los glicoconjugados de superficies celulares.
Algunas estructuras de oligosacáridos de la leche humana se encontraron en tejidos
oncofetales e incluso uno de ellos dio lugar a uno de los primeros anticuerpos
monoclonales utilizados como herramientas de detección de adenocarcinoma
pancreático y tumores colo-rectales. Paradójicamente el estudio de los oligosacáridos
en la nutrición pediátrica se limitó por mucho tiempo a estudios in vitro y algunos
experimentos in vivo debido a la disponibilidad limitada de oligosacáridos puros.
Durante la década de los 90 y los primeros años del presente siglo, un grupo de
científicos e ingenieros patrocinados por Abbott Nutrition se dieron a la tarea de
explorar métodos para la síntesis a gran escala de oligosacáridos de la leche humana.
Esto resultó en la primer síntesis de productos genéticos secundarios en ratones
transgénicos y en la elucidación de un sistema de fermentación basado en levaduras y
la acción de glicosiltransferasas selectas. La disponibilidad de oligosacáridos permitió la
conducción de un estudio clínico que arrojó resultados que no han sido fáciles de
interpretar mientras que la síntesis en animales transgénicos generó una serie de
preguntas al utilizarse otras especies (además de ratones) para sintetizar
glicosiltransferasas exógenas durante la lactancia. La expresión de una
fucosiltransferasa humana en conejas lactantes indujo la suspensión de la lactancia tal
vez afectando el estado de diferenciación de la glándula mamaria activa. Si se
consideran estos resultados en su totalidad, no es atrevido considerar que ya hay
métodos para sintetizar oligosacáridos en gran escala y que los animales transgénicos
que resultaron de este proyecto pueden ser modelos útiles tanto para el estudio de la
lactancia como para la elucidación de sus efectos en la salud.
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3
Insights into evolutionary history of animal sialyltransferases: a
phylogenetic approach.
Anne Harduin-Lepers1*, Jean-Michel Petit2, Philippe Delannoy1, Rafael Oriol3 and Daniel
Petit2
1
Structural and Functional Glycobiology Unit, Université Lille Nord de France, Lille1,
UMR CNRS 8576, 59655 Villeneuve d’Ascq, FRANCE; 2 Laboratoire de Génétique
Moléculaire Animale, Université de Limoges, INRA UMR 1061 ; 87060 Limoges,
France ; 3 Unité de Microenvironnement et physiologie de la différenciation, Université
de Paris Sud XI, INSERM U1004, 94807 Villejuif, France
*[email protected] The animal sialyltransferases (ST) are Golgi type II transmembrane glycosyltransferases
that catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of
glycoconjugates. The ST superfamily comprises 4 families (ST3Gal, ST6Gal, ST6GalNAc
and ST8Sia) and 20 subfamilies have been identified in both human and murine genomes.
Despite low overall protein sequence identities, ST share four conserved peptide motifs (L,
S, motif III and VS) that are hallmarks for sialyltransferase identification. Genome
sequencing programs offer a new route into understanding multigene families both within a
single species and across different species. We have identified and assembled in silico
hundreds of new putative ST genes in vertebrate and in invertebrate genome databases.
Based on four lines of evidence, (i) multiple sequence alignments (ii) molecular phylogeny
(iii) exon-intron organization of the genes and (iv) phylogenomic studies, we suggested
ancient occurrence of the four ST families early in the Metazoan lineage and subsequent
divergent evolution of STs [1]. Focusing on the st6gal gene family, we showed that the two
subsets of st6gal genes (st6gal1 and st6gal2) arose from the second round of whole
genome duplication that took place early in vertebrate evolution, about 450 MYA. We
evidenced an accelerated evolution of the st6gal1 genes both in their genomic regulatory
sequences and in their coding sequence in reptiles, birds and mammals known as
amniotes. On the other hand, st6gal2 genes conserved an ancestral profile of expression
throughout vertebrate evolution. Altogether, our recent studies suggest that the vertebrate
ST6Gal-I progressively acquired functions in the immune system from a probable ancestral
role in embryonic and adult central nervous system that has been maintained by ST6Gal-II
[2].
[1] Harduin-Lepers A et al. 2005 Glycobiology 15, 805; [2] Petit et al. 2010 J. Biol. Chem. 285, 38399.
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4
Interactions of cell wall carbohydrates of Histoplasma capsulatum
with host molecules: their possible roles in the immunomodulation
response of histoplasmosis.
1
Patricia Gorocica*1, Lucia Taylor 2, Armando Pérez-Torres 3, Edgar Zenteno4.
Depto. de Bioquímica, Instituto Nacional de Enfermedades Respiratorias. 2.Depto. de
Microbiología y Parasitología. 3Depto. de Biología Tisular. 4Depto.de Bioquímica,
Facultad de Medicina, UNAM
*[email protected]
Histoplasma capsulatum is a dimorphic fungal pathogen that causes respiratory and
systemic disease by proliferating within phagocytic cells. The entry to the host cell
involves the exchange of mycelium to yeast dimorphic (morphotypes M and L) and
structural and antigenic changes of some carbohydrates such as cell wall glucans. The
major difference between yeast and mycelia lies in the amount and type of glucan,
which consist of glucose homo and hetero-polymers and whose glycosydic linkage
types differ between the L and M morphotypes (the α-and β –glucans). The glucans
have different biological functions. The α-glucan present mainly in the L morphotypes is
considered an important factor in the virulence of the fungus while the β-glucan found in
the M morphotypes has an important role in initiating the innate immune response
against the fungus. The binding of H. capsulatum to phagocytes may be mediated by
the pathogen’s cell wall glucans. The β–glucan binds to CR3 receptor on CD18
(CD11b/CD18) and CR4 (CD11c/CD18) in macrophage and Dectin-1 in dendritic cells.
This molecular interaction benefits the entry of the fungus to the host cell because it
triggers the production of toxic oxygen metabolites. At present, the specific receptors
recognizing H. capsulatum α-1,3-glucan have not been identified. It has been suggested
that H. capsulatum α-1,3-glucans block host Dectin-1 from recognizing β-glucans
present during the morphotype L. Surface carbohydrates are the major fungal structure
involved in interactions with the host. They are important for the early activation of the
innate immune response and the subsequent control and destruction of these
pathogens. The β-1,3-glucan is antigenic and participates in the modulation of the host
immune response whereas the α-1,3-glucan is considered relevant for H. capsulatum
virulence and they seem to provide H. capsulatum with the capacity to survive in
macrophages. Another lectin-like activity mediated interaction between H. capsulatum
yeast and host cells, in which lectin activity is associated with a component present on
the yeast cell surface. H. capsulatum cell wall components with lectin-like activity seem
to interact with the host cell surface, while host membrane lectin-like receptors can
recognize a particular fungal carbohydrate ligand. This lectin-like activity is specific to
galactosylated surface molecules (mainly β-anomer) on murine macrophages. It is a
highly dynamic entity and changes in its composition or structure may trigger critical
consequences for the host-parasite relationship.
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5
Identification of CD4+ T cell surface receptors recognized by
Amaranthus leucocarpus lectin.
María del Carmen Arenas del Angel1, Blanca Espinosa1, Edgar Zenteno2, Ricardo
Lascurain2*
1
Departamento de Bioquímica, Instituto Nacional de Enfermedades Respiratorias
Ismael Cosío Villegas, SS. 2Departamento de Bioquímica, Facultad de Medicina,
Universidad Nacional Autónoma de México, México.
[email protected]
Cell surface glycosylated molecules play an important role in processes of cell
maturation, activation and death; remain essential in the immune response. Antibodies
have been used for study of these biological processes, but lectins are also useful tools,
which bind in a specific and reversible way to carbohydrates, either soluble molecules or
forming part of more complex saccharide structures. Amaranthus leucocarpus lectin
(ALL) is a homodimeric 35 kDa glycoprotein specific to T antigen (Galβ1-3GalNAcα1-OSer/Thr) and its precursor Tn antigen (GalNAcα1-O-Ser/Thr) present in O-glycans. The
study of the molecule recognized by ALL in mononuclear cells from mouse lymph
nodes, showed a 70 kDa glycoprotein, containing 20% of carbohydrates mainly Gal and
GalNAc. The composition of aminoacids corresponded to Serine residues, which is
common in O-glycosylated proteins type mucins. The sequencing by matrix assisted
laser desorption ionization - time of flight (MALDI-TOF) method, showed that there is not
significant homology with other described proteins. Analysis by flow cytometry has
shown that ALL can bind to CD4+ T cells from mouse axial lymph nodes. The stimulation
by anti-CD3 antibodies plus ALL (1/5 μg/mL) induces cell proliferation (81.8% ± 0.8)
similar to T cells stimulated by anti-CD3 plus anti-CD28 antibodies. In contrast, CD28stimulated or ALL-stimulated or no-stimulated CD4+ T cells exhibited 14.6%, 16.4% and
16.3%, respectively. Preliminary studies have shown that ALL binds mainly to a cell
membrane 70 kDa protein from mouse lymph node CD4+ T cells stimulated via anti-CD3
and anti-CD28 antibodies and analyzed by western blotting. The protein bound by ALL
is found in a region in turn recognized by cholera toxin subunit-beta, suggesting that
ALL-recognized membrane protein is derived from lipid rafts.
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6
T cells glycosylation pathway regulated by STAT-6 in a model of
STAT-6 -/- knock out mice.
Janette Arias1, Blanca Ortiz1, Jaime Chávez2, Federico Ávila3, Leonel Armas3, Ricardo
Lascurain4, Edgar Zenteno4, Francisco Urrea1*.
1
Depto. de Bioquímica, Instituto Nacional de Enfermedades Respiratorias. 2 Depto. de
Hiperreactividad Bronquial, Instituto Nacional de Enfermedades Respiratorias. 3
UBIMED, FES Iztacala, UNAM-INER. 4 Depto. de Bioquímica, Facultad de Medicina,
UNAM.
*[email protected]
STAT-6 is a transcription factor that is essential for the IL-4 signaling pathway and the
development of CD4+ T cells TH2 type. It has been reported that STAT-6/GATA-3 affect
the expression of fucosyltransferase and sialyltransferases, and recent studies have
showed that these glycosyltransferases play a key role in the function and susceptibility
to apoptosis of TH2 cells. These data suggest that STAT-6 could regulate several
pathways of glycosylation that are essential for TH function. However, little is known
about which glycosylation pathways are regulated by this transcription factor. The aim of
this work is the identification of glycosylation pathways regulated by STAT-6 in T cells
and their participation in the immune response, by evaluating the glycosyltransferase
gene expression and surface glycosylation pattern using RT-PCR in real time and flow
cytometry analysis (lectins) in a model of asthma in BALB/c and BALB/c STAT-6 -/knock out mice. The bronchial asthma pathogenesis is mediated by TH2 cells and it is
well documented the involvement of STAT-6 in this disease. This study will elucidate a
possible association between glycosylation pathways regulated by STAT-6 and
asthmatic immune response.
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7
Galectin-1-glycan interactions in the modulation of immune cell
functions.
Marta A. Toscano*, Juan M. Ilarregui, Diego O. Croci, Mariana Salatino, Gabriel
Rabinovich
Laboratorio de Inmunopatología. Instituto de Medicina y Biología Experimental. Consejo
Nacional de Investigaciones Científicas y Técnicas. Buenos Aires, Argentina.
*[email protected]
Recent efforts toward decoding the glycosylation signature of immune cells have
revealed substantial changes in N- and O-glycan structures during T cell activation and
differentiation. These alterations have also been detected during the course of dendritic
cell differentiation and maturation, suggesting that protein-glycan interactions may have
a decisive role in the control of immune cell responsiveness and tolerance. The
responsibility of interpreting these glycosylation changes is assigned in part to
endogenous glycan binding proteins. Galectin-1 and endogenous lectin which
recognizes galactose-β1-4-N-acetylglucosamine (LacNAc) units present on the
branches of N- and O-linked glycans, elicits a broad spectrum of anti-inflammatory and
immunomodulatory effects. Blockade of galectin-1 expression in tumor tissues results in
heightened T cell-mediated tumor rejection and increased secretion of T helper (Th)
type-1 cytokines. Moreover, galectin-1-deficient (Lgals1-/-) mice exhibit augmented Th1
and Th17 responses and are considerably more susceptible to autoimmune disease
than their wild-type counterparts. The mechanistic bases underlying these antiinflammatory effects are still under study. We will discuss the relevance of differential
glycosylation of T helper cell subsets in regulating susceptibility to galectin-1-induced
cell death and the role of galectin-1 signaling in the generation of tolerogenic dendritic
cells which contributes to the resolution of autoimmune inflammation. Strategies to
manipulate galectin-glycan interaction may be capable of influencing immune tolerance
versus inflammation with broad therapeutic implications in immunopathology.
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8
Lymphocyte glycosylation and its role in chronic inflammatory
disorders.
José Luis Montiel Hernández. Laboratorio de Citocinas y Autoinmunidad, Facultad de
Farmacia, UAEM, Cuernavaca, Morelos.
[email protected]
Chronic inflammatory disorders are characterized by persistent inflammation because the
immune system has an inappropriate response to its own molecules (autoimmune disease),
infection response or environmental factors. The better understanding of the immune regulatory
pathways should be important for the development of a more effective therapy. By the other
side, the glycobiology constitute one of the more challenging fields, since a vast array of
proteins could be structural and functional modified by the addition of carbohydrates, doing
possible its participation in the regulation of the immune response as well as in the development
of chronic inflammatory disorders. The aim of the present talk is to present the major alterations
of the lymphocyte surface glycoproteins and its potential immunological implications. 1)
Sialylation of lymphocyte glycoproteins is continuously regulated, according its activation or
differentiation state; resulting in changes of adhesion, surviving or activation capacity.
Modifications in expression of sialyltransferases or sialidases could result in immunological
alterations. 2) Biological functions of the membrane proteins on the surface of lymphocytes are
strongly influenced by N-glycosylation alterations (CD22, CD43, CD45, integrin, L-,P- or Eselectin and chemokine receptors), suggesting immunological implications as consequence of
its alteration. 3) Soluble lectins, Siglecs and C-type lectin receptors play important
immunological homeostasis. 4) Cytokines as TNF-α induced a tissular increase in fucosylation
and expression of sialyl-Lewis x epitopes, whereas inflammation protocols stimulate sialylation
of soluble glycoproteins. 5) Mammalian N-glycan branching could limit no self-signals between
immune cells and autoimmune disorders. 6) Aberrant galactosylation of antibodies or membrane
proteins, implicating an active role of β-1, 4-GalTI, are associated with inflammation diseases. In
conclusion, a better knowledge of the diverse glycosylation alterations in the course of
inflammatory diseases may help not only to found new therapies, but to understand the
development of the immunological disorders.
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9
Glycobiology of viral infections.
Julio Reyes Leyva
Centro de Investigación Biomédica, Instituto Mexicano del Seguro Social
Km 4.5 Carretera Atlixco-Metepec, CP 74360 Metepec, Pue.
[email protected]; [email protected]
Notable improvements in the study of virus disease pathogeny have been reached.
These include identification of crucial biochemical process involving virus-cell
interactions that are mediated by lectins, and their glycan ligands. It is well known that
several viruses possess surface glycoproteins with lectin activity to specifically
recognize cell glycoconjugates to adhere and penetrate permissible cells, indeed cells
that permits virus replication. At the same time, during the initial recognizing process,
cells use lectin-like receptors to identify molecular patterns of virion structural
components to start an innate response according with, and to block the infectious
process, arrest virus replication, lead to viral protein degradation, presentation and
induction of a cytotoxic immune response that protect the infected body.The short time
that viruses spend to complete a replicative cycle together with their great genetic
diversity and load inside each cell, give rise to a fast adaptive phenomenon that permits
most viruses to survive in adverse environments, to evade immune response and in
some cases to use defense mechanism to favor viral replication and spread all around
the organism and host populations.Virus-cell interactions mediated by glycans are not
limited to simple molecular recognition at cell surface. Several cell signaling pathways,
transcription factors, transmembrane or soluble communication molecules, as well as
differentiation and maturation process may be involved or affected during virus
infectious process. In this talk, I will give an account of the role played by several lectins,
glicans, transferases, and glycosidases in the infectious process and disease produced
by viruses.
GRANTS: CONACYT 25617 y PAICYT.
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10
Cell surface glycan engineering through metabolic labeling:
Implications for cell adhesion and cancer cell metastasis
Ruben T. Almaraz,1* Yuan Tian,2 Rahul Bhattarcharya,3 Konstantinos Konstantopoulos,1
Hui Zhang,2 and Kevin J. Yarema3
1
Department of Chemical and Biomolecular Engineering, 2 Department of Pathology, 3
Department of Biomedical Engineering, The Johns Hopkins University, Baltimore,
Maryland, USA
*[email protected]
In the last few years metabolic glycoengineering has emerged as a novel technology to
understand the glycobiology of the cell. This technology is based on the ability of certain
non-natural sugar analogues, such as ManNAc, GlcNAc,and GalNAc derivatives, to be
incorporated into the cells metabolic pathways to generate non-natural glycan
structures. In this talk, we present several examples demonstrating how these nonnatural sugar analogues have been implemented in our laboratory. In the first example
we discuss the modification of the sialic acids repertoire on pancreatic cancer cells. We
used a butyrated sugar, 1,3,4-O-Bu3ManNAc, a compound that dramatically increases
flux through the sialic acid pathway with minimal side effects on overall cellular
physiology, and renders a pancreatic cancer cell line more migratory using adhesion
assays that reproduce aspects of the metastatic process in an in vitro environment. We
demonstrated that surface expression of cancer-associated sialyl carbohydrate
determinants such as sialyl Lewis X and sialyl Lewis A increased under treatment of the
high flux sugar analog while others such Lewis X and RCA-lectin ligands decreased
suggesting that the overall number of glycans remained relatively constant and
metabolic modifications were limited to terminal sialic acid residues. Furthermore, we
discuss a new line of azido non-natural sugars for glycoproteomics applications.
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11
The role of glycoconjugates in innate immunity: relevance of the
GlcNAc-specific adhesin from Mannheimia haemolytica in enzootic
septicemia.
1,2
Sujey Larios, 2F. Suárez-Güemes, 2 F. Trigo, 3L. Jaramillo, 1M. Ali Pereyra, 4C.
Agundis, 1,4* E. Zenteno.
