MCB 450Lecture 7OGlycoconjugatesProteoglycansGlycoproteinsGlycan SynthesisExamples of Importanceof Glycans7-17-2 GlycoconjugatesNOTE:THE MODIFICATIONSWITH SUGARS ARE ALLON EXTRACYTOPLASMICPORTIONS OF PROTEINSAND LIPIDSPROTEINGlycoprotein:ProteinOligosaccharide (not GAG)covalently attached to peptide backboneProteoglycan:ProteinUronic acid and/or SO42-containingpolysaccharide (glycosaminoglycan)Glycosaminoglycan (GAG)Protein7-3 Definitions: proteoglycan vs glycoprotein7-4 Glycosaminoglycan repeats-1• One sugar always GalNAc, GlcNAc, or GlcN, often esterified with sulfate• The other usually a uronic acid (e.g. glucuronate)GlcA GalNAcRepeating disaccharide:HYALURONIC ACIDKERATAN SULFATECHONDROITIN SULFATE~50,000# disaccharide repeats/chain:20-60~257-5• Ionized carboxyl & sulfate groups give molecule a -ve charge• Assume e x t e n d e d c o n f o r m a t i o n in solutionGlycosaminoglycan repeats-2GlcAGalNAcGalGlcNAcGlcAGlcNAcHYALURONIC ACIDKERATAN SULFATECHONDROITIN SULFATE7-6 Glycosaminoglycans in vertebratesHighly hydrated.Forms viscous solution& is lubricant in joints;also, structural componentof "extracellular matrix" ofvertebrate cellsNo uronic acid.In cartilage, bone, nails,horn, hoofs.In cartilage, tendonsligaments, aorta wallsCartilage proteoglycanElectron micrographCentral filament of hyaluronateMass ~ 2 x 106daltons7-7SUPRAMOLECULAR COMPLEXOF GLYCOSAMINOGLYCANCHAINS & PROTEINSAggrecanCan spring back after being deformed,so cartilage can cushion compressive forcesCartilage proteoglycan7-8HIGHLY HYDRATEDGlycoprotein:ProteinOligosaccharide (not GAG)covalently attached to peptide backboneProteoglycan:ProteinUronic acid and/or SO42-containingoligosaccharide (glycosaminoglycan)GlycosaminoglycanProtein• Two (major) types of linkage to extracytoplasmic domains of proteins• Occur (with variations) in all eukaryotes• Examples in bacteria and archaea7-9 Definitions: proteoglycan vs glycoprotein7-10 Two major types of protein-saccharide linkagein glycoproteinsGlcNAc ADDED IN ERGalNAc ADDED IN Golgi7-11 N-linked glycoproteinsHUGE VARIETYOF STRUCTURESCAN BE BUILT UP ONN-LINKED GlcNAc:DIFFERENT SUGARS,BRANCHING7-12 Asparagine-linked (N-) glycoproteins• Glycan is added to proteins in lumen of rough ER• The GlcNAc is actually added as partof a larger, branched oligosaccharidea2a6a6a3a2a3a2a2a3a3a2b4b4GlcNAcManGlcN-glycosidic linkageinvolving Asn amide NGlcNAc is the first sugar of abranched oligosaccharide• Oligosacch. trimmed backto varying degrees:• Different sugars added backin many linkages (in Golgi)• Great structural diversitypossible w. different sugars,different linkages betweenthem, and branching• VaRiability between protein, cell & tissue type,developmental stage, organism...important in intermolecularrecognition: information-rich.....also for stability of someproteins7-13a6a3a3a2a2b4b4b2b4b4a6a3b2b6b3b3 b3b3 b3b3 b3b3 b3b3 b3b3NAcNAcFucoseA great variety of N-linked glycan structures canbe generated in the Golgia2a6a6a3a2a3a2a2a3a3a2b4b4OCOO-OHOHOHOHOHH O l llH-C-C-NlH7-14 Complex glycans with sialic acid(a.k.a. N-acetylneuraminic acid)C-2 =anomeric C7-15 N-linked glycans are important• Help promote proper folding of freshly translated polypeptidesin the lumen of the ER• Can help stabilize protein conformation andprotect against proteolysis• Can help direct proteins to various membrane compartments(e.g. lysosome in mammalian cells)• Complete inability to attach any N-linked glycans is"embryonically lethal" in mice• Even subtle underglycosylation of asparagines (i.e. an Asnglycosylation site is missed) is associated with humandevelopmental defects: "Congenital Disorders of Glycosylation")7-16 O-linked glycoproteins• Mostly added to proteins in lumen of Golgi • Single sugar transferred initially• Subsequent ones addedindividually7-17 Mucins are heavily O-glycosylatedRICH IN Ser & ThrVariable number tandem repeats= major components of the mucussecreted by epithelial cells thatline respiratory, digestive& urogenital tracts§ The highly glycosylated domains of mucinsare l o n g e x t e n d e d structures,much less flexible than unglycosylated random coils § Oligosaccharides contribute to stiffness by...ü limiting rotation around peptide bondsü charge repulsion among neighboring-vely charged oligosacch. groups§ Long, extended mucin molecules havea much greater solution volumethan native proteins w. little or nocarbohydrate§ Mucins therefore formviscous solutions or gels thatprotect against infectionby microbes that bindcell surface carbohydrates7-18 Mucins polymerizeS-S bonding &dimerizationO-linkedsaccharidesPerez-Vilar, J. & Hill, R.L. (1999)J. Biol. Chem. 274: 31751covalently attached sugars(O-glycosidically-linked)force protein into extendedconformationcovalently attached sugarchain (N-glycosidically-linked)Glycolipid anchors glycoproteinin membraneProtein7-19 Contribution of carbohydrate to protein structureEnergetics of glycosidic bond formation7-20Formation of a glycosidic bond between two sugars:Sugar + SugarnSugarn+1energetically unfavorableGeneral strategy for making glycans: use an "activated" sugar donor= compounds in which anomeric carbon is activated by attachment to anucleotide through a phosphodiester linkage, e.g. UDP-glucosea-linked!Different sugar nucleotidesserve as sugar donors fordifferent glycosyltransferases:UDP-GlcUDP-GlcNAcUDP-GalGDP-ManCMP-sialic acidLuis Leloir, Nobel Prize in Chemistry 1970for his discovery of sugar nucleotides andtheir role in the biosynthesis of carbohydrates7-21• Nucleophilic displacement of UDP from C-1 of UDPGlcby C-4 -OH of glucose at non-reducing end of chain• UDP = excellent leaving group,facilitates nucleophilic attack by activating the sugarcarbon to which it is attached7-22Elongation of an a1®4 linked glycogen chainGlc added atnon-reducingend of chainUDP“Nucleophile” (from “nucleus loving”or “+ve-charge loving”) =reactant that provides a pair of electrons to form a new covalent bond OCH2OHPPlNENZYMETAKES THE HHO R..aR-O: MAKESNUCLEOPHILIC ATTACK7-23 Nucleophiles and leaving groups“Leaving group”accepts the electron pair from the nucleophile: http://www.masterorganicchemistry.com/2012/06/05/nucleophiles-and-electrophiles/ACTIVATION BY UDP MAKES C-1"WANT" TO ACCEPTELECTRONSDoblin et al. (2002) Plant Cell. Physiol. 43: 1407Cellulose synthase catalyzes repeated Glc transfers & extrudes growing cellulose chain
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