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Glicobiologia

Glicobiologia. El estudio de la estructura, química, bioquímica y función biológica de los carbohidratos complejos (glicanos). Glicobiología. mas del 50% de todas las proteínas conocidas así como mas del 80% de las proteínas de membrana están glicosiladas.

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Glicobiologia

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  1. Glicobiologia El estudio de la estructura, química, bioquímica y función biológica de los carbohidratos complejos (glicanos).

  2. Glicobiología • mas del 50% de todas las proteínas conocidas así como mas del 80% de las proteínas de membrana están glicosiladas. • El contenido de carbohidrato de una glicoproteina varia entre el 1 y el 80% del peso de la misma. • Los glicanos o glicoconjugados son el biopolímero mas abundante en la naturaleza. • Mas del 1.5% de todo el genoma humano codifica enzimas involucradas en la biosíntesis y modificación de glicoconjugados, en ciertos protozoos este porcentaje se eleva a casi el 12%. • La enzima Hidroxilasa del CMP-acido sialico es el único gene identificado que no se encuentra en humanos pero si en chimpance. • La O-acetilglucosaminilacion es una modificación regulatoria mas abundante que la Fosforilación

  3. Glicobiología

  4. GLICOCONJUGADOS Proteoglicanos Glicoproteinas Glicolipidos Glicosilfosfatidil-inositol anclajes Monosacarido Proteina Lipido Etanolamina fosfato Inositol fosfato

  5. Glicobio(pato)logia El estudio de la estructura, química, bioquímica y función biológica de los carbohidratos complejos (glicanos) y su implicación causal en el desarrollo de patologías, diagnosis y terapia.

  6. Diferencias en la composición de isomeros entre oligopeptidos y oligosacaridos Hexasacarido Hexapeptido Nº Isomeros > 1012 64 106 (librería 6 hexopiranosas)(20 aminoácidos) Cantidad > 100 nmol < 100pmol Síntesis 20 semanas 3 h (robot) Esto implica que no hay método analítico que permita un análisis estructural completo (determinación de secuencia) en cantidades de 100 nmol como se puede hacer con 100 pmol de proteínas u oligonucleotidos. No hay método analítico que permita distinguir entre 1012 estructuras con la misma masa.

  7. Al menos 7 monosacáridos distintos son comunes de todos los glicanos, aunque hay mas de 30 que pueden formar parte de glicanos en la naturaleza. NeuAc Gal Glc Man Fuc GalNAc GlcNAc Origen de la Diversidad Estructural y Regulación de Afinidad • Composición de monosacáridos • Enlace glicosidico (secuencia) Entre el carbono aldehido/ceto y cualquier grupo hidroxilo del otro monosacarido • Anomericidad Configuracion a o b del carbono que contiene el grupo aldehido/ceto • Ramificación: numero y posición • Tamaño del anillo: furanosa o piranosa • “Cluster” con cadenas vecinas en la misma glicoproteina (mucinas) o en la superficie celular (“rafts”).

  8. O H H O C H 2 O H O N H H O C H 2 O H O C H C O H O 3 a O 6 O H C H H O 2 a O H O 1 O H C H 2 O O H O C H 2 O O H 3 b H O O H O O H 3 H O O H O H 1

  9. Enzimas DNA RNA Proteína Carbohidratos Célula Glycoconjugados Organismo Complejidad del Glicoma GENOMA PROTEOMA TRANSCRIPTOMA GLICOMA Cáncer Desarrollo Inflamación Diferenciación Activación Respuesta infecciones Dinámico Variaciones espaciales y temporales Respuesta al ambiente

  10. Desarrollo secuencial y cambios de importancia de las funciones de los glicanos Procariotes Eucariotes Unicelular Multicelular Estructural Plegamiento de proteínas Modulación del plegamiento “Tagging” Extracelular

  11. Funciones Biológicas de los Glicanos Físicas/Estructurales Reconocimiento Intrínseco = Propio ReconocimientoExtrínseco = No Propio INTRINSIC RECEPTOR EXTRINSIC RECEPTOR SELF P SELF Mimetismo molecular P = Patogeno OLIGOSACCHARIDE =

  12. Los Glicanos actúan como señales vía Lectinas

  13. ANIMAL AND PLANT GLYCOPROTEINS SHOW VARIOUS SUGAR CONTENTS GLYCOPROTEIN Present in Mr % sugar content ENZYMES Alkaline phosphatase Murin liver 130 000 18 Carboxypeptidase Y Yeast 51 000 17 HORMONES AND CYTOKINES Chorionic gonadotropin Hum. urine 38 000 31 Erythropoietin Hum. Urine 34 000 29 Interferon g Hum. WBC 26 000 20 LECTINS Soybean lectin 50 000 50 Potato lectin 120 000 6

