Essentials of Glycobiology Lecture 5 April 6, 2004 Ajit Varki

1. Essentials of Glycobiology Lecture 5April 6, 2004Ajit Varki N-Glycans Asparagine (N)-linked oligosaccharides N-linked Glycans N-linked Sugar Chains

2. CHONDROITIN SULFATE HYALURONAN GLYCOSAMINO- GLYCANS HEPARAN SULFATE N-LINKED CHAINS GLYCOPHOSPHO- LIPID ANCHOR GLYCOSPHINGOLIPID O-LINKED GlcNAc Major Glycan Classes in Animal Cells P S S S Ser-O- S S S S S -O-Ser NS NS Proteoglycan Ac O-LINKED CHAIN P Etn S P N O N NH Asn Ser/Thr 2 Asn INOSITOL Glycoprotein Ac OUTSIDE P Sialic Acids INSIDE O Ser

3. N- Glycans on Membrane-Bound and Secreted Proteins N-LINKED CHAIN N Asn Secreted Protein O N Membrane Protein Asn OUTSIDE CELL MEMBRANE INSIDE

4. GlcNAc Man Glc “Hybrid” Gal Sia Fuc Major Classes of N-Glycans “High-Mannose” (oligo-mannose) “Complex” Linkages in the Box exactly the same in all three!

5. Subcellular Trafficking Pathways for Glycoproteins Vesicular Stomatitis Virus (VSV) Glycoprotein Short-Term Pulse with 2[3H]Mannose

6. GlcNAc Man Glc Gal Sia Fuc Linkages exactly the same as in N-glycans! “Lipid-Linked Oligosaccharide” (LLO) Structure of the Dolichol-linked Glycan Labelled in a Short-Term Pulse with 2[3H]Mannose EXACT STRUCTURE IS CONSERVED IN PLANTS, FUNGI AND ANIMALS

7. GlcNAc Man Glc Gal Sia Fuc Dolichol Biosynthesis of N-Glycans:Production of GlcNAc-P-P-Dolichol Tunicamycin Blocks - not very specific! Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

8. GlcNAc Man Glc Gal Sia Fuc Biosynthesis of the N-GlycanPrecursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER) Glycosylation mutants in Yeast, CHO cells (obtained by plant lectin resistance) and lymphoma cells missing Thy-1 glycoprotein (obtained by antibody killing) Were useful in elucidating the pathway CDG = Congenital Disorder of Glycosylation in Humans Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

9. GlcNAc Man Glc Gal Sia Fuc Biosynthesis of the N-GlycanPrecursor on Lumenal Leaflet of ER Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

10. GlcNAc Man Glc Gal Sia Fuc Completion of Biosynthesis of N-GlycanPrecursor on Lumenal Leaflet of ER- and Transfer to Protein Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

11. Target “sequon” for N-glycosylation • Necessary but not sufficient • X = any amino acid except proline • Rarely can be Asn-X-Cys • Transfer co-translational/immediate • post-translational before folding • ~2/3 of proteins have sequons • ~ 2/3 sequons actually occupied • (some variably) Yeast OST complex contains nine membrane-bound subunits Oligosaccharyltransferase complex (OST) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteins

12. GlcNAc Man Glc Gal Sia Fuc Initial Processing of N-Glycans in the ER and Golgi ER Golgi Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

13. Improperly folded proteins are re-glucosylated by glucosyltransferase which acts as “sensor” for improper folding 3 Glucose Residues Calnexin (and Calcireticulin) function during glycoprotein folding in the endoplasmic reticulum

14. GlcNAc Man Glc Gal Sia Fuc Completion of Processing of N-Glycans in ER and Golgi Final products often show “microheterogeneity” at each N-Glycosylation site Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

15.           Complex-type glycans Peptide:N-glycosidase F (PNGase F) “N-glycanase” Endo-beta-N-acetyl- glucosaminidase H(Endo-H) Hybrid glycans Enzymes Useful in detecting Steps in N-glycan Biosynthesis High mannose-type glycans Also useful: “PNGase A” and various “Endo-F” enzymes

16. GlcNAc-Transferases Determine Number of“Antennae” of N-glycans

17. Some representative examples of mammalian complex-type N-glycans

18. Slime Yeast Plants Mold Insects 3 a a3 6a a3 6a a 4 N N Asn Asn a 4 b 2 a 4 a3 a6 N N Vertebrates Asn Asn “Pauci- mannose” Evolutionary Variations of the N-glycan Processing Pathway Eubacteriae do not express N-glycans, but Archeabacteriae do. However, the linkage involved may be different (e.g. GalNAc-Asn or Glc-Asn) (Campylobacter jejuni recently reported to do N-glycosylation)

19.           NO 1 = Golgi Mannosidase I Complex and hybrid-type glycans 2 = GlcNAc Phosphotransferase 3 = GlcNAc Transferase I 4 = Phosphodiester glycosidase * * 5 = Galactosyltransferase -P- -P * * +/- 6 = Sialyltransferase(s) 4 E 1 * -P- * 5,6 * + -P * 4 D 1,2,3 1 * * P- -P -P- -P- * * -P- * +++ 4 2 2 A C B BINDING TO MPRs Biosynthesis of Phosphorylated N-glycans of Lysosomal Enzymes: Recognition by Mannose 6-Phosphate Receptors (MPRs) NO NO +/-

20. -6P -1P Congenital Disorders of Glycosylation -6P Ib Ia Dol-P- Dol-P- If Ie GDP- Id Dol-P-P Ig Ic Dol-P-P Dol-P-P Dol-P-P Dol-P-P Dol-P-P Dol-P-P LLO Dol-P-P On Protein IIb UDP -N-X-T/S -N-X-T/S -N-X-T/S -N-X-T/S -N-X-T/S IIc Golgi UDP UDP GDP- CMP Cytosol IIa IId GDP- -N-X-T/S -N-X-T/S -N-X-T/S -N-X-T/S H.Freeze -N-X-T/S

21. Golgi ER High Mannose a2 Hybrid Complex a3 a3 a2 a2 a2 b2 a2 a6 a3 b2 a3 a6 b4 GlcNAc-TI GlcNAcT-II -mannosidase II a b4 Mgat2 gene Mgat1 gene Asn Asn Asn Asn Dol Gpt, then others CDG-IIa phenocopy Lethality E9-10 a -M III Lethality E4-5 GlcNAc-TI Asn Mouse Mutants in N-Glycosylation J.Marth

22. Biosynthesis and folding Stability in the ER - targeting to proteosomes Secretion rate Intracellular trafficking Cell surface expression Intracellular stability and turnover rate Range or specificity of function Activity of enzymes, hormones & cytokines Signal transduction function of receptors Susceptibility to proteases or denaturants Recognition by antibodies (important for viruses) Circulatory half-life Targeting to specific cell types or organs FUNCTIONAL EFFECTS OF MODIFYING OR ELIMINATING N-LINKED CHAINS ON GLYCOPROTEINS

23. N-glycosylation appears to be carefully titrated and there is only one set of genes for the pathway...why?