1
Dep. Bioquímica, Fac Medicina UNAM, México; 2Fac. Medicina Veterinaria y Zootecnia,
UNAM, México; 3INIFAP-SAGARPA, México; 4.Centro de Investigaciones
Multidisciplinarias UNAM-UABJO, Oaxaca, México
*[email protected]
M. haemolytica is the causative of acute pleuropneumonia in cattles, it contained a 68
kDa GlcNAc-specific adhesin (MhA) which participate in adhesion to respiratory tract of
bovines. This work is aimed at identify the role of this adhesion in the enzootic
septicemia and neumonitis. The FITC-MhA recognizes mainly neutrophils (80%) and
macrophages from healthy bovines. MhA induces increased oxidative burst, determined
by NBT reduction of bovine neutrophils in a dose dependent form. The MhA receptor
from bovine granulocyes (MhANr) purified by affinity chromatography on MhASepharose, is an 83 kDa glycoprotein mainly composed by Glx, Asx, Ser, Gly and no
Cys. The glycannic portion (25%), contained NeuAc, GlcNAc, Man, and Gal. Nano LCESI-MS/MS analysis of tryptic peptides from MhAr showed homology with metavinculin
(29%), a membrane-associated protein which participates in the cytoskeleton ordering.
After 45-60 min neutrophils activation, MhA also recognizes from cell lysate, two 26 kDa
glycoproteins with homology to bovine haptoglobin, which should down-regulate
deleterious effect of oxidative burst. Our data confirm that MhA should participate as
pro-inflammatory by activating neutrophils oxidative metabolism and membrane
organization allowing steric hindrance of pathogenic molecules, such as lymphotoxins,
favoring acute lung injury.
Financed in part by PAPIIT-UNAM and CONACyT, México.
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12
Sialylation in brain metastasis.
Miguel Mayoral1,2*, Daniel Rembao3, Edgar Zenteno1, Jorge Guevara1.
1
Departmento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma
de México, Facultad de Medicina, UNAM, C.P. 04510, Mexico.
2
Centro de Investigación en Ciencias Médicas y Biológicas, Facultad de Medicina y
Cirugía, Universidad Autónoma Benito Juárez de Oaxaca, Oaxaca, C.P. 68000, México.
3
Departamento de Neuropatología, Instituto Nacional de Neurología y Neurocirugía
(INNN), C.P. 14269, México.
*[email protected]
Alterations in membrane cell glycosylation seem to be the hallmark of malignant
transformation. By histochemistry, using sialic acid (SA)-specific lectins and polysialic
acid (PSA) antibodies, we evaluated SA expression in primary breast adenocarcinoma
tissues and brain metastasis. Our results indicated that in healthy tissues, epithelial
glycoconjugates showed higher interaction with Maackia amurensis agglutinin (MAA,
specific for SAα2,3), than Sambucus nigra agglutinin (SNA, SAα2,6) that interacts
mainly with connective tissue and Macrobrachium rosenbergii lectin (MRL, O-acetylSA)
showed no recognition. In ductal breast tumors, MAA and MRL recognized glandular
structures; whereas, SNA showed increased recognition of the periglandular and
glandular structures. In metastasis was identified higher reactivity of pseudoglandular
structures by MAA and MRL, than by SNA. PSA antibodies showed high
immunoreactivity in pseudoglandular–like structures exclusively in brain metastasis.
After trypsin or sialidase treatment of all tissues, reactivity of the lectin and PSA
antibodies was abolished in tumor and metastasis cells. Our results showed increased
expression of O-acetylated SA and PSA in brain metastasis that seems to be
associated to malignant potential in tumor cells.
.
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13
O-glycans in the development of breast cancer.
1
Itandehui Belem Gallegos1 and Pedro Hernandez1*
Centro de investigación en Ciencias Médicas y Biológicas. Facultad de Medicina y
Cirugía UABJO
*[email protected]
Breast cancer is a serious threat to the health of women globally and an unrecognized
priority in middle-income countries. It is the second cause of death among women aged
30 to 54 and affects all socioeconomic groups. Data on detection, although
underreported, show 6000 new cases in 1990 and a projected increase to over 16500
per year by 2020. Genetic, aging, gender and glycosylation changes are risk factors to
develop breast cancer. O-glycosylation plays an important role in biological activity of
glycoproteins involved in control of cell differentiation[1,2]. Alterations in glycosylation of
cell membranes glycocongugates in neoplastic lesions from a variety of organs
including lung, stomach, ovary, skin and endometrium has been reported [3,4].
Abnormal O-glycosylation, specially in mucin and mucin type glyproteins, results in
exposure of peptide core, as well as in the exposure of the normally cryptic core Tn
(GalNAcα1-O-ser/Thr), sialyl Tn(NeuAα2-6GalNAcα1-O-Ser/Thr T (Galβ1-3GalNAcα1O-Ser/Thr) antigens[5], which are distributed discontinuously along the peptide
backbone. Tn antigen is the precursor of T antigen by action of galactosyltranserase
which be deficient in cells and premature sialylation can occur leading to formation of
antigens relationated with cancer progression[5]. Lectins are proteins or glycoproteins
that recognize carbohydrates or precipitate glycoconjugates. They are important tools
for oligosaccharide characterization as well as isolation of cellular population[6].
Lectins with specify for O-glycans are excellent tools in the study of expression
oligosaccharides. In this work we used lectins with different specificity for O-glycans to
determinate O-glycosylation in breast cancer
1. Bulmer, J.C. J Anat 188 (1996).
2.Tanda, N.; Mori, S.; Nose, M.; Saito, T.; Song, S.T.; Sato, A. Pathol Int 46 (1996)
3. Eckart, L.; Haleh, N. Am J Pathol 160 (2002)
4. Kamura, V.; Shanthi, P.; Madhavan, M. J Pathol Microbiol 35 (1992).
5.- Yamashita, Y.; Chunng, Y.S.; Horie, R.; Kanagi, R.; Sowa, M. J Natl Cancer Inst 87 (1995).
6.- Shuman, J.; Dongxu, Q.; Koganty, R.; Longenecker, R.; Campbell, P. Glyconconj J 17 (2000)
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14
F-18-Fluorodeoxiglucose and diagnosis in oncology: Quo vadis
México?
Martínez Duncker, C.
Coordinator of the Molecular Imaging Commission of the Mexican Academy of Surgery
[email protected]
The D-glucose analogue 2-(fluorine-18) fluoro-2-deoxy-D-glucose (FDG) is the most
commonly used radioactive molecule in PET. Increased glucose metabolism by malignant
cells allows physiologic differentiation between benign and malignant abnormalities. FDG
PET is consequently useful in characterizing indeterminate lesions such as pulmonary
nodules and brain, breast and colon tumors, as well as in staging and assessing the
response to treatment. FDG PET is accurate in classification of tumors as malignant or
benign as well as in determining the extent of disease and thus the best therapeutic
approach. The accuracy of FDG PET in demonstrating metastatic disease is greater than
that of computed tomography or magnetic resonance imaging. Whole-body PET is useful
in detection of metastases. Conventional imaging often does not allow differentiation of
tumor from post-treatment scarring. Increased FDG uptake at the sites of residual
radiographic abnormalities is indicative of persistent or recurrent tumor.
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15
Heparan sulphate proteoglycans and congenital disorders of Oglycosylation: biochemical and molecular bases of multiple hereditary
exostoses.
Asteggiano, Carla G.1,2,3
Centro de Estudio de las Metabolopatías Congéntias (CEMECO), Facultad de Ciencias
Médicas, Universidad Nacional de Córdoba, Argentina.2 Cátedra de Química Biológica,
Facultad de Medicina, Universidad Católica de Córdoba, Argentina.3 Consejo de
Investigaciones Científicas y Técnicas (CONICET), Argentina.
E-mail: {HYPERLINK "mailto:[email protected]"} Web page: {HYPERLINK 1
"http://www.cdgargentina.com.ar"}
Heparan sulphate (HS) proteoglycans are ubiquitously expressed at cell surfaces and in
extra-cellular matrices. They are composed of a core protein and one or more heparan
HS glycosaminoglycan chains that interact with numerous proteins, including growth
factors, morphogens and extra-cellular-matrix proteins. The HS proteoglycans play
crucial roles in physiology. On the other hand, under certain conditions, they also
contribute to pathophysiology. In this sense, the truncated HS chains in the
proteoglycans cause the loss of binding to specific growth factors in chondrocytes,
resulting in abnormal signaling that leads to altered endochondral ossification. This
produces a multiple osteochondroma formation leading to multiple osteochondromatosis
/ multiple hereditary exostosis, an autosomal dominant O-glycosylation disorder recently
classificated as EXT1/EXT2-CDG. Products of the EXT genes are involved in HS
biosynthesis. Three loci have been identified to date, EXT1, mapped in 8q24, EXT2,
11p11-p13, and EXT3, located in chromosome 19. The existence of this third locus is
not clear, since most of the cases are caused by mutations in either EXT1 (65%) or
EXT2 (35%). EXT1 and EXT2 genes code for two glycosyltransferases called exostosin
1 and 2, both are part of a hetero-oligomeric complex in the Golgi apparatus. They are
involved in the HS biosynthesis. O-xylosylation initiates the synthesis of a tetrasacharide
linker consisting of xylose-galactose-galactose-glucuronic acid (Xyl-Gal-Gal-GlcA) that
links glycosaminoglycan moieties with the protein backbone in proteoglycans such as
HS. Recent studies presented a hypothetical model for hereditary exostoses formation,
when a chondrocyte bearing a germline mutation in either EXT1 or EXT2 develops an
inactivating “hit” in the second copy of the same gene. This event leads to an impaired
HS biosynthesis and expression, resulting in abnormal endochondral ossification, during
the process of bone remodeling in childhood and early adolescence. Multiple
osteochondromatosis has an estimated occurrence of about 1 to 50,000 in Western
populations and it is clinically characterized by cartilage-capped tumors developed from
the growth plates of long bones or the surface of flat bones. Some common
manifestations are orthopedic deformities of forearm, ankle, varus or valgus of knee,
arthritis, vessels and nerves compression, but the most important complication is
malignant transformation to chondrosarcome. This work presents the biochemical and
genetic basis of EXT1/EXT2-CDG, and the clinical manifestation of multiple
osteochondromatosis (EXT1/EXT2-CDG), a particular disease in the broad spectrum of
Congenital Disorders of Glycosylation.
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16
The COG complex and Golgi glycosylation.
F. Foulquier
UMR8576 CNRS Laboratoire de Glycobiologie Structurale et Fonctionnelle, USTL,
Villeneuve D’Ascq, France.
[email protected]
The basic principle underlying the evolution from the prokaryotic to the eukaryotic cell is
the segregation of cell functions into distinct organelles. This compartmentalization has
considerably increased efficiency, but also generated the need for rapid communication
among these organelles to ensure the correct coordination of cellular functions. The
COG complex is a cytosolic heteromeric Golgi complex constituted of 8 subunits (Cog1
to Cog8) and involved in retrograde vesicular Golgi trafficking. The involvement of this
complex in glycosylation and more specifically in Golgi glycosyltransferases localization
has been highlighted with the discovery of COG subunit deficiencies leading to CDG
(Congenital Disorders of Glycosylation), a group of inherited disorders of glycosylation.
To date, many COG deficient CDG patients have been discovered and this article
reviews the birth and rise of this group of defects. The architecture of the COG complex
and its cellular functions in Golgi.
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17
A new profile of CDG type IIx.
Rosella Mollicone1*, Célia Chambrion1, Jérôme Guéchot2, Hamidou Ryma1, Ivan
Martinez-Duncker3 and Rafael Oriol1.
1
Inserm U1004 and Paris XI University, Paul Brousse Hospital, 16 Ave Paul VaillantCouturier, Villejuif, 94800, France. 2 Saint-Antoine Hospital, GHU-est, Biochimie A
department, 184 rue du Faubourg Saint-Antoine, 75571 PARIS Cedex 12, France. 3
Facultad de Ciencias, Univ. Aut. Edo. Mor., Cuernavaca, 62210, Mexico.
*[email protected]
In 2005, we discovered the first CDG-IIf [1] with a genetic defect in the CMP-sialic acid
transporter gene. It showed megathrombocytopenia, with giant platelets and severe
thrombocytopenia, repeated hemorrhagies and neutropenia with complete lack of sialylLewisx in leukocytes. The patient died at 37 month. With this first case we established a
clinical profile permitting the recruitment of new patients with congenital
thrombocytopenia and lack of sialyl-Lex in leukocytes. A new patient died at 8 months
with this clinical profile. We checked for the CMP-sialic acid transporter transcript
mutations reported for the first patient and only one of the two mutations was found in
this new patient. A profile with a majority of truncated CMP-sialic acid transporter
transcripts is observed, but at variance with the first case a small proportion of wild type
transcripts with normal size is expressed. A heterozygous insertion of 4 bases (CACT)
in the intron 6 of the gene creates a new U2 snRNA site ACTCAAT situated at 5’ from
the regular GAGTGAT. This new U2 is in competition with the putative normal site and
can destabilize the regular splicing mechanism of the gene. Histo-blood group antigen
distribution of the patient fibroblasts shows a dysfunction of the synthesis or the traffic of
the terminal sialylated and/or fucosylated antigens as sialyl-Lex and sialyl-Lea and a 20
times excess of production of hyaluronic acid in cell culture medium and in plasma. In
tissues we observe an increase of macrophages in the connective tissue with aberrant
expression of CD68 and of structure recognized by PNA.
1. Martinez-Duncker I, Dupré T, Piller V, Piller F, Candelier JJ, Trichet C, Tchernia G, Oriol R and
Mollicone R. Blood, 105 2671 (2005)
.
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18
Tools for identifying new human glycosylation disorders.
Ping He1, Marie-Estelle Losfeld1, Mie Ichikawa1, Bobby Ng1, Hudson Freeze1
1
Genetic Disease Program, Sanford-Burnham Medical Research Institute
La Jolla, CA, USA
[email protected]
Over 30 Congenital Disorders of Glycosylation (CDG) were identified in 15 years. The
alliance of savvy clinicians and patient-oriented glycobiologists applying biochemical
approaches to demonstrate mutations in familiar glycosylation genes drove this
progress. Many patients have defects in unidentified glycan-modifying genes, but broad
searching was impractical. Now, falling costs of genomic sequencing and improved
informatics brings analysis of 100’s of possible glycosylation-related genes within reach.
Demonstrating impaired glycosylation narrows the candidate genes, but it seldom
provides a single candidate. Moreover, proving the putative mutated gene is impairs
glycosylation requires complementation of the phenotype. CDG patient fibroblasts
seldom show impaired glycosylation, and indirect methods are needed. We used a
proteomic approach search for proteins that were under expressed in glycosylationdeficient CHO cells. We identified several proteins, but the best candidate was ICAM-1.
When examined in a large series of CDG cell lines, ICAM-1 was deficient based on
FACS, immuno-staining and Western blot. CDG-Ib cells, which are deficient in MPI
(mannose-6-P Å>Fructose-6-P), normalize ICAM-1 staining when provided with
mannose, similar to mannose therapy in CDG-Ib patients. As a second marker we
modified Green Fluorescent Protein (GFP) with an ER-retention signal and a potential
N-glycosylation site. When it is occupied, GFP does not glow, but it does when the site
is unoccupied. In control cells, glow appears only in the presence of tunicamycin, which
blocks N-glycosylation. CDG cells fluoresce due their decreased N-glycosylation.
Addition of mannose to CDG-Ib cells corrects glycosylation and GFP fluorescence
disappears. Both of these markers can be used to test putative defective genes and
have already been used to identify new types of CDG from whole exome or targeted
exome analysis. In the future, we will use these markers in concert to identify other new
types of CDG and for high throughput screening of molecular libraries to search for
potential therapies.
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19
Transcriptional response of genes associated to N-glycosylation and
cold stress during mab production by CHO cells grown under
moderate hypothermia.
Martha Hidalgo-Morales1, Norma A. Valdez-Cruz1, Francisco Bolívar2, Noemí Flores2,
and Octavio T. Ramírez1*
1. Departamento de Medicina Molecular y Bioprocesos. 2. Departamento de Ingeniería
Celular y Biocatálisis. Instituto de Biotecnología, Universidad Nacional Autónoma de
México. Avenida Universidad 2001, Cuernavaca, Morelos, 62210 México.
*Corresponding author: Octavio T. Ramírez. Phone: +52-777-3291646. Fax: +52-7773138811.
*[email protected]
Hypothermic culture of mammalian cells has been a common strategy for improving the
productivity of many recombinant therapeutic glycoproteins. However, little is known
about the molecular changes and response mechanisms involved during cellular
adaptation to hypothermia. In this work, Chinese hamster ovary (CHO) cells were
cultured for 48 or 72 h at 37ºC and then shifted to 30ºC. Twenty-four hours after
temperature downshift, quantitative real time reverse transcriptase polymerase chain
reaction was used to characterize the expression of selected genes associated to Nglycosylation and genes coding for a monoclonal antibody (MAb), endoplasmic
reticulum (ER) chaperones, and cold shock proteins. Results were compared to control
cultures maintained at 37ºC. Genes coding for enzymes localized in the Golgi, such as
β(1,4)-galactosyltransferase I, α(2,3)-sialyltransferase IV, UDP-galactose transporter,
glucosidase, and sialidase, were down regulated in hypothermic cultures. In contrast,
genes involved in lipid-linked oligosaccharides synthesis in the ER and those coding for
selected cold shock proteins were up regulated. Important alterations on genes
involved in galactosylation and sialylation, and in selected genes coding for ER
chaperons were observed. Genes coding for the heavy and light MAb chains were up
regulated in all hypothermic cultures, however, only a slight increase in specific
recombinant antibody productivity was observed when temperature was down shifted at
72 h. Extracellular activity and production of hexosaminidase, mannosidase, and
galactosidase decreased as much as 70% during reduced temperature cultures. The
results of the present work indicate that mild hypothermia improved MAb productivity,
reduced CHO cells metabolism and decreased the potential for extracellular
degradation of N-glycans attached to MAb, confirming the beneficial effects of such
culture strategy. However, hypothermic conditions triggered the cold stress response
and caused an unbalance at the transcriptional level between the initial and branching
steps of the N-glycosylation pathway.
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20
Candida albicans cell wall glycobiology.
Héctor Manuel Mora-Montes
Departamento de Biología, Universidad de Guanajuato, México
[email protected]
The cell wall of the opportunistic pathogen Candida albicans is composed of chitin, glucans and glycoproteins, which are enriched with N-linked and O-linked mannans [1].