  14. EXAMPLES OF GLYCOPROTEINS IN ANIMALS AND PLANTS Cell-surface and viral coat glycoproteins Serum glycoproteins Structural glycoproteins

  15. oligosaccharide integral protein hydrophobic  helix glycolipid integral protein phospholipid cholesterol Oligosaccharides of the cell surface are attached to membrane embedded proteins and certain lipid

  16. Molecular model, based on the crystal structures of IgG Fab and IgG Fc. Fab glycosylation sites are in the hyper-variable region and occupied approx. 40% of the time. (Dweck, 1995) X-ray structure of murine antibody against canine lymphoma Textbook presentation 1998

  17. New methods revolutionised the study of glycoconjugate structures Many proteins bear a sugar attachment such as 13.6 kDa T Lymphocyte Adhesion Domain of Human CD2

  18. Successful fertilisation depends on binding of spermal lectins to glycoproteins of egg membrane Specific glycoprotein - lectin recognition defines many physiological processes • Fertilisation • Development and differentiation • Activity regulation of hormones • Immune response and inflammation • Memory consolidation • and many more Cel-cell recognition relies on the specific glycoprotein-lectin interaction capable to decode information carried in the presented oligosaccharide motive.

  19. Glycoproteins are found responsible for • Xenografts rejection • Cancer progression and metastasis • Storage diseases • Immune response towards recombinant therapeutics - and many more

  20. STUDYING GLYCOCONJUGATES EVOLUTION DIAGNOSIS THERAPY PHARMACEUTICAL INDUSTRY ANALYTICAL TOOLS

  21. ALDOSE: CH2(OH)(CHOH)n-2HCOKETOSE: CH2(OH)(CHOH)n-3COCH2OH D-Glucose Cyclic hemiacetal forms

  22. UDP GDP CMP GalNAc Fucose Sialic a. GlcNAc Mannose Galactose Glucose Glucuronic a. Xylose Glycosidic link 16 kJ ENZYME Sugar1-OH + Sugar2-OH Sugar1-O- Sugar2 + H2O Enzyme catalyzed reaction seals the anomeric configuration.

  23. Laine (1988) Pentasacarido lineal, monosacaridos no repetidos. n! x 2na x 2nr x 4n-1 n=numero de monosacaridos a=anomericidad r=tamaño del anillo 4n-1= enlaces Con cinco monosacáridos distintos el total es: 31 457 280 estructuras posibles Con seis monosacáridos distintos el total es: 819 200 000 Para Proteínas el total es 20n. Si n=5 3 200 000 Para oligonucleotidos el total es 4n . Si n=5 1024 Si incluimos posibles ramificaciones el total para un n=6 llega a: 2 633 600 000 = 2.6 x109 sin considerar las formas D o L. El total de formas posible para un hexasacarido (el tamaño medio involucrado en reconocimiento) es de 1.05 x 1012. Esto implica que no hay metodo analitico que permita un análisis estructural completo (determinacion de secuencia) en cantidades de 100 nmol como se puede hacer con 100 pmol de proteinas u oligonucleotidos. No hay metodo analitico que permita distinguir entre 1012 estructuras con la misma masa.

  24. Comparable sequences of oligopeptide and oligosaccharide differ drammatically in isomeric possibilities R.R. Schmidt, Angew. Chem. 1986, 212-235

  25. Diversidad de enlaces sacárido-proteína en las glicoproteínas

  26. Oligosaccharides are linked to proteins by O- or N-glycosidic bonds In N-linked oligosaccharides N-acetylglucosamine at the C1 position of reducing end of sugar binds to Asn within Asn-X-Ser/Thr sequence. O-glycosidic bonds are formed between C1 position of reducing terminus of monosaccharide or oligosaccharide and hydroxyamino acid, Ser or Thr. (N-acetylgalactosamine is often at the non-reducing end.)

  27. BIOSYNTHESIS OF GLYCOPROTEINS STARTS IN ER In ER oligosaccharide is preassembled on a lipid carrier and transferred “en block” to the nascent protein. Glycan processing and tagging occurs in Golgi. Glycosylated product is packed and exported to final destination.

  28. EndoH sensitive EndoH resistant Biosintesis compleja pero conservada evolutivamente …en principio no diana terapeutica

  29. Lectin-like chaperons mediate and controlproper protein folding Calnexin and calreticulin bind misfolded or unfolded monoglucosylated glycoproteins to prevent final deglucosylation by -glucosidase II, which signalises the export to ER, in case of impaired protein folding.

