Williams BCH 444 2014 Lectures 3-6

Over all kingdoms 30000 glycosyltransferases are

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Unformatted text preview: donor and the acceptor substrate - leading to the one enzyme-one-linkage hypothesis. Over all kingdoms 30,000 glycosyltransferases are known, e.g. UDP-Gal:GlcNAc β1,4 galactosyltransferase Naming based on: donor + acceptor + linkage Catalytic Domain 2)  The vast majority of glycosyltransferases elongate glycan chains to give linear or branched glycan structures and do not recognize the underlying polypeptide. Those that initiate saccharide attachment to form glycoproteins or glycolipids use polypeptides or lipids as acceptor. 3) Many glycosyltransferases are type II membrane proteins. Those involved in Asn-linked glycosylation have active sites directed into ER and Golgi lumen. TM segment N 4) Despite the large number of sequence families the vast majority of glycosyltransferases fall into two fold types. GT-A bound nucleotidesugar donor Golgi Lumen Neck Region GT-B Cytosol 37 Biosynthesis of Asn-linked glycoproteins - I Asn-linked oligosaccharides are all derived from a common precursor that is assembled on a lipid carrier called dolichol -P-P-dolichol Glcα1-2Glcα1-3Glcα1-3 Glc3Man9GlcNAc2-PP-dolichol blocked by tunicamycin Assembly of the oligosaccharide-dolichol precursor in the ER lumen (all reactions catalyzed by glycosyltransferases) 38 Biosynthesis of Asn-linked glycoproteins - II Oligosaccharyltransferase (OST or OT) Asn-linked glycosylation occurs co-translationally glycosylation signal Human subunit Yeast Subunit Essential in yeast? OST48 Ribophorin II STT3A and STT3B Ribophorin I DAD1 N33 DC2 IAP WBP1 SWP1 STT3 OST1 OST2 OST3 OST4 OST5 OST6 YES YES YES YES YES NO NO NO NO Function recognizes dolichol-PP-oligosaccharide unknown active site of the OTase bears peptide binding site of the OTase aids Wbp1p in recognition of the dol-PP-oligosacc associates with translocon recruits Ost3p and Ost6p into OTase complex unknown associates with translocon 39 Biosynthesis of Asn-linked glycoproteins - III Asn-linked glycoprotein processing - formation of complex structures * (1)  Monoglucosylated oligosaccharide is an important signal for glycoprotein folding assisted by the molecular chaperones, calnexin and calreticulin (see later). (1) Other specific oligosaccharide structures are important for export from the ER and for degradation of misfolded glycoproteins (see later). (2) (2)  Mannose-6-phosphate signal is attached to lysosomal enzymes. Critical signal for delivery to lysosomes * * * (3) Processing from a Man4 to a Man3 structure results in resistance to cleavage by the bacterial enzyme, endoglycosidase H. A nice experimental tool for monitoring rates of glycoprotein traffic from ER to medial Golgi. (3) Endo Hs Endo Hr * * Sialic acid * nucleotide sugar antiporter Essentials of Glycobiology Second Edition 40 Endo H as a tool to monitor traffic along the seretory pathway shift in mobility due to oligosaccharide removal 120 min post synthesis, ~50% of glycoprotein has trafficked past medial Golgi Endo Hres Endo Hsens Endo H Radiolabel cells for 5 min with [35S]Met (pulse) Add large excess of unlabeled Met and incubate cells for periods up to 120 min (chase) Remove cells at various times lyse in detergent Immunoisolate with Ab against protein of interest Digest -/+ endo H SDS-PAGE 41 Control of oligosaccharide structure Examples of oligo...
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This note was uploaded on 03/27/2014 for the course BCH 444 taught by Professor Mccallan during the Spring '14 term at University of Toronto.

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