Protein mannosylation starts in the endoplasmic reticulum, where the N-mannan core or
the first O-linked mannose are attached to the nascent proteins, and finalizes in the
Golgi complex, where mannosyltransferases further modify the mannans. Despite
mannan biosynthesis has been extensively studied in the non-pathogenic yeast
Saccharomyces cerevisiae, the study of glycosylation pathways in C. albicans has
allowed to assess the importance of these protein modifications for fungal virulence and
interaction with the host immune system. We have generated a collection of mutant cells
lacking key steps in N-linked mannosylation processes and have found that disruption of
both N-linked mannan core trimming and outer chain extension, are required for
virulence, proper cell wall composition, dimorphism, and interaction with components of
the immune system; though the disruption of N-linked mannosylation pathway led to
increased virulence in C. glabrata. Human monocytes produce high cytokine levels
when stimulated with S. cerevisiae, whereas C. albicans barely stimulates cytokine
production. Comparative studies on the cell wall porosity of these organisms have
allowed us to demonstrate that this physical property is tightly controlled by the N-linked
mannan size, and contributes to the differential recognition of these organisms by
human monocytes: In S. cerevisiae the N-linked mannan layer is porous enough to
allow exposure of -glucan at the cell wall surface, thus stimulating a strong cytokine
response, while in C. albicans, mannans form a glycoshield that protects the inner cell
wall components from recognition by immune cells. Finally, we have also found that loss
of protein phosphomannosylation is dispensable for virulence and stimulation of
cytokines by human monocytes, but is required for phagocytosis and the effect of
cationic antimicrobial peptides.
1. Mora-Montes, HM, et al. Future Microbiol 4 1167 (2009)
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21
Glycosylation of proteins in the pathogenic fungus of human
Sporothrix schenckii.
Dr. Arturo Flores Carreón, Universidad de Guanajuato, México
Departamento de Biología, División de Ciencias Naturales y Exactas, Campus
Guanajuato, Universidad de Guanajuato, México
[email protected]
Sporothrix schenckii has largely been known as the ethiological agent of the
sporothricosis which is an acute or chronic subcutaneous mycosis of humans and other
mammals [1]. It is a dimorphic fungus that grows as filamentous and yeast-like forms
during saprophytic and parasitic phases, respectively. The cell wall, the outermost part
of fungi and other organisms, is important to mediate all host-pathogen interactions [2].
In S. schenckii this organelle is constituted of β-glucans and glycoproteins such as
peptidorhamnomannan and peptidorhamnogalactan [3]. The peptidorhamnomannan
contains α-D-glucuronic acid residues that are important as antigenic determinants [4].
In addition, glycoproteins are essential molecules for fungal virulence, cell wall integrity
and host immune recognition. Our group has been interested in the study of
glycosylation pathways in the pathogenic fungi Candida albicans and S. schenckii.
However, little is known about the biosynthesis of glycoproteins (N-linked and O-linked
glycans) in S. schenckii. We have isolated SsCWH41 and SsROT2 genes that encode
for α-glucosidase I and II, respectively (unpublished data). The S. schenckii ER α1,2mannosidase that belongs to glycosyl hydrolases family 47 has been purified and
characterized from mycelia and yeast forms [5]. This 75 kDa membrane-bound enzyme
process the N-linked glycan core Man9GlcNAc2 oligosaccharide in Man8GlcNAc2 isomer
B. Another group of enzymes, mannosyltransferases that participate in N-linked or Olinked glycan biosynthesis are under study. S. schenckii MNT1 has been expressed in a
heterologous system and the resulting recombinant protein has been biochemically
characterized in order to know its role in the biosynthesis of glycoproteins in this fungus.
1. Rippon, WJ Tratado de Micología Médica 351 (1990)
2. Bates, S J Biol Chem 281 90 (2006)
3. Previato JO Exp. Mycol 3 92 (1979)
4. Lopes-Alves LM Glycobiology 4 281 (1994)
5. Mora-Montes HM Mem Inst Oswaldo Cruz 105 79 (2010)
Finnancial support: SEP-CONACYT, CB-2007 No. 83414; DAIP, Universidad de Guanajuato, México.
{PAGE }
22
Synthetic glycoconjugates: A tool for exploring and exploiting the
molecular recognition in medicine.
Vicente Verez Bencomo*, Violeta Fernandez Santana, Maria del Carmen Rodriguez
Montero and Yury Valdes Balbin
Centro de Quimica Biomolecular, Calle 21 y 200, Playa, La Habana, Cuba.
[email protected]
The molecular recognition is on the basis of several biological process. Natural and
synthetic glycoconjugates play a pivotal rol in understanding that principle and also in
their use in medicine. The development of a diagnosis for leprosy1, a monoclonal antihuman group B serotyping reagent2, a glycoconjugate vaccine against Haemophilus
influenzae type b3 using a synthetic antigens, and Very Small Size Particles4 as a potent
adjuvant in cancer treatment will be discussed. 1-Marino-Albernas J, Verez-Bencomo V, Gonzalez L, et al, (1988) Carbohydr. Res 183:175
2-Campos-Valdes MT, Perez-Figueroa S, Verez Bencomo V (1999) Carbohydrate Letters 3: 369
3-Verez-Bencomo V, Fernandez-Santana V, Hardy E, et al (2004) Science 305: 522
4- Labrada Mayrel, Clavell Marilyn, Bebelagua Yanín, León Joel de, Alonso Daniel F, Gabri Mariano R,,
Veloso Roberto C, Vérez Vicente, Fernández Luis E (2010) Expert Opinion on Biological Therapy 10:
153
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PRESENTACIONES EN CARTEL
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SESIÓN DE INMUNOGLICOBIOLOGÍA
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IN1
Regulation of synthesis and transport of CMP-sialic acid during
activation of human CD4+ T naive lymphocytes
Villanueva-Cabello Tania María1,2, Martínez-Duncker R. Iván1*
Laboratorio de Glicobiología Humana, Facultad de Ciencias, Universidad Autónoma
del Estado de Morelos, Av. Universidad 1001, Col.Chamilpa 62209, Cuernavaca, Mor.
2
Instituto de Biotecnología-Universidad Nacional Autónoma de México.
*[email protected]
1
The CD4+ T helper immune cells (Th) are responsible for orchestrating the adaptive
immune response [1]. During an infection, naïve Th cells recognize antigens exposed on
the surface of antigen presenting cells resulting in their activation and differentiation
towards Th1, Th2 or Th17 polarized states that are characterized by secretion of
specific effector cytokines [2,3]. The naïve, activated and polarized cells are
characterized by differential expression of genes that code for α2,6 (ST6Gal1) and α2,3
sialyltransferases (ST3Gal1) resulting in different sialophenotypes that play an important
role in their survival and consequently in the regulation of the immune response
mediated by the apoptotic effects of endogenous lectins like Galectin-1 [4,5]. In order to
have a more integrative view of how the sialylation machinery is regulated, the aim of
this work is to study the extent of gene regulation during Th activation at 0, 24 and 48
hours post-activation (p.a.) beyond STs genes and towards other genes that code for
key proteins involved in the synthesis and transport of CMP-Sia, the donor substrate of
STs. Methods: We determined through real time PCR assays the difference in
expression levels between human naïve and anti-CD3/CD28 activated T CD4+ cells of
genes that code for ST6Gal, ST3Gal1, UDP-N-acetylglucosamine 2-epimerase/Nacetylmannosamine kinase (GNE), CMP-Sia synthetase and the CMP-Sia transporter.
Results: It was observed that although downregulation of the ST3Gal1 and ST6Gal1
genes in activated vs. non activated cells is detected at 24, 48 and 72 hours, the
ST6Gal1 gene is markedly downregulated at 72 hours p.a.. Interestingly, this marked
ST6Gal1 downregulation at 72 hours p.a. was accompanied by marked downregulation
of the GNE and CMP-SiaTr genes pointing towards a possible en bloc downregulation
of these three genes. The CMP-Sia synthetase gene was the only gene that showed
upregulation at all studied times.
1. Zhu, J., H. Yamane. Annu Rev Immunol 28 445 (2010).
2. Krawczyk, C. J Leukoc Biol 69 317 (2001).
3 Nakayama, T. Semin Immunol 22 303 (2010).
4. Daniels, M. A. Nat Immunol 3 903 (2010).
5. Toscano, M. A. Nat Immunol 8 825 (2007).
6. Bi, S. Biochim Biophys Acta 1790 1599 (2009).
{PAGE }
IN2
Association between the glycosylation profile of peripheral blood
CD4/CD8 T cells of rheumatoid arthritis patients and its clinical status.
Marisol Sandoval,1 Ana L. Hohlatcheff,1 Daniel Xibillé,2 Iván Martínez Duncker,3 y José
Luis Montiel.1* 1Laboratorio de Citocinas y Autoinmunidad, Facultad de Farmacia,
UAEM, Cuernavaca, Morelos. 2Hospital General de Cuernavaca, SSA. 3Laboratorio de
Glicobiología Humana, Facultad de Ciencias, UAEM, Cuernavaca, Morelos.
*[email protected]
INTRODUCTION: Rheumatoid Arthritis (RA) is an autoimmune disease characterized by a
chronic inflammatory arthropathy. Classical studies have observed a significant increase in the
galactosylation of IgG in RA patients, however the glycosylation profile of the T cells in RA
patients (RAp) is poorly understood. OBJECTIVE. To compare the glycosylation profile of
mononuclear cell (MNC) and CD4/CD8 T cells from peripheral blood of healthy donors (HD) and
RAp and to associate it with the clinical activity of the disease. METHODOLOGY. All RAp
complied with the 1987 ACR criteria for classification and HD were recruited from a local blood
bank. MNC were obtained from the blood sample by Ficoll-Paque. The glycosylation profile and
CD3/CD4/CD8 phenotyping was performed using specific lectins and antibodies by FACS. The
results were analyzed by FlowJo 7.5 software, significant differences were determined with the
Mann-Whitney test and correlations were performed with Pearson’s test >0.05%. RESULTS.
One hundred and sixteen clinically active RAp and 72 HD were included. Comparing the
glycosylation profile of MNC of HD and RAp, we observed a significant reduction in galactose
β1-3 (60%, PNA), galactose β1-4 (50%, ECL), sialic acid α2-3 (40%, MAA) and NAcGal β1
(30%, Gal-1) in RAp. Additionally, an increase in sialic acid α2-6 (45%, SNA) was seen in RAp.
Gating analysis for CD4+T cells of RAp showed a significant increased binding for lectins ECL,
SNA and Gal-1 with respect to CD8+T cells, while in the HD these changes were not evident.
Correlation analysis between the clinical activity of RAp and its glycosylation alterations, suggest
that a decrease in galactose β1-4, galactose β1-3 and sialic acid α2-3 are associated with high
RA activity, whereas the increase of sialic acid α2-6 seems to associate with low inflammatory
activity. CONCLUSION. Our results suggest that during the development of RA there are
important changes in the glycosylation profile of MNC and CD4+/CD8+T cells, modifications that
could influence the patient’s clinical status. ACKNOWLEDGMENT. This work was supported by
grants from the Consejo Nacional de Ciencia y Tecnologia (SALUD-2007-C01-71161) and
PROMEP-SEP/UAEM (2005-07).
{PAGE }
IN3
Physiological state and hemagglutinating activity in marine
organisms: fish, crustaceans and molluscs
Cristina Pascual1*, Ariadna Sánchez1, Honorio Cruz1, Adolfo Sánchez1, Rossanna
Rodríguez-Canul2, Nancy Herrera-Salvatierra1, Carlos Rosas1. 1Facultad de Ciencias,
Universidad Nacional Autónoma de México, 2 Laboratorio de Inmunología y Biología
Molecular. Centro de Investigación y de Estudios Avanzados del Instituto Politécnico
Nacional, Unidad Mérida.
*[email protected]
The study of marine species has received a great attention by its commercial
importance. Between the main groups are the crustaceans, molluscs and fish. A better
knowledge of the homeostatic mechanisms of these organisms can have diverse
implications in its management, as they are the study of the health state of wild and
culture populations. With this perspective we have studied the physiological indicators in
the hemolymph of shrimps, of the spiny lobster of the Caribbean, the sea bass and the
red octopus. In the different biological models the hemagglutinating activity has shown
to be a very consistent indicator with the nutritional state, the immunological condition
and the response to stress conditions. In Panulirus argus the hemagglutinating activity
significantly decreases in the advanced states of the disease by PaV1, viral infection (p
< 0.05). In the reproducers of Litopenaeus setiferus and juveniles of L. vannamei the
hemagglutinating activity increases significantly with a better nutritional condition (p <
0.05). In Centropomus undecimalis the activity significantly decreases before the acute
exposure to lauril sodium sulphate (40 mg/l for 8 days). The study of wild populations
has allowed obtaining values of reference for Octopus maya, Centropomus undecimalis
and Panulirus argus. The seasonal variation in the hemagglutinating activity presents
interesting association with growth and reproductive process. These results indicate the
participation of the lectins in the immune response and the maintenance of the
homeostasis of the diverse species.
{PAGE }
IN4
Binding of lectins to carbohydrate moieties on hemocytes of the
whiteleg shrimp Litopenaeus vannamei and giant lions-paw scallop
Nodipecten subnodosus.
Norma Estrada Muñoz*, Edwin Velázquez Espinoza1, Carmen Rodríguez Jaramillo,
Felipe Ascencio Valle
Centro de Investigaciones Biológicas del Noroeste. La Paz, B.C.S., Mexico.
*[email protected]
Hemocytes
innate
{HYPERLINK
"http://en.wikipedia.org/wiki/Immune_system" \o "Immune system"} of {HYPERLINK
"http://en.wikipedia.org/wiki/Invertebrates" \o "Invertebrates"}, recognizing and subsequently
eliminating or sequestering invading pathogens or unwanted cells through processes
that include phagocytosis and encapsulation, where lectin-carbohydrate interactions are
important in cell-cell recognition, e.g. phagocyte-pathogen. Lectins are proteins that
form reversible complexes having mono- or oligosaccharide structures; they have been
extensively used for characterization of glycoconjugates in a variety of research areas.
We investigated the specificity and distribution of carbohydrate moieties that act as
receptors for lectins on the hemocytes of two marine invertebrates, the whiteleg shrimp
(Litopenaeus vannamei) and the giant lions-paw scallop (Nodipecten subnodosus).
Circulating hemocytes were isolated and labelled with FITC-conjugated lectins (ConA,
PNA, LEA, UEA, and WGA), and the carbohydrate moieties were analyzed by
fluorescence microscopy. The lectin-binding assays indicated that hemocytes of L.
vannamei, possesses N-acetyl-D-glucosamine, β-galactose, and α-D-mannose
moieties, whereas hemocytes of N. subnodosus have N-acetyl-D-glucosamine, α-Dmannose, and L-fucose determinants. Circulating hemocytes express carbohydrates in
a heterogeneous manner, and the variation on labelling pattern observed among
different cells could help to identify different hemocyte populations. Binding between
lectins and the carbohydrate receptor in the hemocytes were specific, since labelling
was inhibited by a high concentration of the corresponding carbohydrate. Moreover, in
the scallop hemocytes, the L-fucose determinant is only present in dying or dead
hemocytes, suggesting that carbohydrate moieties on the hemocytes could be
recognized as a target to eliminate damage cells in the bloodstream.
play
an
important
role
in
the
{PAGE }
IN5
Caracterización de una lectina sérica y determinación de su
participación en la respuesta inmune de la langosta Cherax
quadricarinatus
José Luis Sánchez Salgado1,3*, Juan José Alpuche Osorno1, Guadalupe
Maldonado Mercado1, Ali Pereyra Morales1, Claudia Sierra Castillo2, Vivanco
Rojas Oscar1, Edgar Zenteno Galindo1,Concepción Agundis Mata1.
1
Laboratorio de Inmunología, Departamento de Bioquímica, Facultad de Medicina,
Universidad Nacional Autonoma de México, México, 2Laboratorio de Biología
celular, Facultad de Ciencias Biológicas, UAEM, México.3 Estudiante del posgrado
de ciencias del mar y limnología, UNAM.
{ HYPERLINK "mailto:*[email protected]" }
Introducción. Los crustáceos representan un grupo económicamente importante para
el acuicultivo. Sin embargo, estos organismos son afectados por una gran variedad de
enfermedades ocasionando graves pérdidas económicas. Las lectinas, presentes en
estos decápodos, participan en los mecanismos de defensa por su capacidad de
reconocimiento a azucares específicos que se encuentran en la superficie de los
patógenos, sin embargo, no se ha esclarecido completamente las rutas o eventos
involucrados. En nuestro laboratorio determinamos las características bioquímicas de la
lectina y su participación en algunos mecanismos de defensa en la langosta Cherax
quadricarinatus (CqL). Metodología. Se obtuvo la hemolinfa y el paquete celular
conteniendo los hemocitos a partir de organismos machos. La lectina del suero se
purificó por cromatografía de afinidad en una columna con estroma de eritrocitos de
rata [1]. Se determinó la concentración de proteínas por el método de Bradford y el
peso molecular por electroforesis (SDS-PAGE), el peso molecular nativo por FPLC por
filtración en gel en una columna de Sephadex-HR-300. La especificidad a azucares y la
dependencia a cationes divalentes se midió por hemaglutinación. Por otro lado, se
determinó la participación de la lectina en los mecanismos de fagocitosis, estallido
respiratorio, y el sistema de la profenoloxidasa. Resultados. Se obtuvo una lectina
(CqL) de 295 kDa conformada por cuatro subunidades, con especificidad a GalNH2,
GlcNH2. Presentó dependencia a los cationes divalente Mn2+ Ca2+. Se observó un
incremento significativo en el índice fagocítico en presencia de CqL, así mismo, se
observó un aumento significativo en la producción de ROI durante el estallido
respiratorio y actividad de la profenoloxidasa en presencia de CqL e
inmunoestimulantes. Los resultado presentados indican que Ch. quadricarinatus posee
una lectina tipo C que participa en la regulación de la actividad fagocítica de los
hemocitos. PAPIIT IN226211 (UNAM, México).
1.- L. Vázquez, L. Jaramillo, R. Lascurain, E.L. Cooper, P. Rosas, E. Zenteno (1996). Bacterial
agglutination by the sialic acid specific serum lectina from Macrobrachium rosenbergii, Comp. Biochem.
Physiol. 113B: 355–359.
Financiado por PAPIIT IN226211 (UNAM, México).