  30. Evolucion de complejidad Levaduras Hongos Plantas Insectos Vertebrados

  31. Origin of Glycoheterogeneity O-linked peptide Ser/Thr N-linked peptide A-X-S/T Oligosaccharide transfer Branching enzymes Processing enzymes Initial monosaccharide Terminal transferases Branching enzymes Glycoforms Terminal transferases

  32. Glycosaminoglycans attached to proteoglycans are formed from repeating disaccharide units

  33. PROTEOGLYCAN STRUCTURES are predominantly glycosaminoglycans O-linked to Ser Secreted or extracellular Cartilage proteoglycan *Mr 220 952 Versican *Mr 264 048 Decorin *Mr 38 000 Intracellular granule Serglycin *Mr 10 190 Membrane intercalated Syndecan *Mr 38 868 *Protein Mr

  34. O-linked saccharides extend functional protein domains above membrane surface

  35. PROTEOGLYCAN STRUCTURE OF BOVINE CARTILAGE Hyaluronate (up to 4 m), coated with proteo-glycans, forms the backbone of a complex polyanionic structure (Mr>2x106) which is highly hydrated and enmeshed in a network of collagen fibers. Link protein assists the noncovalent attachment of proteoglycan core proteins.

  36. ADVENTITIOUS General propertiesand structural roleN-linked cores are added earlyin the life of glycoproteins. INTERMEDIATE Sorting roleTransient glucose signals for sorting. Oligomeric proteins assemble before entering Golgi. COLLATERAL and RECENT Substitutional roles for amino acids Extensions and O-linked sugars Recognition markers Potential for adaptation Terminal elaborations Supporting facts for the Hypothesis of sequential evolution of glycosylation: Two strategies : “En bloc” vs.., stepwise ER-Golgi shuttling protein ERGIC-53, similar to L-type lectins, recognizes the same structures as calnexin/calreticulinCore sugar bound to a proximal Asn in the vicinity of a hydrophobic region of trypanosomes coating protein makes redundant the protective -helix segment. EVOLVING FUNCTIONS OF OLIGOSACCHARIDES “Structural effects arose adventitiously and were selected for. Direct effects of glycosylation on behavior of glycoproteins evolved recently.”T. Drickhamer, 1998

  37. Diversidad Evolutiva de las Lectinas

  38. Oncogenic transformations are associated with numerous changes in glycosylation • different cancers are characterized by different changes • appearance of novel carbohydrate structures • changes in the activity of glycosyltransferases • appearance of novel lectins • novel carbohydrate structures and lectins are promising targets for cancer vaccines and drug targeting

  39. NON-SPECIFIC ROLES OF GLYCANS BOUND TO PROTEIN • Folding, assembling Hepatic lipase and secretion Erythropoietin Vascular endothelial growth f. immature GP aggregation HBV glycoproteins • Conformational effects Yeast acid phospfatase increased thermal stability t-Plasminogen activator decreased backbone mobility Cell-surface CD2 Mucins (terminal Sia) • Protective effects Fibronectin proteolytic degradation Ribonuclease B LDL-receptor (ovary cells) circulatory life time ceruloplasmin • Functional effects  and  antithrombin binding Glycoprotein hormones kinetics Ribonuclesase A and B

  40. CONDROITIN SULFATO HIALURONANO GLICOSAMINO- GLICANOS HEPARAN SULFATO N- CHAINS O-LINKED CHAIN ANCLAJE GLICOFOSFO- LIPIDO GLICOESFINGOLIPIDO O-GlcNAc Glicanos mas comunes en células animales P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglicano Ac P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glicoproteina Ac EXTERIOR P Acido Sialicos INTERIOR O Ser

  41. GLYCOSPHINGOLIPID LETHAL CHONDROITIN SULFATE HYALURONAN Eliminacion de los glicanos mayoritarios permite la viabilidad celular in vitro GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

  42. GLYCOSPHINGOLIPID CHONDROITIN SULFATE HYALURONAN Eliminación de los glicanos mayoritarios causa letalidad embrionaria in vivo GLYCOSAMINO- GLYCANS P S S S Ser-O- HEPARAN SULFATE S S S S S -O-Ser NS NS Proteoglycan N-LINKED CHAINS Ac O-LINKED CHAIN GLYCOPHOSPHO- LIPID ANCHOR P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O-LINKED GlcNAc O Ser

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