PowerPoint PresentationSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Slide 25Slide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32Slide 33Slide 34Slide 35Slide 36Slide 37Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43MCB 450Lecture 7OSome ExplanationsGlycoconjugatesProteoglycansGlycoproteinsGlycan SynthesisExamples of Importanceof Glycans7-17-2 GlycoconjugatesNOTE:THE MODIFICATIONSWITH SUGARS ARE ALLON EXTRACYTOPLASMICPORTIONS OF PROTEINSAND LIPIDSGlycoprotein:ProteinOligosaccharide (not GAG)covalently attached to peptide backboneProteoglycan:ProteinUronic acid and/or SO42- containingpolysaccharide (glycosaminoglycan)Glycosaminoglycan (GAG)Protein• Two (major) types of linkage to extracytoplasmic domains of proteins• Occur (with variations) in all eukaryotes• Examples in bacteria and archaea7-3 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 GalNAc7-5• 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 solutionHighly hydrated:forms viscous solution& is lubricant in jointsGlcAGlcNAcGlcAGalNAcGalGlcNAcGlycosaminoglycan repeats-2Cartilage proteoglycanElectron micrographCentral filament of hyaluronateMass ~ 2 x 106 daltons7-6SUPRAMOLECULAR COMPLEXOF GLYCOSAMINOGLYCANCHAINS & PROTEINSAggrecanCan spring back after being deformed,so cartilage can cushion compressive forcesCartilage proteoglycan7-7HIGHLY 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 archaea7-8 Definitions: proteoglycan vs glycoprotein7-9 Two major types of protein-saccharide linkagein glycoproteins7-10 Asparagine-linked (N-) glycoproteins• Added to proteins in lumen of rough ER• Branched oligosaccharide transferredfrom lipid carrier (= glycan assembly platform)2663232233244GlcNAcManGlcN-glycosidic linkageinvolving Asn amide NGlcNAc is the first sugar of abranched oligosaccharideOligosaccharide is built up on and transferred froma carrier lipidDolichol,a "polyisoprenoid"(C80-100)7-112663232233244HTransfer to selected asparaginesin lumen of endoplasmic reticulumHelenius, A. & Aebi, M. (2001) Science 291: 2364-2369 7-12 N-glycosylation is a highly conserved pathwayfor modification of secretory proteins in the rough ER14-sugar oligosaccharide transferredto protein post-translationallyMEMBRANE7-13 N-linked saccharides are trimmed further,and new sugars are added back in the GolgiHelenius, A. & Aebi, M. (2001) Science 291: 2364-2369• 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 someproteins7-14 A great variety of N-linked glycan structures canbe generated in the Golgi2663232233244244632633 33 33 33 33 33NAcNAcFucoseOCOO-OHOHOHOHOH H O l llH-C-C-N l H7-15Complex glycans with sialic acid(a.k.a. N-acetylneuraminic acid)C-2 =anomeric C7-16 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)•Even subtle underglycosylation of asparagines (i.e. an Asnglycosylation site is missed) is associated with humandevelopmental defects: "Congenital Disorders of Glycosylation")7-17 O-linked glycoproteins• Mostly added to proteins in lumen of Golgi • Single sugar transferred initially• Subsequent ones addedindividually7-18 Mucins are heavily O-glycosylatedRICH IN Ser & ThrVariable number tandem repeats 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...limiting rotation around peptide bondscharge 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 that protect against infectionby microbes that bindcell surface carbohydrates7-19 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)GPI anchors glycoproteinin membraneProtein7-20 Contribution of carbohydrate to protein structureEnergetics of glycosidic bond formation7-21Formation 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 a nucleotide through a phosphodiester linkage, e.g. UDP-glucose-linked!Different sugar nucleotidesserve as sugar donors fordifferent glycosyltransferases:UDP-GlcUDP-GlcNAcUDP-GalGDP-ManCMP-sialic acidLuis Leloir, Nobel Prize in Chemistry 1970“for his discovery of sugar nucleotides andtheir role in the biosynthesis of carbohydrates” 7-22• Nucleophilic displacement of UDP from C-1 of UDPGlc by C-4 -OH of glucose at non-reducing end of chain• UDP = excellent leaving group, facilitates nucleophilic attack by “activating” the sugar carbon to which it is attached7-23Elongation of an 14 linked glycogen chainGlc added atnon-reducingend of chainUDP“Nucleophile” (from “nucleus loving” or “+ve-charge loving”)
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