{PAGE }
IN6
1
Identificación de estructuras sacarídicas de la pared celular de
Ustilago maydis reconocidas por Concanavalina A
Soberanes Gutiérrez Cinthia V., 2 Villa-Tanaca Lourdes, 1* Pérez Santiago Alma
D.1Unidad de Bioquímica e Inmunología, Instituto Tecnológico de Oaxaca;
2
Departamento de Microbiología, Laboratorio de Genética Microbiana, Escuela Nacional
de Ciencias Biológicas-IPN
*[email protected]
La pared celular de los hongos es una estructura que da forma a la célula, controla la
permeabilidad celular y constituye el lugar de interacción con el medio externo,
localizándose en ella las adhesinas y un gran número de receptores. Se ha reportado
el uso de Con A como herramienta de prueba para la identificación de receptores
sacarídicos presentes en la superficie de basidiosporas de U. maydis, tanto en células
vivas como en células previamente fijadas con p-formaldehído[1]. La distribución de
azúcares específicos en la superficie de U. maydis en la fase dimórfica, podría ser
importante en el desarrollo biológico y en el proceso infectivo. Por lo tanto, el aspecto
central de esta investigación fue identificar las glicoproteínas de la pared celular
reconocidas por la lectina Concanavalina A directamente en geles de poliacrilamida en
condiciones desnaturalizantes. Se identificaron por microscopía de fluorescencia
estructuras glicosídicas de la pared celular de Ustilago maydis, se aislaron paredes
celulares de levaduras y se liberaron las proteínas de la pared celular utilizando SDS,
liticasa y quitinasa. Se analizaron por secuenciación, las bandas de mayor peso
molecular de las glicoproteínas identificadas.
1.
Santiago M. A. Identificación de receptores glucosídicos para Con A en levaduras de U. maydis.
(2002).
2.
Téllez–Jurado A., Cruz Ramírez M.G, Mercado Flores Y., et al. Mecanismos de acción y respuesta
en la relación de hongos entomopatógenos e insectos. Rev. Mex. Mic. v.30 Xalapa Dic. (2009).
{PAGE }
IN7
Identificación de glicoproteínas antifúngicas en plántulas de teosinte
(Zea diploperennis)
Pérez-Díaz Martha, Castillo-Hernández Rommel, Pérez-Santiago Alma Dolores*
Unidad de Bioquímica e Inmunología, Instituto Tecnológico de Oaxaca
*[email protected]
El maíz y el teosinte guardan una estrecha relación, tanto fenotípica como genotípica [1].
Reportes previos muestran que la lectina de teosinte (TCL), al igual que la del maíz (CCL), se
encuentran en un complejo molecular con la enzima β-glucosidasa [2]. La función principal de la
β-glucosidasa en maíz, está relacionada con la defensa de la planta contra insectos y
patógenos sintetizando agliconas tóxicos (ácidos hidroxámicos) de sus glúcidos. Numerosos
estudios han demostrado un alto grado de correlación entre el contenido de DIMBOAGlc de
maíz y el nivel de resistencia a insectos y patógenos [3]. El objetivo de este trabajo es identificar
estas glicoproteínas en plántulas de teosinte (Zea diploperennis) y estudiar su efecto sobre el
crecimiento y desarrollo de Ustilago maydis. En charolas de plástico con tierra abonada, se
sembraron semillas de teosinte (Zea diploperennis) y a partir de la germinación se monitoreó la
presencia de lectina y beta glucosidasa. Extractos crudos de teosinte preparados con PBS pH
7.2 fueron empleados cada tercer día para medir aglutinación celular y porcentaje de inhibición
en bioensayos en microtitulación. Los resultados indican la presencia de lectina y enzima beta
glucosidasa durante los primeros 5 días de crecimiento, posteriormente la lectina desaparece
mientras que la enzima se incrementa. El complejo molecular lectina-beta glucosidasa de
teosinte presenta actividad antifúngica contra Ustilago maydis.
1. Aragón-Cuevas F., Suketoshi T., et. al. Libro Técnico Núm. 6. 2006.
2. { HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Mart%C3%ADnezCruz%20M%22%5BAuthor%5D" }, { HYPERLINK
"http://www.ncbi.nlm.nih.gov/pubmed?term=%22P%C3%A9rez-Campos%20E%22%5BAuthor%5D" }, {
HYPERLINK "http://www.ncbi.nlm.nih.gov/pubmed?term=%22Zenteno%20E%22%5BAuthor%5D" }, {
HYPERLINK
"http://www.ncbi.nlm.nih.gov/pubmed?term=%22C%C3%B3rdoba%20F%22%5BAuthor%5D" }. {
HYPERLINK "javascript:AL_get(this,%20'jour',%20'J%20Agric%20Food%20Chem.');" \o "Journal of
agricultural and food chemistry." } (13):3783-9. 2003.
3. Esen, A. Plant Physiol.98, 174-182(1992)
Palabras clave: Teosinte, glicoproteínas, Ustilago maydis
{PAGE }
IN8
Partial characterization of glycosylations of an immunodominant
protein of Entamoeba histolytica.
Adriana Obregon Cardenas1, Katiushka Arevalo1, Luis Galan1, Maria S. Flores1*
1
Instituto de Biotecnologia, Facultad de Ciencias Biologicas,
Universidad Autonoma de Nuevo Leon, Mexico
*[email protected]
Amebiasis is caused by Entamoeba histolytica. According to the World Health
Organization, there are 50 million people infected with ambiasis, and approximately
40.000 to 110.000 patients die annually. There is no vaccine against amoebiasis, and
antiamebic drugs have numerous side effects. The development of an effective vaccine
against amebiasis could improve health in developing countries. In our laboratory, we
are working with glycoproteins of E. histolytica, to search better options in the diagnosis
and treatment of amebiasis [1-4]. We identified an immunodominant glycoprotein
recognized by 99% of the sera of patients with amebic liver abscess. We demonstrate
the carbohydrates are immunogenic. The aim of this study was to characterize partially
the carbohydrates by FACE and find out the O-linked and N-linked glycosylations by
Electrospray ionization tandem mass spectrometry (ESI-MS/MS).Five structures were
identified as N-glycosylations and four O-linked glycosylations. This is the first report of
O-glycosylations in Entamoeba histolytica. It is important to know the carbohydrate
structures because they would be used for a vaccine development, or as a drug target
to prevent the synthesis of carbohydrates to damage the parasite.
1.
Flores de Castañeda M. (1995) Procedure to preserve antigens of E. histolytica without enzymatic
inhibitors. USA Patent 5459042.
2.
Flores de Castañeda M. (1999) Preparation of preserved antigens of E. histolytica without
enzymatic inhibitors and their use of in immunological methods. USA Patent 5861263.
3.
Flores de Castañeda M. (2002) Procedimiento para la preservación de moléculas antigénicas, sin
el uso de inhibidores enzimáticos. Patente 209646 IMPI. México
4.
Flores de Castañeda M. (2002) Procedimiento para la preservación de moléculas antigénicas, sin
el uso de inhibidores enzimáticos y su aplicación en métodos inmunológicos. Patente 209648. IMPI.
México.
{PAGE }
IN9
Virulent and non-virulent variants of E. histolytica HM1:IMSS.
Comparison of the terminal sugar in the heavy subunit of the
Gal/GalNAc lectin
Corona Coronel A., Pérez de la Cruz J., Ortega Vargas E., Soto Fajardo R.C., González
Canto A., López Vancell D., López Vancell R*. & Pérez Tamayo R.
Depto. de Medicina Experimental, Facultad de Medicina, U N A M.
*[email protected]
Human infection by Entamoeba histolytica results in different clinical outcomes: many
subjects remain asymptomatic, a few develop acute colitis with diarrhea, and even fewer
show extraintestinal disease, such as amoebic liver abscess. The underlying reasons
for such different outcomes are unclear. Some studies have attempted to uncover
differences in several amoebic virulence factors using various amoebic species and
strains of the parasite (Shah et al, 2005, MacFarlane & Singh, 2006, MacFarlane &
Singh, 2007). The amoebic strain most commonly used in many experiments is
HM1:IMSS. When axenically cultured for prolonged periods, such amoebic strain loses
its original virulence, defined as its ability to produce liver abscesses in susceptible
experimental animals. The existence of two variants: virulent (vEh) and non-virulent
(nvEh), derived from the same strain HM1:IMSS presents a convenient system to
search for differences in the expression of several virulence factors. In a previous study
(López Vancell et al 2010) we showed that Gal/GalNAc lectin (a protein implicated in
several amoebic processes) is equally expressed and distributed in both virulence
variants. Nevertheless, the sugars present in one of the subunits of the heterodimeric
lectin could be different in the two variants. In the present study we have purified the
Gal/GalNAc lectin from each variant, transferred them to a pvdf membrane and
analyzed the terminal sugar present in the heavy subunit using lectins of different
specificity. We found that the terminal sugar in both amoebic variants is a mannose.
References
López-Vancell, R., Arreguín Espinosa, R., González-Canto, A., Néquiz Avendaño, M., García de León,
M.C., Olivos-García, A., López-Vancell, D. & Pérez-Tamayo, R. 2010. “Entamoeba histolytica: expression
and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain”.
Experimental Parasitology 125: 244-250
MacFarlane, R.C. & Singh, U., 2006. “Identification of differentially expressed genes in virulent and
nonvirulent Entamoeba species: potential implications for amebic pathogenesis”. Infect. Immun. 74: 340351.
MacFarlane, R.C. & Singh, U., 2007. “Identification of an Entamoeba histolytica serine-, threonine-, and
isoleucine-rich protein with roles in adhesion and cytotoxicity”. Eukaryot. Cell 6: 2139-2146.
Shah, P.H., MacFarlane, R.C., Bhattacharya, D., Matese, J.C., Demeter, J., Stroupe, S.E., & Singh, U.,
2005. “Comparative genomic hybridization of Entamoeba strains reveal unique genetic fingerprints that
correlate with virulence”. Eukaryot. Cell 4: 504-515.
{PAGE }
IN10
Immunodominant Epitopes of Escherichia coli O157
Lipopolysaccharide Identified by the Phage Display Procedure
Armando Navarro1*, Ulises Hernández1, Luis León, Gabriel Pérez, Delia Licona1, Carlos
Eslava1. 1Departamento de Salud Pública, Facultad de Medicina, Universidad Nacional
Autónoma de México, México.
*[email protected]
The O-antigen repeating units of carbohydrates are related to the antigenic diversity of
Gram-negative bacteria lipopolysaccharides (LPS). Escherichia coli O157 strain is
responsible for outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome
(HUS), which are similar clinical manifestations that have been reported recently in the
European outbreak associated with E. coli O104. The aim of this study was to identify
the immunodominant sites of E. coli O157 LPS using the Phage Display procedure.
Methods: IgG from immunized rabbits with the E. coli O157 LPS were used to select
phages that express peptides (mimotopes) from a library obtained from the surface
phage protein III. Results. Fifteen phagotopes (phage-expressing peptides) were
selected, and the nucleotide bases and corresponding peptide sequences obtained.
Four phagotopes (Fgtp-5, Fgtp-11, Fgtp-12, Fgtp-16) were selected to immunize rabbits
in order to obtain antibodies. The IgG reactivity of S-Fgtp-5, S-Fgtp-11, S-Fgtp-12, SFgtp-16 was analyzed by ELISA assays, and a positive response against each of the
respective phagotopes (p <0.05) was identified. To confirm that the observed response
was directed against the selected peptide mimotopes, rabbits were immunized with PS5, SPS-11, PS-12 and SPS-16 synthetic peptides. The rabbit serum samples were
evaluated by ELISA test against both the peptides and E. coli O157 LPS. The results of
this assay showed that the sera anti-peptides SPS-11 and SPS-12 reacted with the
homologous peptides in a 1:3200 serum dilution, and with E. coli O157 LPS in a 1:800
dilution. The anti-LPS O157 serum reactivity was evaluated against PS-11 and PS-12
peptides, with a positive result being identified in a 1:1600 serum dilution (p <0.05).
Conclusion. The results showed that Phage Display is a suitable method for selecting
phagotopes that express peptide sequences, which are mimotopes of immunodominant
epitopes of E. coli O157 LPS.
[1].
[2].
Hernandez-Chiñas U., et al. Peptides 30: 2127-2135.
Smith G. P. Science 1985; 228: 1315-1317.
{PAGE }
IN11
Naegleria fowleri uses D-mannose residues in N-linked glycans to
adhere and infect the nasal mucosa of mice.
M. Maricela Carrasco-Yépez1, Arturo Contis-Montes de Oca1, X. Liliana OlivaresLópez1, Lucio Rivera-Santiago1, Ma Concepción Peña-Juàrez1, Marco RodríguezMonroy2, Patricia Bonilla-Lemus2, Rafael Campos-Rodríguez1, Saùl Rojas-Hernández1.
1. Departamento de Investigación y Postgrado, Escuela Superior de Medicina, Instituto
Politécnico Nacional, Plan de San Luís y Díaz Mirón, México, D.F., México,
2. UIICSE-POOYECTO CyMA, FES-Iztacala, Universidad Nacional Autónoma de
Mèxico, Avenida de los Barrios. Los Reyes Iztacala, Tlalnepantla, México.
Naegleria fowleri is an etiological agent of primary amoebic meningoencephalitis (PAM)
in humans and laboratory animals. Pathogenic Naegleria have been isolated from the
environment, but the determinant of virulence and pathogenicity are unknown. The
mechanisms by which N. fowleri adheres to nasal epithelium have not been reported. In
this contribution, we analyzed the composition of glycoconjugates containing Dmannose on the surface of N. fowleri and their role in the infection used specific lectins
(Con A, GNL and PSA) that recognize D-mannose on glycocunjugates. When the
trophozoites were incubated with 50 μg of each lectins and were inoculated by i.n. route
in mice Balb/c, with Con A was observed 90% of survival, while GNL and PSA the
survival was of 80%. For considerer that lectins are not toxic for N. fowleri, we incubated
the trophozoites with 50 μg each lectins for two hours and we observed than lectins are
not toxic for N. fowleri. Con A GNL and PSA were able of adhere on the surface of N.
fowleri, although Con A recognize greater amount of glycoconjugates than GNL and
PSA. When the surface proteins of N. fowleri were incubated with these lectins, Con A
and GNL recognized many glycoconjugates from 250 to 10 kDa and PSA only
recognized two glycoconjugates of 257 and 73 kDa, but the band of 73 kDa was
recognized by the streptoavidin peroxidaxe, suggest than this protein is a
transcarboxilase has been reported than protein biotinilated are transcarboxilases. Nglycoconjugates with terminal α (1-3)-linked mannose residues participate in the
adhesion and subsequent infection and cytotoxicity of N. fowleri in mice.
{PAGE }
IN12
Role of glycans in the recognition of sialic acid by the 2009 pandemic
influenza virus hemagglutinin.
1
Juan A. Castelán-Vega1*, Alicia Jiménez-Alberto1, Rosa M. Ribas-Aparicio1
Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, México
*[email protected]
The influenza virus hemagglutinin is a glycoprotein that binds sialic acid residues
present on cells from the respiratory tract. It has been shown that removal of glycans in
the vicinity of the receptor binding site increases sialic acid binding affinity [1, 2]. To
elucidate the mechanism that modulates receptor affinity, we performed molecular
dynamics simulations with the structure of the 2009 pandemic virus hemagglutinin
complexed with sialic acid receptor. We found that glycans interact with an aspartic acid
residue (D222) from the receptor binding site; this interaction prevented D222 from
making proper contacts with sialic acid. Binding free energy of sialic acid with the
glycosylated hemagglutinin was higher than that of sialic acid with a non-glycosylated
variant, which confirmed the destabilizing role of glycans in receptor binding.
1. Wang, CC et al. Proceedings of the National Academy of Sciences U S A. 106 18137 (2009)
2. Yen, HL et al. Proceedings of the National Academy of Sciences U S A. 106 286 (2009)
{PAGE }
IN13
Papel del glicocalix celular en las interacciones con el virus del
Dengue
Gabriela Fuentes-García, Sandra Ortega-Francisco, Jorge Omar Pozo-Aguilar, Janet
García-Pillado, Jacqueline Barrios-Palacios, Verónica Monroy-Martínez, Blanca RuizOrdaz*.
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México
*[email protected]
La fiebre por Dengue es la enfermedad viral más prevalente transmitida por mosquitos
(Aedes aegypti, Aedes albopictus) en el mundo. Aproximadamente el 55% de la
población se encuentra en riesgo de padecerlo debido a que habitan en zonas donde el
vector está presente. Más de 100 millones de casos ocurren anualmente con un rango
de mortalidad del 5%. El Dengue es un problema de salud pública a nivel mundial, en el
90-95% de los casos el dengue es una enfermedad autolimitante pero en un 5-10% se
pueden presentar las manifestaciones severas de la enfermedad. Los mecanismos
patogénicos involucrados en el establecimiento de los casos más severos, hasta la
fecha no han podido ser dilucidados (ya que no existe un modelo animal para su
estudio) a pesar de numerosos estudios que se han llevado a cabo en los últimos años
[Halstead, 2007]. Actualmente no existe una vacuna segura y efectiva ni un tratamiento
antiviral específico, por lo que el conocimiento de las interacciones tempranas entre el
virus y su célula blanco nos podría permitir proponer una alternativa para su control
(antiviral). Estas interacciones entre el hospedero y los microorganismos patógenos
tienen un papel importante durante la invasión tisular. Se ha observado que los
carbohidratos superficiales son moléculas esenciales en el reconocimiento de
diferentes agentes virales ya que su localización les permite ser el primer contacto con
el agente patógeno [Smith and Helenius, 2004]. Por lo que nosotros proponemos
evaluar el papel del glicocalix celular de monocitos y hepatocitos que son células
importantes en la patogenia y la progresión del virus del Dengue.
1. Halstead, S. Lancet 370: 1644–52 (2007).
2. Smith, A.E. and Helenius A. Science 304 237-242 (2004)
{PAGE }
IN14
Glycosylation alterations of the A549 Cell Line Infected By Influenza
Virus
Genoveva Bustos Rivera B.,1 Marisol Sandoval,1 and José Luis Montiel.1* 1Laboratorio
de Citocinas y Autoinmunidad, Facultad de Farmacia, UAEM, Cuernavaca, Morelos.
*[email protected]
INTRODUCTION. Flu is an acute respiratory disease caused by the infection of the
influenza virus. Several studies suggest an association between the severity of the
disease and the presence of a massive local inflammatory response, also known as
“cytokine storm”. However, it’s still poorly characterized the series of cellular events that
occurs during the influenza infection, which can modulate the inflammatory response.
Recent studies have shown that influenza virus is able to enter into N-glycosilation
deficient cells but with a diminished replication capacity. OBJECTIVE. To compare the
glycosylation profile of cells in vitro infected by the influenza virus and mock cells.
MATERIAL AND METHODS. In vitro cell infection was done employing the influenza
virus H1N1 (New Caledonia/99) in A549 cell lines. The cell infectivity was monitored by
hemagglutination and lytic plaque-assays. Cells only with culture media were employed
as control of glycosylation. The presence of NS1 was done by western blot and FACS.
The glycosylation profile was performed using specific biotinylated-lectins by FACS and
the results were analyzed by FlowJo v.7.5 software. PRELIMINARY RESULTS. In both
cell systems, infection by the influenza virus (MOI 5.0) at 24 hrs infection was confirmed
by hemagglutination assay, lytic-plaque assay and expression of the non-structural
protein NS1. In comparison with control cell, we observed a significant reduction in
galactose β1-4 (69.3%, ECL), sialic acid α2-3 (72.1%, MAA), sialic acid α2-6 (37.55%,
SNA) and galactose β1-GlcNAc (31.1%, Gal-1) on the surface of infected cells.
CONCLUSION. Our preliminary results suggest that during the influenza infection in
vitro occur important changes in the glycosylation profile of infected cells. Some
modifications could be responsible by the viral neuraminidase, but others could results
of the intracellular cell signaling changes induced by the virus. ACKNOWLEDGMENT.
This work was supported by grants from SEP/CONACYT (82772) and PROMEPSEP/UAEM (2006-08).
{PAGE }
IN15
Acetylcholinesterase glycosilation on leucemic leucocytes and REH
cells.
1*
Palomec-Sánchez Guillermina, 1*Pérez-Aguilar Benjamín 1*Salinas-Arreortua Noé,
3
Fabiola Mujica Guzmán, 3Lina T. Romero Guzmán, 2Patricia Pérez Vera, 1GómezOlivares José Luis. *Posgrado en Biología Experimental. 1Departamento de Ciencias de
la Salud. Universidad Autónoma Metropolitana-Iztapalapa. 2Laboratorio de cultivo de
tejidos, Instituto Nacional de Pediatría. 3Departamento de análisis clínico y estudios
especializados, Instituto Nacional de Pediatría.
*[email protected]
Introduction. Acetylcholinesterase (AChE) classic function is the acetylcholine
hydrolysis during the nervous and neuromuscular transmission besides it presents noncholinergic enzymatic activity in other tissues. AChE has been suggested as direct
participant on hematopoietic cells development on bone narrow using AChE as a
megacariopoietic marker. AChE gene suppression on 7q22 locus is associated to
mielodisplasic syndromes, as myeloid leukemia (MLA). AChE need to be processed
post-traductional (glycosilated) to function optimally.Objective. Determine the enzymatic
activity and glycosilation of the acetylcholinesterase on leukemic leucocytes and REH
cells.Methods. We obtained peripheral blood of 50 healthy kids (LcC) and from 30 kids
with some type of leukemic (LcH). Leukocytes were separated by density gradient. We
realize the propagation of the REH line cell of type Pre-B leukemia. AChE was obtained
from lymphoid cells using a buffer solution Herpes-salt with and without Triton X-100 on
a 1:10 (P/V) relation. Enzymatic activity was estimated by the Ellman method. AChE
glicosilation changes on distinct groups were studied using supernatants and let it
interact with lectins of distinct specificity.Results and discussion. The interaction
percentage with lectin showed that AChE is incorporated to the secretion way that
requires the rough endoplasmic reticulum and Golgi complex. AChE from leukocytes
control showed an average interaction with lectins; with-A LCA that show the
incorporation of manose, glucose and manose associated to fucose centers,
respectively. We found a low incorporation of N-acetylglucosamine or galactose (WGA)
and a null incorporation of sialic acid (RCA). In the other hand, AChE from REH cells
showed a greater incorporation on the first three cases probably related to an AChE
activity increment. We suggest to study if the post-traductional modifications are related
to AChE gene mutations or alterations on specific glycosiltransferases activity.
{PAGE }
IN16
Galectina-10 en Enfermedad Respiratoria Exacerbada por Aspirina
Jiménez-Torres CY, Cruz-García ML, Montes-Vizuet R, Velázquez-Rodríguez JR,
Terán-Juárez LM, Negrete-García MC.
Depto. Inmunoalergia y Asma, Instituto Nacional de Enfermedades Respiratorias.
*[email protected]
INTRODUCCIÓN: La Enfermedad Respiratoria Exacerbada por Aspirina (EREA) es un
padecimiento que se caracteriza por presentar asma, intolerancia a la aspirina,
rinosinusitisis crónica y poliposis nasal [1]. Las galectinas son una familia de 15
lectinas que tienen una alta afinidad por oligosacáridos que contienen betagalactósidos. Algunos miembros de esta familia se encuentran involucrados en la
respuesta inmune y en procesos de tipo inflamatorio [2]. La Galectina-10 es el principal
componente de los gránulos de eosinófilos y basófilos, pero a la fecha no se le conoce
alguna función biológica. Sólo existen algunos datos sobre su expresión en la Rinitis
alérgica [3,4]. Actualmente no existe algún reporte sobre la posible participación de
esta proteína en enfermedades de tipo inflamatorio como la EREA, por lo que
decidimos analizar su expresión local en este padecimiento. OBJETIVO: Analizar la
posible liberación de galectina-10 en lavados nasales de pacientes con EREA pre y
post reto nasal con L-aspirina. METODOLOGÍA: El grupo de estudio estuvo
conformado por 15 pacientes con EREA, 10 Asmáticos Tolerantes a la Aspirina (ATA) y
15 sujetos sanos. Se les realizó un lavado nasal (LN) pre y pos reto nasal con Laspirina [5]. La galectina-10 se determinó por ELISA. RESULTADOS: En condiciones
basales, en los pacientes con EREA (0.045±0.046 vs 0.015±0.011p<0.001), y en los
ATA (0.034±0.048 vs 0.015±0.011, p<0.05) se encontró mayor expresión de Galectina10 comparado con el grupo de sujetos sanos. Posterior al reto nasal con L-ASA éste
incremento resultó más evidente en los EREA. CONCLUSIONES: Este es el primer
estudio donde se reporta una inmunoreactividad exacerbada de galectina-10 en
pacientes con EREA en lavados nasales, por lo que suponemos una posible
participación de ésta proteína en la patogénesis de esta enfermedad. Sin embargo se
requieren más estudios para determinar su función biológica específica.
[1] Farooque S, Lee T. Aspirin-Sensitive Respiratory Disease. Annu. Rev. Physiol. 2009; 71: 465-487.
[2] Rabinovich GA, Rubinstein N, Toscano MA. Role of galectins in inflammatory and immunomodulatory
processes. Biochimica et Biophysica Acta. 2002; 1572: 274-284.
[3] Ghafouri B, Irander K, Lindbom J, Tagesson C, Lindahl M. Comparative Proteomics of Nasal Fluid in
Seasonal Allergic Rhinitis. Journal of Proteome Research. 2006; 5: 330-338.
[4] Benson M, Langston MA, Adner M, et al. A network-based analysis of the late-phase reaction of the
skin. J Allergy Clin Immunol. 2006; 118 (1): 220-225.
[5] Nizankowska-Mogilnicka E, Bochenek G, Mastalerz L, Swierczynska M, et al. EAACI/GA2LEN
guideline: aspirin provocation tests for diagnosis of aspirin hypersensitivity. Allergy. 2007; 62: 1111–1118.
{PAGE }
SESIÓN DE GLICANOPATÍAS
{PAGE }
GP1
Characterization of P1 promoter region of the human beta-galactoside
alfa2,6-sialyltransferase gene in cervix carcinoma cell line
Verónica Vallejo-Ruiz1*, Lorena Milflores-Flores1, Lourdes Millán-Pérez-Peña2, Gerardo
Santos-López1, Julio Reyes-Leyva 1.
1
Centro de Investigación Biomédica de Oriente, IMSS; Mepetec, Puebla; 2Centro de
Química, Benemérita Universidad Autónoma de Puebla.
Benemérita Universidad Autónoma de Puebla.
*[email protected]
The addition of sialic acid in alpha2,6-linkage to the galactose residue of lactosamine
(type 2 chains) is catalyzed by the beta-galactoside alpha2,6-sialyltransferase (ST6Gal
I). This enzyme shows a complex pattern of regulation which allows its tissue- and
stage-specific modulation. ST6Gal I gene is regulated through the use of alternative
promoters that originate in mRNA isoforms with different 5’ untranslated regions. The
hepatic transcript result of the promoter I activity and an increased level of this isoform
has been reported in cervical cancer. We characterized the P1 promoter region using
luciferasa assays in cervical and hepatic carcinoma cell lines. Mutation of the HNF1 site
reduced luciferasa activity in hepatic cell line but not in C33A cell line, supporting that
regulation is cell type specific. Deletion of a 5’ fragment of 900 bp increased the
promoter activity more than 600% in C33A cell line and more than 200% in HepG2 cell
line. The minimal promoter was defined by the nt -13 to +20 region in the C33A cell line,
but in the HepG2 cell line the minimal region with transcriptional activity was the nt -13
to +66. We identified an initiation site at the position +7, mutation.
Project financed by Mexican Institute for Social Security: FIS/IMSS/PROT/MD10/865.
{PAGE }
GP2
Over expression of mucine-type glycosylation in cervical cancer
recognized by the Machaerocereus eruca agglutinin
Carlos J. Solórzano Mata1, Concepción Agundis1, América Gutiérrez2, Jaime Berumen2,
Jorge Guevara1, Guillermo Mendoza1, Edgar Zenteno1*
1
Facultad de Medicina, Universidad Nacional Autónoma de México, México,
2
Hospital General de México, Secretaria de Salud, México
*[email protected]
The invasive capacity of cervical cancer is the most relevant feature of this cancer type.
It has been suggested that glycosylation is involved in carcinogenesis and the invasive
capacity of cervical cancer [1]. In this work, we analyzed mucin type O-glycosylation in
biopsies of invasive cervical cancer in FIGO stage II B through histochemistry using
lectins specific for O-glycosidically linked glycans. Our results showed that the lectin
Machaerocereus eruca (MeA, specific for Fucα1,2 (GalNAcα1,3) Galß1,4 in complex
mucin structure) [2], showed increased recognition of tumoral cells and tumoral tissue
stroma as compared to other lectins with similar specificity for O-glycans; healthy
cervical tissue, was negative for MeA. The treatment with trypsin of recognized tissues
abolished MeA’s recognition; moreover, interaction of MeA was inhibited with
oligosaccharides from porcine stomach mucin. As demonstrated by western blot of 2-D
electrophoresis, this lectin recognized in cancer cervical lysate a complex of 10
glycoproteins from 122 to 42 kDa. The LC-ESI-MS/MS analysis of the MeAs’ recognized
peptides matched with the amino acid sequences of 6 proteins , Lamin A/C, Vimentin,
Elongation factor 2, keratin 1 and beta actin; the POTE (an ankyrin domain family
member E) showed the lower rate of matched peptides. Our results suggest that MeA
recognizes a complex of overexpressed O-glycosidically-linked proteins that play a
relevant role in cervical cancer’s invasive capacity.
1.
2.
.
Santaella A. and cols., Prep Biochem Biotechnol 37 (2007)
Zenteno E. and cols., Glycoconj J 12 (1995)
{PAGE }
GP3
Characterization of saccharide epitopes presents in MUC1 expressed
in tumor cells of patients with lung adenocarcinoma.
Ana Karina Saldaña1* José Agustín Atzin1, , María Eugenia Vázquez2, Patricia
Gorocica. Depto. Bioquímica1, Depto Patología2. Instituto Nacional de Enfermedades
Respiratorias
*[email protected]
Mucins are high molecular weight glycoproteins characterized by carbohydrate attached
via O-glycosidic linkages to serine or threonine. Alterations of mucin-associated
carbohydrates expression can be detected in neoplastic epithelial tissues in patients
with adenocarcinomas. These alterations accompany the development of cancer and
influence cellular growth, differentiation, transformation, adhesion, invasion and immune
surveillance. MUC1 is expressed in the apical side of normal epithelia. MUC1 changes
their distribution, structure and immunogenicity in adenocarcinomas, presenting specific
glycoforms in each type of tissue. MUC1 is hypoglycosylated on the entire surface of
adenocarcinoma cell membranes. So, some saccharide epitopes are exposed as the
Thomsen–Friedenreich antigen (TF or T) (β-Gal-[1-3]-α-GalNAc-O-serine) and its
precursor Tn (GalNAc-O-serine) are exclusively expressed in carcinomas but not in
normal tissues, as would be expected to have a better antitumoral response. The
immunogenic properties of the glycosylated peptides in comparison to their nonglycosylated counterparts have not been determined clearly. It has been suggested that
mucins and their carbohydrate-associated antigens may be implicated in tumour
spreading, which may be also influenced by an anti-MUC1 immune response.
OBJETIVE: Identify saccharide epitopes in MUC1 expressed in tumor cells of patients
with lung adenocarcinoma. METHODS. Histological sections embedded in paraffin were
obtained from patient’s primary lung tumors and metastatic tissues. The patients were
diagnosed as having pulmonary adenocarcinoma in clinical stages III and IV and died in
the INER. It was determined the pattern of saccharide tumor antigens and phenotypes
of infiltrating T cells by lectin histochemistry and immunohistochemistry with monoclonal
antibodies. The qualitative and quantitative analyses were performed by light
microscopy and digital image analysis using IMAGE J software. We used the Spearman
correlation coefficient for data analysis. RESULTS. The tumor cells either primary tumor
or tissue with metastasis expressed hypoglycosilated MUC1 in high intensity and
distribution throughout the membrane and cytoplasm. T antigen glycoforms were
identified using 3 lectins: Peanut lectin recognizes 12.65% MUC1 expressingneoplastic cells, Amaranthus lectin 9.21%, and Jacalina lectin 7.96%. In addition,
MUC1 correlated with Tn antigen (r = 0.95), but not with T antigen (r = 0.16) or with LeX
and SLEX. No correlation was found among Tn antigen-bearing MUC1 and CD8+ T cells
(r = -0.17), CD4+ T cells (r = -0.25), and CD57+ T cells (r = 0.48). CONCLUSION.
Hypoglycosilated MUC1 contain Tn antigen in tumor cells from lung adenocarcinoma
patients
Project financed by CONACYT: SNI-2008/90243
{PAGE }
GP4
Reduction of the metastatic potential of a human hepatocellular
carcinoma cell line by knockdown of the CMP-Sialic acid transporter
gene.
Roberta Salinas Marín, Iván Martínez Duncker*
Laboratorio de Glicobiología Humana, Facultad de Ciencias, Universidad Autónoma
del Estado de Morelos, Av. Universidad 1001, Col.Chamilpa 62209, Cuernavaca, Mor.
[email protected]
1
Introduction: The metastasic potential of Hepatocellular carcinoma (HCC) has been
correlated with overexpression of the sialylated tumor antigen SLeX 1,2,3, which allows
binding of circulating tumor cells to E- and P-selectins expressed by the vascular
endothelium causing eventually their extravasation and establishment of secondary
tumor sites4. The reduction of the metastasic potential of HCC is crucial to improve
patient prognosis. We propose to obtain this reduction by decreasing expression of the
SLeX antigen using antisense oligos to knock down the expression of the SLC35A1
gene. The SLC35A1 gene codes for the Golgi cytidine monophosphate-sialic acid
transporter (CMP-SiaTr), responsible for subministering CMP-Sia to the Golgi lumen
where sialyltransferases use it to synthesize sialylated glycans, including the SLeX
antigen. Methodology: HepG2 cells, a human HCC cell line, were used as an in vitro
model of HCC. HepG2 cells were treated with a morpholino antisense oligo that was
designed to induce a non functional alternative splicing (del177 isoform) of the
CMPSiaTr gene. The splicing effect was determined by conventional PCR and the
reduction in wt functional transcripts was quantified by qPCR. The SLeX cell surface
profile was determined by flow cytometry. Results: we obtained a 40-60% reduction in
the expression of the wt transcript of the CMP-SiaTr gene by inducing non functional
alternative splicing using morpholino antisense oligos. Phenotipically this caused a 40%
reduction in the expression of the SLeX antigen. Conclusions: The knockdown of the
SLC35A1 gene seems a promising alternative for reducing metastasic potential,
particularly by using morpholino antisense technology that is being validated for its use
in humans.
1.-Guo, P. J Exp Clin Cancer Res 22 135 (2003)
2.-. Fei, L., Eur J. Biochem 268 3501 (2001)
3.- Liu, F. British Journal of Cancer 84 1556 (2001)
4.- Zhang, B. H. Hepatobiliary Pancreat Dis Int 1 80 (2002)
{PAGE }
GP5
Hepatitis C virus capside protein alters the expression of Lewis and
sialyl-Lewis antigens in hepatic cells
Jacqueline Solares Tlapechco1,2, Nora H. Rosas Murrieta2, Verónica Vallejo Ruiz1,
Francisca Sosa-Jurado1, Julio Reyes Leyva1, Gerardo Santos López1*
1
Centro de Investigación Biomédica de Oriente, IMSS; Mepetec, Puebla; 2Centro de
Química, Benemérita Universidad Autónoma de Puebla.
Benemérita Universidad Autónoma de Puebla.
*[email protected]
Hepatitis C virus (HCV) represents an important public health problem world wide. It
cuases chronic hepatitis, cirrhosis and hepatocellular carcinoma [1]. Expression of HCV
capside protein is associated with malign transformation in liver cells [2]. Malign
transformation involves several biomolecular changes included alterations in cell
glycosylation, with notable increases in the distribution and amounts of fucose- and
sialic acid-containing glycoconjugates [3]. The objective of this work was to evaluate the
expression level of Lewis antigens (Lea, sLea, Lex, sLex) and sialic acids (α2,3- and
α2,6-linked) in the human liver cell line HepG2 transfected with the HCV capside
protein. The open reading frame coding for the capside protein was amplified from a
sample of the HCV genome, inserted in an expression vector pCDNA3.1, and
transfected in HepG2 cells. Tumoral Lewis antigens and sialic acids were identified by
means of flow cytometry using specific antibodies and lectins. We found that
transfection of HCV capside protein enhanced the expression of sLex antigen and sialic
acids in HepG2 cell membranes. These results suggest that capside protein is involved
in the origins of glycosylation changes associated with chronic liver disease and
development of hepatocellular carcinoma.
1. Te HS y Jensen DM. Clin Liver Dis 14 1 (2010)
2. Hu Z, et al. Cancer Science 100 2465 (2009)
3. Chrostek L. et al. Ann Hepatology. 10 150 (2010)
{PAGE }
GP6
Isolation, characterization and function of lectins from the coronary
luminal membrane: A mechanism for flow sensing
Sandra Perez-Aguilar, David Torres-Tirado, Juan Ramiro-Diaz, Maureen Knabb, Rafael
Rubio*.
Physiology, Universidad Autonoma de San Luis Potosi, San Luis Potosi, Mexico
*[email protected]
Vascular endothelial vascular luminal membrane (VELM) is coated with a variety of
proteoglycans, glycosaminoglycans, glycoproteins and glycolipids. We and others have
established that EVLM glycosilation is a requirement for flow sensing, so these
glycosylated structures may mediate myocardial flow-induced responses. Also several
VELM lectinic proteins, such as VCAM-1, ICAM-1, PECAM-1, integrins, have been
shown to be implicated in the response to flow, and we have shown that lectinic nature
is also a requirement for flow sensing. Evidence indicates a) G-protein coupled
structures are flow sensors and b) some hormone receptors are flow sensitive. Is
because they are lectins?
VELM were isolated and this fraction passed through a lectin affinity column with 3
bound sugars: N- acetylglucosamine, mannose, galactose and retain lectins eluted. 2SDS shows that 15% of the EVLM proteins are lectins. Several individual lectins were
identified by immuno-dot blots. VCAM-1, ICAM-1, PECAM-1 and G-protein coupled
receptors: AT1, α1a, B2, ETA, TxA2, PRL, A2, β1, βENaCs. Preliminary results show that
the effects of α1a, B2 agonists are flow-dependent. Since oligosacharides and lectins
coexist in the ELM, likely form complexes. We propose the following hypothesis.
Oligosacharide: O
Lectin: L,
Flow/Stress
O+L
O-L
Flow-induced signaling could result from formation of O-L. The extracellular domain of
L, a transmembrane protein, interacts with specific endothelial O and if L is a hormone
receptor, its hormonal gain must depend on the oligosacharide environment and flow.
These results indicate that there are important flow sensitive proteins that are lectinic in
nature and are present on VELM.
Funded by CONACyT: SEP-42567, SEP-101850 y SALUD 2004-C01-156, Fulbright-Garcia Robles 9583
{PAGE }
GP7
Luminal endothelial lectins with affinity for N-acetylgucosamine
determine flow-induced cardiac and vascular paracrine responses
Juan Ramiro-Diaz1, Alma Barajas-Espinosa1, Erika Chi-Ahumada1, Sandra PerezAguilar1, David Torres-Tirado1, Jesus Castillo-Gonzalez1, Maureen Knabb2, Ana Barba
de la Rosa3, Rafael Rubio1,
1Physiology, Universidad Autonoma de SLP, San Luis Potosi, SLP, Mexico, 2Biology,
West Chester University, West Chester, PA, 3 Inst Potosino Ciencia y Tecnologia, San
Luis Potosi, SLP, Mexico
*[email protected]
Coronary endothelial luminal membrane (CELM) oligosaccharides (O) and lectins (L)
are involved in flow detection. Flow may induce a reversible L-O interaction causing
flow-induced endothelial (FIE) cardiac effects. Because N-Acetylglucosamine (GlcNac)
is a component of glycocalyx O, we isolated CELM GlcNac-binding L (using GlcNacaffinity probe) and show their role in cardiac and vascular FIE responses. The GlcNacaffinity probe is a 460 kDa GlcNac polymer (GlcNac-Pol). In the heart: 1) Intracoronary
given GlcNac-Pol upon binding to CELM, reduces the FIE inotropic and dromotropic
effects. 2) GlcNac-Pol used as an affinity probe isolated CELM GlcNac-recognizing L;
35 individual L were identified, some of which likely participate in FIE and in GlcNac-Polinduced effects. 3) In isolated blood vessels perfuse at controlled flow rates; FIE
vasodilatation (FIEV) is blocked by: binding GlcNac-Pol to CELM, Hyaluronidase,
LNAME plus Indomethacin and Amiloride; a ENaC blocker. We also show ENac is
present in CELM and is a L. These suggest FIEV is due to: a) FIE ENaC-hyaluronidate
(a GlcNac polymer) interaction, b) release of NO and prostaglandins and c) GlcNac-Pol
upon binding to ENaC inhibits release of paracrine mediators.
Funded by CONACyT SEP-42567 , CONACyT-SALUD 2004- C01-156.
{PAGE }
GP8
Role of luminal endothelial hyaluronidate (HA) on the inhibition by
vasoinhibins (Vi) of Bradikinin (BK) effects
1
Carmen González 1*, 2 Sandra Perez-Aguilar, Rafael Rubio 2
Facultad de Ciencias Químicas, 2Facultad de Medicina, Universidad Autónoma de San
Luis Potosí,México
*[email protected]
Vi are prolactin fragments that on endothelial cells inhibit the BK effects; nitric oxide
release, vasodilatation and angiogenesis. But, the nature and identity for Vi “receptor”,
remains unknown. In addition, we found that luminal endothelial HA hydrolisis upon
treatment with hyaluronidase (Hyalse), reverted the Vi inhibitory effects. We also found
that Vi blocked the NO production induced by flow; an HA dependent process, and this
blockade was also reverted by the Hyalse. These findings have leaded us to
hypothesize that luminal endothelial cell glycosidic structures, specifically HA may act as
a “receptor” for Vi. As a further support to this hypothesis we have isolated and
identified the B2 BK receptor among the endothelial luminal membrane proteins from
guinea pig perfused coronary vessels and showed that B2 BK receptor is a lectin with
affinity to N-acetyl-glucosamine; a 50% component of HA suggesting that B2 and HA in
situ form HA-B2 and the equilibrium: [HA + B2
HA-B2] by a mechanism still not
clear determine Bk effects, thus, Vi upon binding to HA disrupts this chemical
equilibrium, consequently the BK effects: NO production, proliferative and the
vasodilatation. Our results show that for Vi to act require the presence of HA but
whether it binds to it remains to be demonstrated. Is HA Vi “receptor”. is Vi a lectin?.
Also, at the level of the luminal endothelial glycocalix, open novel potential avenues on
the mechanism of action of hormones like prolactin proteolytic fragments.
This work was Funded by CONACyT: SEP- 1945, SEP-42567, SEP-101850 y SALUD 2004-C01-156.
{PAGE }
GP9
Glycation and glycosylation of seric cholinesterases on diabetic rats
treated with C. ficifolia.
2
Dr. Francisco Javier Alarcón Aguilar, 2Dr. José Luis Gómez Olivares,
1*
Biol. Noe Salinas Arreortua,
1
Universidad Autónoma Metropolitana –Iztapalapa. Profesor Titular C. Departamento de
Ciencias de la Salud. 2Posgrado en Biología Experimental. UAM-I
*[email protected]
High glucose levels on Diabetes mellitus favor protein glication. On the other side, on
endoplasmic reticulum occurs a stress that gives as result an anomalous posttraductional processing of proteins. Traditionally, there exist drugs that may reduce high
glucose levels but there a lot of people that make use of medical plants as Cucurbita
ficifolia, which has hipoglucemic activity.In this work, we determinate activity, glication
and glicosylation levels of seric cholinesterases using CD-1 strain rats. We separated
the rats on 3 groups: 1) control, 2) diabetic (streptomycin 137.5 g/kg) and 3) extract of
C. ficifolia. Glycation was estimated by binding to a matrix of boronic acid and
carbohydrate incorporation by its interaction with lectins. AChE specific activity was
higher on diabetic rats (1.0 U/mg prot) with respect to healthy cases (0.7 U/mg prot). In
the other hand, we found a decrement on those rats treated with C. ficifolia (0.8 U/mg
prot). BuChE activity showed no decrement on diabetic rats treated with the plant. We
observed an increment on glication on both enzymes. AChE showed an 6% activity
increment on treated diabetic rats and 3% on healthy ones and for BChE showed a
0.5% and 1.2% for healthy and treated diabetic rats, respectively. Finally, ChEs
interaction pattern with lectins showed that DM on both enzymes present an alteration
on oligosaccharides incorporation, Man or Glc (with A), Man associated to fucosilation
centers (LCA), GlCNAc (WGA) and Gal or NANA (RCA), probably related to stress on
endoplasmic reticulum.
{PAGE }
GP10
Glycosylation pattern and expression of trophic factors are induced
by the 25-35 of β-amyloid fragment (Aβ25-35) in C6 in vitro.
Minerva Calvillo1*, Blanca Espinosa2, Aurora Sánchez.3, Edgar Zenteno4, Jorge
Guevara4.1Lab. de Enfermedades Neurodegenerativas. I. N. N. N.; 2Dpto. de
Bioquímica, I. N. E. R., 3Lab.de Patología Experimental, I. N. N. N., 4Dpto. de
Bioquímica Fac. de Medicina U. N. A. M.
*[email protected]
In Alzheimer’s disease (AD), deposits of amyloid beta peptide (Aβ), are observed in
cerebral tissue. It is known that the Aβ is toxic or trophic, both in vivo as in vitro. Aβ
toxicity is dependent on it´s aggregation[1,2]. In addition, in AD the glycosylation pattern
of proteins are altered. The lectins allowed recognized that this alteration is due to
increase on the production of O-glycosylated protein, indicating that there is a reactive
plasticity[3]. Because of this, it is suggested that mechanisms of dendritic and neural
regeneration in AD are exist. These mechanisms are linked to the activation of trophic
and neural growth factors[4]. For this reason, it is important to evaluate the effects of the
Aβ in vitro. Therefore, the aim of this work was to evaluate the effect of Aβ25-35 on the
expression of N and O glycosylated sites in culture cell. Rat C6 glioma cells were
seeded onto eight wells chambers (700 cells per well), three days before incubation with
Aβ25-35. Previously, a stokc solution of Aβ25-35, (1 mM) was prepared in sterile water was
incubated for 24 hours at 37° C. Later, in 4 wells 25 μl (50μM) of Aβ25-35 were added to
the cells and other 4 wells without Aβ25-35, were control group. After 72 hours, cells were
washed with PBS and fixed with 4% paraformaldehyde. Then, were processed to
perform immunocytochemistry with lectins ALL, ConA, MAA, MRL, PNA, SNA and
WGA. The images were captured with a microscope Leica with IM1000 sofware. Aβ25-35
administration modifies the glycosylation pattern increasing the immunoreactivity in this
form: PNA > ConA, WGA > MRL, SNA > ALL, MAA. This enhance of immunoreactivity
was depent of time. Proliferatives and adhesives properties of C6 cells are dependent
on the expression of surface glycoproteins[5], therefore we needed to evaluate to
assess biochemical assays.
1.
2.
3.
4.
5.
.
Cotman C. W. Ann. N.Y. Acad. Sci. 1997; 814: 1-16.
Howlett R. D., et al. Neurodegeneration. 1995; 4: 23-32.
Guevara J. et al. J. Neuropathol.Exp. Neurol. 1998; 57(10): 905-914.
Espinosa B. J. Neuropathol. Exp. Neurol. 2001; 60(5): 441-448.
Broquel P. Biochem. Biophys. Res. Comm. 1991; 178(3): 1437-1443.
{PAGE }
GP11
Effect of hyperglycemia in the glycosylation of tau protein in one
experimental model of alzheimer's disease.
Liliana Lozano(1,3)*, Tony Lefebvre(2), Yobana Pérez (3); Noe Alvarado(3), Ma.Guadalupe
Maldonado(1), Edgar Zenteno(1).(1) UNAM, Facultad de Medicina Departamento de
Bioquímica; (2) l'Université Lille, Unité de Glycobiologie Structurale et Fonctionnelle
CNRS-UMR; (3) Instituto Nacional De Enfermedades Respiratorios “Dr. Ismael Cosío
Villegas” Departamento de bioquímica.
*[email protected]
Alzheimer's disease is a neurodegenerative disease, characterized by the presence of
extracellular amyloid beta (Aβ) plaques, and neurofibrillary tangles. In experimental
models of neurotoxicity and Alzheimer disease using amyloid β peptide 25-35 (Aß2535), has been observed an augment in the synthesis of reactive oxygen species (ROS),
entrance of Ca++, impaired mitochondrial membrane potential and increase in glucose
uptake. Also, an increased phosphorylation of tau protein associated with a reduced OGlcNAcylation (O-GlcNAc) has been described. Diminution of O-GlcNAc favored the
hyperphosphorylation of tau protein and as consequence intensifies this
neurodegenerative effect. The O-GlcNAc is catalyzed by the enzyme O-GlcNAc transfer
(OGT). Actually, three isoforms of the enzyme, the nucleocytoplasmic (ncOGT), the
mitochondrial (mOGT) and the short (sOGT) isoform has been reported. The isoforms
ncOGT and mOGT, moreover, having relevance in the metabolism of lipids and
carbohydrates. Recently, the mOGT isoform has been linked with the beginning of
cellular apoptosis.
For the mentioned, we decide to evaluate the O-GlcNAc and tau protein
phosphorylation in Kelly (which express constitutively at protein tau) and neuroblastoma
SH-SY5Y cells, previously incubated with the peptid Aß25-35. Our results showed and
diminution in the O-GlcNAc linked with a increase in the phosphorylation of tau protein
in the fifth day of incubation with the Aß25-35. Also, our results suggest a major
expression of mOGT isoform in the cells incubated with Aß25-35.
Financiado en parte por PAPIT-UNAM y ECOS México-Francia.
.
{PAGE }
GP12
Alteration of the sialylation patterns and memory deficits by the Aß (2535) injection into hippocampus of rats.
Ramírez Eleazar1, Díaz Alfonso1, Mayoral Miguel2, Guevara Jorge3, Edgar Zenteno3
and Limón I. Daniel1.1Lab. Neurofarmacología, Benemérita Universidad Autónoma de
Puebla, Puebla, México.2Fac. de Med., Centro de Investigación en Ciencias Médicas y
Biológicas, Universidad Autónoma Benito Juárez, Oaxaca, México.3Bioquímica,
Facultad de Medicina, Universidad Nacional Autónoma de México, D.F., México.
[email protected], [email protected], [email protected]
The cell surface is covered with glycoproteins and glycolipids that differ significantly
among the cell types and different stages of mammalian development even they
contribute to homeostasis into organism. The glycosylation of proteins is an enzymatic
co- and post-translational process that takes place mainly in the Endoplasmic Reticulum
and Golgi apparatus [1]. Sialic acid is typically located at the non-reducing terminal
position of oligosaccharide chains on glycoproteins and glycolipids. It participates at
important cellular functions associated with normal development, growth,
communication and survival [2]. Abnormalities in the expression of sialic acids are
related with certain disorders, such as Alzheimer Disease, so the abnormal pattern of
glycosylation appears in the brain as neurodegenerative marker [3,4]. The objective of
this work was evaluated the sialylation patterns and memory deficits caused by the
injection of Aβ(25-35) into the hippocampus of rats [5]. Our results showed that Aβ(25-35)
injection into CA1 subfield of the hippocampus (Hp) impaired both spatial learning and
memory at long-term. After that the altered sialylation patterns were examined using
sialic acid-specific lectin: Maackia amurensis agglutinin (MAA; specific for
Neu5Acα2,3Gal), Macrobrachium rosenbergii lectin (MRL;specific for Neu5,9,7Ac),
Sambucus nigra agglutinin (SNA; specific for Neu5Acα2,6Gal/GalNAc). About the
sialylation patterns we found that the vehicle and Aβ(25-35) increased the MAA reactivity
in the CA1 and the dentate gyrus (DG), whereas another changes on altered sialylation
patterns assessed by MRL and SNA were found increased in the CA1 and DG in the
Aβ(25-35)-treated group. The immunorreactivity for homopolymer of α2,8 linked to sialic
acid increased in the CA1 and DG subfields only for the Aβ(25-35)-treated group
compared to the control group. These results suggest that Aβ(25-35) injection cause
impairments in the spatial learning and memory related with changes on the sialylation
pattern, because its results during the cellular response for the compensatory
reorganization even for the sprouting of dendrites and axons after Aβ(25-35) treatment.
[1] Yuji S. Neuroscience Letters. 262 49-52, (1999).
[2] Jimenez D. Birth defects research (Part A). 73 980-988, (2005).
[3] Guevara J. J Neuropathology Exp Neurology. 57 905-914, (1998).
[4] Mikkonen M. Europea J Neuroscience. 11 1754-1764 (1999).
[5] Limón ID. Neuroscience Research. 63 129-137, (2009).
[6] Espinosa B. J Neuropathology Exp Neurololgy. 60 441-448 (2001).
[7] Méndez-Otero R and Cavalcante L. Neuroscience Letters. 204 97-100 (1996).
[8] Cremer H. Int J Devel Neuroscience. 18 213-220, (2000).
{PAGE }
GP13
The role of nitric oxide in the alteration of glycosylation patterns and
spatial memory deficit induced by injection of Amiloid-β (25-35) in rat
temporal cortex
Díaz Alfonso1, Ramírez Eleazar1, Mayoral Miguel2, Guevara Jorge3, Edgar Zenteno3
and Limón I. Daniel1.
1
Lab. Neurofarmacología, Benemérita Universidad Autónoma De Puebla, Puebla,
México. 2Fac. de Med., Centro de Investigación en Ciencias Médicas y Biológicas,
Universidad Autónoma Benito Juárez, Oaxaca, México. 3Bioquímica, Facultad de
Medicina, Universidad Nacional Autónoma de México, D.F., México.
[email protected], [email protected], [email protected]
The amiloid-β plays an important role in the neurodegeneration process of Alzheimer´s
disease, but its neurotoxic mechanisms are not clear [1]. The Aβ (25-35) fraction mimics
the toxic effects of the complete peptide Aβ(1-42) because this decapeptide is able to
cause memory impairment and neurodegenerative events [2,3]. The Aβ(25-35) increase
oxidative stress, cause neuronal damage, alteration of glycosylation patterns and
decrease the spatial memory in rats [4,5]. Recent evidence has shown that the injection
of Aβ (25-35) into the temporal cortex (TCx) of rats increase the nitric oxide (NO)
pathways, and changes the patterns of glycosylation in response to damage produced
by the peptide Aβ (25-35)[6,7]. The objective of this work were evaluate the role of NO in
the alteration of glycosylation patterns and spatial memory deficit induced by injection of
Amiloid-β (25-35) in rat temporal cortex. Methodology: the animals were tested for their
spatial learning and memory in the eight-arm radial maze. The eight-arm radial maze
test indicated that the injection of Aβ(25-35) into TCx impaired both learning and memory
of rats. The control and Aβ (25-35)-treated group were examined for immunorreactivity for
neuronal or inducible nitric oxide synthases (nNOS and iNOS) in the TCx. We found an
increase for nNOS and iNOS in the Aβ (25-35)-treated group respect to control group. The
altered glycosylation patters was examined using lectin Amaranthus leucocarpus lectin
(ALL, specific for Galβ1,3GalNAcα1,0 Ser/Thr), Macrobrachium rosenbergii lectin (MRL,
specific for Neu5,9,7Ac), Triticum vulgare (WGA, specific for O-GlcNAc). Our results
showed an increase on the ALL, MRL, WGA reactivity in the CTx in the Aβ(25-35) injected
group. Abnormalities in the expression of O-glycans are related to a number of
biological functions that become altered in certain disorders, such as neurodegenerative
disease. Modification in the o-glycosidically linked glycans that is likely to regulated cellto-cell and cell-to matrix interactions, in response to effect neurotoxic of Aβ (25-35). These
data suggest that the Aβ (25-35) injected into the TCx might contribute to understanding
the role of NO on the biological changes related to the neuropathological progression
and the memory impairment in AD.
[1] Selkoe DJ. Biol Chemical. 271 18295-18298, (1996).
[2] Pike CJ. Journal Neurochemical. 64 253-265, (1995).
[3] Stepanichev M. Behavior Brain Research. 175, 352-361.
[4] Guevara J. J Neuropathology Exp Neurology. 57 905-914, (1998).
[5] Espinosa B. J Neuropathology Exp Neurololgy. 60 441-448 (2001).
[6] Limón ID. Neuroscience Research. 63 129-137, (2009).
[7] Diaz A. Pharmacology Biochemistry and Behavior. 98 67-75 (2011).
{PAGE }
GP14
Anomalous glycosylation of cholinesterases on HepG2
hepatoblastome cells.
1*
Pérez-Aguilar Benjamín, 1*Palomec-Sánchez Guillermina, 2Luna-López Armando,
3
Gutiérrez-Ruiz Concepción, 4García-Dolores Fernando, 5Vidal-Moreno Cecilio, 1MillanPacheco Cesar, 1Jardón-Valadez Eduardo 1Gómez-Olivares José Luis. *Posgrado en
Biología Experimental. 1Laboratorio de Biomembranas, 2Bioenergetica y Envejecimiento
Celular, 3Fisiología Celular. Departamento de Ciencias de la Salud. UAM-I. 4SEMEFO,
TSJDF. 5Departamento de Bioquímica y Biología Molecular-A-, Universidad de Murcia,
España.
Introduction. Acetylcholinesterase (AChE) gen suffers an alternative splicing process
giving as result three distinct mRNA: AChE-T (nervous system and muscle cells), AChEH (erythrocytes and lymphocytes) and AChE-R (pathological states). Every protein
variant hydrolyzes acetylcholine (Ach) on nervous transmission of signals and
neuromuscular joint. It has been determinate a low AChE activity when compared with
control tissues on multiple tumors. Butyrylcholinesterase (BuChE) gen suffers no
alternative splicing. BuChE is expressed on liver and its function is to detoxify. Its
participation on cancerous process is unknown. A remarking issue is that both
cholinesterases activity depends on a correct glycosilation. Objetive. In this work, we
evaluate the genetic and protein expression and post-traductional processing of
cholinesterases in HepG2 line cell and human livers. Methods. We extract AChE and
BChE activity using Salt-Hepes buffer with and without Brij-97 on a 1:10 (P/V) relation.
Enzymatic activity was obtained using Ellman method. RNA isolation was made using
Trizol method and using a two-step RT-PCR with specific primers for AChE (H, T, R)
and BuChE. The post-traductional processing was studied using the supernatants
interacting with lectins of distinct specificity. A histopathological analysis was made to
establish healthy control livers. Results. Lower activities of AchE and BuChE were
found on HepG2 than those found on human liver. Human liver and HepG2 cells
expressed all the AChE mRNAs and BuChE transcripts, as expected. Lectin interaction
studies showed that HepG2 had an anomalous post-traductional behavior that appears
to be related to a low enzymatic activity. These results rise multiple questions about
what phenomena (extracellular enzyme transportation, for example) is responsible for
the low enzymatic activity found in this work.
{PAGE }
GP15
Argentinean program for the diagnosis and research of congenital
disorder of glycosylation
Bistué Millón MB.(1); Delgado MA.(1) ;Sturiale L.(2) ;Matthijs G.(3) ;Jaeken J.(4); Dodelson
de Kremer R.(1); Asteggiano CG.(1,5,6).. CEMECO (Centro de Estudio de las
Metabolopatías Congénitas), Facultad de Ciencias Médicas, Universidad Nacional de
Córdoba, Córdoba, Argentina; (2) CNR Institute per la Chimica e la Tecnologia dei
Polimeri, Catania, Italy; (3) Department for Human Genetics, and (4) Center for
Metabolic Diseases Katholieke Universiteit Leuven, Belgium; (5) Cátedra Química
Biológica, Facultad de Medicina, Universidad Católica de Córdoba, Argentina; (6)
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.
*E-mail: [email protected] / *Web page: www.cdgargentina.com.ar
“Congenital Disorders of Glycosylation” (CDG) are a growing group of hereditary
diseases caused by abnormal protein or lipid glycosylation defects. The phenotypic
spectrum is highly variable, ranging from severe multisystem disorders to alteration of
specific organs such as multiple osteochondromatosis (EXT1/EXT2-CDG). There is still
a large group of unsolved cases with altered glycosylation patterns classified as CDGx.Aim: Communicate the advances for CDG research program, as a reference center in
Argentina. Methodology: A) Analysis of serum N-glycoproteins by Wb, IEF and HPLC;
B) Lipid-linked-oligosaccharide analysis in fibroblast; C) MALDI-TOF Mass Spectrometry
and D) Genetic analysis. Results: Glycosylation defects were investigated by a complex
algorithm to detect altered profiles. We found several patients CDG-x that showed
transferrin patterns CDG-Type I (n = 2) and CDG-Type II (n = 3). Their characterizations
are in progress to identify the basic defect. As regards O-glycosylation defects, we have
studied 25 EXT1/EXT2-CDG patients (from 2 to 50 years-old). PCR and direct
sequencing of EXT1 and EXT2 genes were performed. Clinical and radiological
assessments, allow us to detect 70% of the patients with a severe phenotype and a total
of 10 exonic changes. The 80% were novel mutations in the EXT1 gene. One patient
suffers a malignant transformation to chondrosarcoma. Discussion: The extremely wide
clinical spectrum of CDG makes a broad screening for these disorders in children as
well as in adults. The diagnosis of CDG should be considered in each patient with
hypotonia, dysmorphic features and developmental delay with frequent cataract.
Multiple osteochondromatosis seems to have a high frequency in Argentinean
population and this interdisciplinary study represents the first genotype-phenotype
investigation in our country. Likewise, this first Argentinean CDG Program allowed us to
start a new genetic and metabolic chapter in our country. We hope to work in a future
Latin American CDG Network that would make possible the diagnosis and possibility the
research of human glycosylation disorders.
{PAGE }
SESIÓN DE GLICOTECNOLOGÍA
{PAGE }
GT1
Síntesis de oligosacáridos fucosilados por vía enzimática mediante
reacciones de fucosilación
Yolanda Escamilla-Lozano1*, Lorena Gómez-Ruiz1, Gabriela Rodríguez-Serrano1,
Mariano García-Garibay1, Ivonne Figueroa-González1 y Alma Cruz-Guerrero1
1
Universidad Autónoma Metropolitana-Iztapalapa Departamento de Biotecnología.
Ciudad de México
*[email protected]
La Leche humana tiene más de 130 oligosacáridos (OS) [1], de estos, los fucosilados
se encuentran en mayor concentración. Son sintetizados en el aparato de Golgi de las
glándulas mamaria mediante glicosiltransferasas, en estudios in vitro se demostró que
su estructura es semejante a la de receptores de patógenos, actuando como
receptores competitivos en las células intestinales de la superficie del huésped [2] son
inhibidores de la adherencia de bacterias a la superficie de células epiteliales [3]. y son
prebióticos. In Vitro se han estudiado las glicosiltransferasas para la síntesis de fucosiloligosacáridos por su selectividad además no necesitan protección química del sustrato
como la síntesis química, una desventaja es el uso de un donador glicosil activado que
lo hace poco viable en producción a gran escala. Recientemente se ha considerado el
uso de glicohidrolasas para la síntesis de OS por su bajo costo y no poseen las
desventajas mencionadas, sin embargo la dificultad de separación y la poca
especificidad ha generado bajos rendimientos.El objetivo de este trabajo ha sido
estudiar la síntesis de oligosacáridos fucosilados a partir de reacciones enzimáticas de
fucosilación empleando glicosil-hidrolasas. El trabajo se divide en dos pasos: la
comprobación de la viabilidad de la síntesis por esta vía y el establecimiento de las
condiciones de reacción y la síntesis del donador fucosilado para la posterior síntesis
de fucosil-lactosa. Resultados. 1.-Síntesis de fucosil-lactosa: Se sintetizó
un
trisacárido fucosilado empleando PNP-F (0.1%) como donador de fucosa y lactosa
(0.5%) como aceptor, la α-L-fucosidasa se encargó de catalizar la reacción. 2.-Síntesis
de disacáridos fucosilado: se sintetizaron dos disacáridos fucosilados: el β-disacárido
fucosilado que con análisis posteriores ha probado tener un potencial prebiótico y un αdisacárido fucosilado. Conclusiones. Se comprobó la viabilidad de la reacción para la
síntesis de fucosil-lactosa y se logró sintetizar α y β-disacáridos fucosilados.
1.
2.
3.
Miñana V. Acta Pediátr Esp. 65 3 (2007)
Gudiel M y Goñi I. Archivos Latinoamericanos de Nutrición. 51 4 (2001)
Kunz C y col. Annu Review Nutr. 20 (2000)
{PAGE }
GT2
Gene expression mannosyltranferase gene of Sclerotium cepivorum
Berk in different culture medium
Ma. Socorro Chávez Ramírez1*, José Ascensión Martínez Álvarez1, Alberto Flores
Martínez1, Patricia Ponce Noyola1. Universidad de Guanajuato. División de Ciencias
Naturales y Exactas. Departamento de Biología. Guanajuato, México1.
*[email protected]
Sclerotium cepivorum Berk, the causal organism of white rot disease, is an important
soil-borne plant pathogen of edible Allium species such as garlic and onion worldwide.
In Mexico, the disease was unknown until 1975; nowadays it has been detected
wherever Allium crops are grown, especially on garlic in the Bajio region of Guanajuato
state. In some fields of this area the disease incidence is extremely high, leading to total
loss of the crop. The low effectiveness and/or inconsistency of the disease control
measures utilized, could be attributed to a complex and efficient mechanism involved in
survival and germination of sclerotia, the only reproductive structures produced by the
fungus and responsible for its propagation and dissemination, which are composed of
compact masses of hyphae covered with melanin, which can be viable in the soil and
under adverse environmental conditions up to 20 years. In this study we determined the
gene expression of a mannosyltransferase (MNT) of Sclerotium cepivorum Berk (1),
involved in protein glycosylation and virulence of some plant pathogenic fungi (2, 3). We
determined whether the composition of the culture medium alters the expression of this
gene in the presence of garlic. The results show that MNT gene expression varies
depending medium where the fungus grow, garlic powder also affects the expression of
this gene. These results suggest that the MNT gene must play an important role in the
biology of the fungus.
1. Hernández M. (2010). Tesis de licenciatura. Universidad de Guanajuato, México.
2. Goto M. (2007). Biosci Bothecnol Biochem. 71: 1415-1427.
3. Spiro R. (2002). Glycobiology. 4: 43-56.
{PAGE }
GT3
Functional analysis of Candida albicans MNN43
González-Hernández, Roberto, Díaz-Jiménez, D. F., Flores-Carreón, A., and MoraMontes, H.M.*Departamento Biología, DCNyE, Campus Guanajuato, Universidad de
Guanajuato, México
*[email protected]
C. albicans is an opportunistic human pathogen that represents an important cause of
nosocomial infections, being its cell wall the immediate point of contact with the host
cells. C. albicans cell wall is formed by chitin, β1,3- and β1,6 glucans, and proteins
enriched with N– and/or O–linked glycans, generally known as mannoproteins. N– and
O-linked mannans can be modified with mannosylphosphate residues, which confer
negative charge to the cell surface, influencing the interaction of the cell with its
environment. Cell wall phosphomannan is important for the recognition and
phagocytosis of C. albicans by macrophages. S. cerevisiae Mnn4 is known as a positive
regulator of phosphomannosylation, and disruption of ScMNN4 blocked
phosphomannan production. Similarly, C. albicans Mnn4 is a key positive regulator of
phosphomannosylation, but at difference of S. cerevisiae, there are 7 genes with
significant similarity degree to CaMNN4, which form the MNN4-like gene family. In this
work we aim to disrupt MNN43, a member of the C. albicans MNN4-like gene family,
and further characterize the null mutant. In order to generate the heterozygous mutant,
a disruption cassette containing the URA3 gene flanked with 70 bp of the 3´and 5´ends
of MNN43 ORF, was amplified by PCR and used to transform C. albicans CAI4, a urastrain. Thus far, we have disrupted the first copy of MNN43, and experiments are in
progress to generate the mnn43Δ null mutant.
Funding: Work supported by CONACYT, Ref. 83414.
{PAGE }
GT4
Biochemical characterization of recombinant Candida albicans
mannosyltransferases Mnt1 and Mnt2 reveals new functions in Omannans biosynthesis.
Díaz-Jiménez Diana Fabiola1, Mora-Montes H.M.1, Hernández-Cervantes A.1, LunaArias J.P.2, A.R Gow N.3, and Flores-Carreón A1*.
1
Departamento Biología, DCNyE, Campus Guanajuato, Universidad de Guanajuato,
Noria Alta s/n, Guanajuato Gto., C.P. 36050, México. 2Departamento de Biología
Celular, Cinvestav-IPN, México. 3Institute of Medical Sciences, University of Aberdeen,
Foresterhill, Aberdeen, Scotland, UK.
*[email protected]
The cell wall of the opportunistic fungus Candida albicans is the first contact point with
the host cells. The cell wall outer layer is formed by mannoproteins, which are modified
polypeptides with mannose oligosaccharides linked to asparagine (N-linked mannans)
and/or serine/threonine (O-linked mannans) residues. C. albicans O-linked mannans are
linear oligosaccharides containing up to five alpha 1,2-mannose moieties. Their
biosynthesis begins in the endoplasmic reticulum, where members of the PMT family
add the first mannose unit to the nascent protein. The chain is further elongated in the
Golgi complex where Mnt1 and Mnt2, members of the KRE2/MNT1 family, add the
second and third mannose units in a redundant manner (Munro et al., 2005). The other
members of KRE2/MNT1 family have not participation in O-linked mannan biosynthesis
(Mora-Montes et al., 2010). Thus far, the mannosyltrasferase(s) adding the fourth and
fifth mannose unit have not been identified. Here we performed the biochemical
characterization of recombinant Mnt1 and Mnt2, obtained as secreted proteins in the
heterologous system Pichia pastoris. Both enzymes showed Mn2+-dependent
mannosyltransferase activity, but they could also use to a less extend cobalt, and exhibit
high ability to use alpha-methyl manoside and alpha 1,2-mannobiose as acceptors,
correlating with their in vivo function. No enzyme activity was found when mannose,
alpha 1,3- and alpha 1,6- mannobioses, Man5GlcNAc2 and Man9GlcNAc2 were used as
acceptor molecules, indicating both enzymes recognize specific elements in the
acceptor structures. Using O-linked mannans isolated from Saccharomyces cerevisiae
as acceptors, we showed that recombinant Mnt1 and Mnt2 have the ability to elongate
the O-linked mannans beyond products with more than 3 mannose residues, suggesting
that Mnt1 and Mnt2 could be the mannosyltransferases adding the fourth and fifth
mannose units to the O-linked mannan.
Mora-Montes et al., J Biol Chem. (2010) 16;285(16):12087-9
Munro et al., J Biol Chem. (2005) 280, 1051-1060.
Work supported by CONACYT, Ref. 83414. México and The Wellcome Trust
{PAGE }
GT5
Isolation of Sporothrix schenckii MNS1
Nahúm Valente Hernández, Arturo Hernández-Cervantes, Carlos A. Rangel-Alfaro,
Arturo Flores-Carreón and Héctor Manuel Mora-Montes*. Departamento de Biología,
DCNyE, Universidad de Guanajuato, Guanajuato, Guanajuato.
*[email protected]
Sporothrix schenckii is a dimorphic fungus, responsible for sporotrichosis, a chronic
granulomatous infection of the skin and subcutaneous tissues. The infection is
distributed worldwide, although is more common in tropical and subtropical areas.
Previous studies have demonstrated that a complex of proteins named adhesins is
capable of binding to collagen and fibronectin. The adhesins are glycoproteins, which
contain O- or N-linked oligosaccharides. The N-linked glycosylation begins in the
endoplasmic reticulum (ER) where the N-linked glycan core is transferred to nascent
polypeptide, and then is processed by two glucosidases and the α1,2-mannosidase that
removes the three glucose and one mannose residues, respectively. In Candida
albicans the study of the N-linked glycosylation pathway has revealed that this protein
modification is required for virulence and recognition by the host immune system.
Disruption of C. albicans MNS1, the encoding gene for the ER α1,2-manosidase,
affected the cell wall composition, the interaction with innate immune cells and virulence
[1]. In S. schenckii the α1,2-manosidase was isolated and biochemically characterized,
although no gene sequence has been thus far reported. Therefore, in order to isolate
the S. schenckii encoding gene for the α1,2-manosidase activity we amplified a 960 pb
DNA fragment using degenerate primers, whose design was based on conserved
regions of MNS1 genes reported in other fungal species. Comparative bioinformatics
analysis showed this DNA fragment corresponds to part of the S. schenckii MNS1 gene.
Next, we generated a partial genomic DNA library and used the MNS1 fragment as a
screening tool. This allowed identifying a 4 Kpb genomic DNA fragment that contains
the MNS1 gene. We are currently sequencing this DNA fragment to obtain the whole
sequence of S. schenckii MNS1.
1. Mora-Montes, H.M. et al., Eukaryotic Cell 6 2184 (2007).
Financial support: SEP-CONACYT, CB-2007 No. 83414.
{PAGE }
GT6
Folding and homodimerization of wheat germ agglutinin.
María del Carmen Portillo-Téllez, Enrique García-Hernández*
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior,
Ciudad Universitaria, 04360 México, D. F.
*[email protected]
Wheat germ agglutinin (WGA) is emblematic among proteins specialized in the
recognition of carbohydrates. It was the first lectin reported with a capacity for
discriminating between normal and malignant cells. Since then, it has being a recurrent
model for basic research, as well as being considered in the development of a number
of biomedical and biotechnological applications. In spite of that, the molecular basis of
the structural stability of this homodimeric lectin remains largely unknown, a situation
that limits the rational manipulation and modification of its function. Herein, we present a
thermodynamic characterization of WGA folding and self-association processes as a
function of pH and temperature, by using differential scanning and isothermal dilution
calorimetry. WGA is monomeric at pH 2, with one of its four hevein-like domains
unfolded at ambient temperature. Under such conditions, the agglutinin exhibits a fully
reversible thermal unfolding, composed of three two-state transitions. At higher pH
values, the protein forms weak transient dimers. This behavior contrasts dramatically
with that observed for other oligomeric plant lectins, indicating distinctly different
molecular bases for WGA function. For dimer formation, the four domains must be
properly folded. Nevertheless, depending on the solution conditions, self-association
may be coupled with the folding of the labile domain. Therefore, dimerization may
proceed as a rigid-body-like association or as a folding-by-binding event. This hybrid
dimerization behavior is unseen in other plant lectins. The emerging molecular picture of
WGA assembly helps further understand some in vitro and in vivo results reported
previously in the literature.
{PAGE }
GT7
Effect of culture conditions on the O-mannosylation of recombinant
APA 45/47 kDa from Mycobacterium tuberculosis in Streptomyces
lividans.
Ramses A. Gamboa-Suasnavart 1,3, Norma A. Valdez-Cruz 2, Luz D. Marin-Palacios 1,3,
Luis Servín-Gonzalez 2, Clara Espitia 3, Mauricio A. Trujillo-Roldán 1,3*
Unidad de Bioprocesos1, Departamento de Biotecnología y Biología Molecular2,
Departamento de Inmunología 3, Instituto de Investigaciones Biomédicas, Universidad
Nacional Autónoma de México.
*[email protected]
Alanine and proline-rich secreted protein APA is a 45/47 kDa glycoprotein from M.
Tuberculosis, with high immunogenicity associated with its mannosylation pattern (1,2).
It has been reported that four threonines are mannosylated on APA, two mannoses
were found in Thr10,18, just one in Thr27 and a variability of 1 to 3 mannoses in Thr277 (3).
APA is an alternative for the production of a new vaccine or a diagnostic kit since
Tuberculosis remains an important public health problem worldwide (4). On the other
hand, S. lividans is a filamentous bacteria used for recombinant protein production (5)
and because of its phylogenetic relationship to M. tuberculosis was chosen as a host for
the production of recombinant APA. This study evaluated the changes induced by the
geometry of three different shaken Erlenmeyer flasks: conventional flasks (CF), baffled
flasks (BF) and stainless steel coiled flask (SSCF), providing different culture
(hydrodynamic-aeration) conditions. S. lividans growth and rAPA production kinetics
were evaluated and related with bacteria morphological changes and mannosylation of
the rAPA C-terminal Thr277. Besides, S. lividans final biomass concentration was similar
in all cultures (3.5 ± 0.1 g/L), the smallest pellets, the higher specific growth and the
higher productivity of rAPA were obtained in SSCF. Moreover, mannosylation in the
Thr277 presents 3 or 4 mannoses in SSCF, compared with 3 mannoses in BF and none
to 2 in CF. Nowadays, determination of the mannosylation pattern in the N-terminal
region (Thr10,18,27) is under study. To our knowledge, this is the first report about the
relationship of morphological changes, productivity and mannosylation in a recombinant
protein produced by bacteria, due to different culture conditions.
1. Lara M et al. App Ennviron Microbiol 70: 679-685 (2004)
2. Horn C et al. J Immunol Methods 197: 151-193 (1996)
3. Dobos K et al. J. Bact. 178: 2498-2506 (1996)
4. WHO Report Report No.: WHO/HTM/TB/2009.411. (2009)
5. Payne et al. Appl. Microbiol. Biotechnol. 33: 395-400 (1990)
Acknowledgments: PAPPIT-UNAM IN228509-3, CONACyT-INNOVAPYME-137854. CONACyT 104951Z.
{PAGE }
GT8
Comparison of the phosphomannosylation levels and relative cell wall
porosity among clinical isolates of Candida parapsilosis
L. Antonio Pérez-García, Arturo Flores-Carreón, Héctor M. Mora-Montes*
Departamento de Biología, División de Ciencias Naturales y Exactas, Universidad de
Guanajuato .
*[email protected]
Candida parapsilosis is commonly associated to nosocomial infections in newborns and
immunocompromised patients. Little is known about the organization and composition of
its cell wall; nevertheless, it is assumed that its composition and organizations are
similar to those found in Candida albicans. The C. albicans cell wall glycoproteins
participate in adhesion, virulence and triggering protective immune response. N- and Omannans linked to glycoproteins, along with β-glucans, represent the major pathogenassociated molecular patterns recognized by the immune system. It has been shown
that cell wall porosity is related to the N-mannan size, and high porosity leads β1,3glucan exposed on the cell surface, and thus with a better recognition by immune
system cells. Phosphomannans, mannose units attached to mannans via a
phosphodiester bond, are required for proper phagocytosis by macrophages, and for the
antimicrobial effect of cationic peptides [1, 2, 3]. Here, we determine the cell wall
porosity and phosphomannan content of six clinical isolates of C. parapsilosis. Our data
showed that C. parapsilosis cell wall exhibits a constant porosity to polycations, but
there are differences on the phosphomannan content among the clinical isolates. These
findings suggest that mannosylation and phosphomannosylation are two independent
processes, since data revealed that cell wall porosity remains unaltered despite
changes in phosphomannosylation levels. In addition it is possible to infer that immune
recognition of this species could be different to that reported for C. albicans.
1.
Netea, MG., Brawn GD, Kullberg BJ and Gow NAR. Nature Reviews Microbiology 6 67 (2008)
2.
MacKenzie CGJ, Koser U, Lewis LE, Bain JM, Mora-Montes HM, Barker RN, Gow NAR and Erwig
LP Infect. Immun. (2010)
3.
Harris M, Mora-Montes HM, Gow NAR and Coote PJ. Microbiology 155 1058–1070 (2009)
Research was supported by grant 83414 from CONACyT
{PAGE }
GT9
N-glycans analysis of two lectins present in tepary beans seeds by
mass spectrometry.
Laura Alondra Ortega de Santiago1, Teresa García Gasca 2, Carlito Lebrilla3, Mariana
Barbosa3, Elizabeth Mendiola Olaya1, Alejandro Blanco Labra1*.
1
Centro de Investigación y de Estudios Avanzados del IPN. Km. 9.6 Libramiento Norte
Carr. Irapuato-León 36821 Irapuato, Gto. México. 2Facultad de Ciencias Naturales.
Universidad Autónoma de Querétaro Av. de las Ciencias s/n. Juriquilla, Querétaro
76230 México. 3University of California Davis, Department of Chemistry. Davis CA,
95616 USA
*[email protected]
The use of mass spectrometry in combination with other molecular tools have allowed
the structural definition of highly complex systems such as glycoproteins due to its high
sensitivity and ability to unravel the molecular composition using tandem MS techniques
[1]. Although legume lectins are the family of carbohydrate binding proteins that has
been studied most extensively, the complet glycosylation patterns of the .glycoprotein
has not been elucidated. In tepary bean. (Phaseolus acutifolius) the presence of lectins
has been reported, however, their structural and functional characteristics have not yet
been fully described.The objective of the present work was to perform a preliminary
analysis of the nature of the glycosylated region of two different samples of purified
lectins isolated from the seeds of tepary bean. For this purpose, the purified lectins were
digested with N-glycans glycopeptidases. The resulting fragments were separated using
an activated carbon cartridge and eluted with acetonitrile. The removed carbohydrates
(N-glycans) were analyzed using a mass spectrometer Pro-MALDI FT-ICR MS [2]. To
analyze the data obtained, the tools of proteomics server; Swiss Institute of
Bioinformatics (ExPASy) and tools Consortium Functional Glycomics (CFG) were
used.N.glycans, mainly oligomannoses were identified to be present in the analyzed
lectin samples, where it was also detected the presence of N-glycans of complex type.
{PAGE }
GT10
Identification of isolated polymer agave fructan angustifolia HAW
Martínez C. Margarito A.1, Martínez C. Jorge Miguel2, Pacheco R. Keyla 1,
1
Unidad de Bioquímica del Instituto Tecnológico de Oaxaca
2
Laboratorio de Análisis Instrumental del Instituto Tecnológico de Oaxaca
*[email protected]
Fructans are a structurally diverse group of polysaccharides consisting of polymers of
fructose, joined together to initial sucrose molecule. Polymers agave fructans consist
mostly of several molecules of fructose and glucose (1). Fructans have great potential
for application in functional food production according to recent studies, due to its
beneficial effects to health (2). Currently agave angustifolia plantations in the state of
Oaxaca are not used in the manufacture of artisan origin drink called "mezcal", but
much of the output is not used for this purpose which requires the search and / or
implementation of an alternative to utilize and exploit the rest for fructan production
similar to what is done with Agave and var. Blue. The aim of this study was to identify
polymers that make up the chain of these fructans from obtaining a crude extract agave
juice which is then subjected to a drying process and finally analysis. The parameters
for identification are the hydrolysis time of samples for both the juice to the dry, drying
temperature, time of ripening of the sample. The identification process was carried out
using a technique of solubility in solvents of different polarity by washing the samples.
Once obtained these purified samples were analyzed by TLC and compared with
different types of fructans mainly derived from chicory, Agave and var. Blue and other
commercial, also perform well for each determination of total reducing sugars, fructose
and glucose quantification. We can conclude that in the sample has characteristics
similar to those of Agave and var. Blue, but different from chicory.
1. Díaz M., “Oligosacaridos en fórmulas infantiles. La inulina y la oligofructosa, nuevos ingredientes
funcionales en alimentos”, Neolac, 2001.
2. Salazar Leyva, J. A., Universidad Politécnica de Sinaloa. “Obtención de fructanos a partir de Agave
tequilana weber var azul. Cultivado en el estado de Sinaloa. 2010
{PAGE }
GT11
Extraction and preliminary crystallization of a lectin from Cucurbita
ficifolia.
Enrique Lemus Fuentes
Universidad Tecnológica de la Mixteca, Huajuapan de León, Oax. 69000.
[email protected]
Previous studies indicates that the Cucurbita ficifolia lectins reduce sugar levels [1,2]. Also
previous reports on the agglutinaton activity of the proteins extrated from Cucurbita
ficifolia seeds have shown that the lectins are effective [3,4]. One of the major concerns
in the lectin research is the elucidation of the structural basis of the protein carbohydrate
interaction. The aim of this study was crystallize a lectin from Cucurbita ficifolia seeds as
previous step to x ray diffraction. The lectin was isolated from Cucurbita ficifolia, purified
and prepared for crystallization trials. Here the author report a approach for the crystallization of
a lectin using the micro batch under oil technique.
[1] Almanza PJC, Cruz E, Román RR, Vázquez CL, Alarcón AFJ. II Congreso Nacional de Química
Médica Dedicado a Investigación en Cáncer y Diabetes. 4-8 de septiembre de 2006. Memorias en CD.
[2] Garibay Bagnis, C.; San Martín Martínez, E. Rev. Salud pública y nutrición. 2006: 11, 1-6.
[3] Castillo-Villanueva, A., Abdullaev, F. Revista de Investigación Clínica. 2005 57: 1, 55-64.
[4] Hernández, P., Pérez-Campos, E. Martínez, L., Ortíz, B., Martínez, G. 2005 REB 24: 1, 21-27.
{PAGE }
GT12
Redesigning the carbohydrate recognition site of hen lysozyme.
1
Gabriel Gutiérrez-Magdaleno*, Enrique García-Hernández.1*
1
Instituto de Química, Universidad Autónoma de México.
*[email protected]
In recent years, our research group has been involved in the study of proteincarbohydrate interactions, trying to clarify the underlying energetic-structural principles
of the phenomenon. As a new stage in our investigation, we have aimed to redesign the
protein’s recognition site, changing its specificity towards other saccharidic structures.
The primary target of the present project is to redesign the recognition site of hen’s egg
lysozyme, to make it specific for glucose β(1-4) oligomers. Using the crystal structure of
lysozyme with chitotriose (GlcNAcβ(1-4)GlcNAcβ(1-4)GlcNAc) as a template, a model of
lysozyme-binding cellotriose (Glcβ(1-4)Glcβ(1-4)Glc) was built. After several cycles of
mutations and energy minimizations using Rosetta, a construct was obtained whose
binding energy was comparable to that of the wild lysozyme-chitotriose complex.
Mutations Ile98Gln, Ile58Gln and Leu75Arg optimized interactions with OH(2) groups in
cellotriose. The optimal conformation of the two new Gln residues was stabilized by
mutations Leu56Ser, Trp108Tyr, which make no direct contact with the ligand.
Furthermore, mutations Ala107Asn and Trp63Tyr yielded increased affinity by improving
interactions with common groups in cellotriose and chitotriose. Recombinant wild
lysozyme was obtained in Aspergillus niger. Recombinant protein was characterized by
means of circular dichroism and fluorescence spectroscopy, and its binding to
chitotriose was measured calorimetrically. Overall, the secondary and tertiary structures,
the thermal stability and the binding properties of recombinant lysozyme were identical
to those of lysozyme purchased from SIGMA. Advances in the expression, purification
and structural and energetic characterization of different lysozyme mutants are
presented here.
{PAGE }
GT13
Development of a conjugate vaccine of Haemophilus influenzae b (Hib) based
on their capsular polysaccharide (PRP)
Francisco Reta Mares, Jorge Gomez-Herrera, Ma Eugenia Jimenez Corona, Ramona Rojas
Galván, Luciana Solano Rodriguez, Patricia Mendez Ortega, Oscar Andrade Correa, Claudia
Jaime Aguilar, Cristian Trejo Sanchez, Samuel Ponce-de-León. Instituto Nacional de Higiene.
Laboratorios de Biológicos y Reactivos de México. BIRMEX, SA de CV. México, DF
[email protected]*.
Introduction: The conjugation of bacterial polysaccharides to carrier proteins is a major technology
in vaccinology; vaccines that use it, have helped control diseases such as meningitis and
pneumonia caused by Haemophilus influenzae b [1]. Objective: Develop the methodology at
laboratory and pilot level for a Hib vaccine, combining their capsular polysaccharide (PRP) to
tetanus toxoid (TT). Material and methods: Hib strain used was a clinical isolate. The production
and purification of PRP was performed using a modification of Robbins methodology [2]. The
conjugation of PRP to TT was performed using a modification of Lee method [3] using CDAP (1cyano-4-dimethylaminopyridinium tetrafluoroborate) as an activator and without spacer. The
purification of conjugate was performed by gel filtration chromatography. For freeze dry, the
vaccine was formulated to 10 μg of PRP per dose. The quality control tests were developed
according to WHO technical report [4].Results: The methodology to production and purification of
PRP to 250-liter fermenter was established. The purified polysaccharide, by national and
international control (NVI, The Netherlands) meets WHO specifications. The methodology to
conjugation of PRP to TT and its purification were developed and were escalated to 10 g of PRP,
with similar yields to literature 15-20% [2]. A freeze drying cycle of 24-36 hours was established,
with 3000 single dose vials. The conjugated polysaccharide produced as purified and freeze dry
bulk, meets with WHO specifications; the efficacy test in rabbit and mouse, were in comply.
Conclusion. A methodology to obtain a vaccine based on the Hib polysaccharide was developed
and the prepared vaccine with it, meets WHO specifications. It is necessary to demonstrate
reproducibility of the conjugation process in a pilot plant with Good Manufacturing Practices by
producing three consecutive batches, and their further evaluation in clinical trials.
1.- Evropi T., Johnson S., Jhass A., Madhi S. A., Andrew C. The effect of Haemophilus influenzae type b and
pneumococcal conjugate vaccines on childhood pneumonia incidence, severe morbidity and mortality. Int J
Epidemiol. 2010.39(1): 172–185.
2.-Scheneerson R, Barrera O, Sutton S., and Robbins J.B. Preparation, characterization and immungenicity of
Haemophilus influenzae type b polysaccharide protein conjugates. J. of Exp. Med 1982, 152:361-357.
3.- Lees A,. Nelson L, B., Mond J.J. Activation of soluble polysaccharides with 1-cyano-4-dimethylaminopyridinium
tetrafluoroborate for use in protein-polysaccharide conjugate vaccines and immunological reagents. 1996. 14:190198.
4.- Requirements for the production and control of Haemophilus type b conjugate vaccines, In: WHO Expert
Committee on Biological Standardization Forty-ninth report. Geneva World Health Organization 2000. Annex 1 (WHO
Technical Report Series, No. 897) {PAGE }
Nombre de archivo: Lista3 Directorio: F:\congreso\abstracts Plantilla: Normal.dot Título: B1 Asunto: Autor: Dr. Ivan M. Duncker Palabras clave: Comentarios: Fecha de creación: 13/07/2011 20:11:00 Cambio número: 32 Guardado el: 09/08/2011 18:47:00 Guardado por: Dr. Ivan M. Duncker Tiempo de edición: 1.937 minutos Impreso el: 09/08/2011 21:22:00 Última impresión completa Número de páginas: 80 Número de palabras: 24.599 (aprox.) Número de caracteres: 135.300 (aprox.)

1 Congreso Latinoamericano de Glicobiología

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