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

Major Glycan Classes in Animal Cells O Ser O Ser/Thr N Asn Ser-O- OUTSIDE INSIDE N Asn S SS -O-Ser S S S SS Etn P INOSITOL P NH Ac P NS Ac S 2 P Glycoprotein ProteoglycanGLYCOPHOSPHO-LIPIDANCHORO-LINKEDCHAIN HYALURONAN GLYCOSAMINO-GLYCANS HEPARAN SULFATE CHONDROITIN SULFATE SULFATE Sialic Acids GLYCOSPHINGOLIPID O-LINKED GlcNAc N-LINKED CHAINS

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

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

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

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

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

Biosynthesis of the N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER) Adapted from Marquardt T, Denecke J. Eur J Pediatr Jun;162(6): GlcNAc Man Gal Sia Fuc Glc 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

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

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

Oligosaccharyltransferase complex (OST) in the ER membrane transfers the dolichol N-glycan precursor to asparagine residues on nascently translated proteins 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

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

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

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

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

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

Some representative examples of mammalian complex-type N-glycans

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) N Asn N  Vertebrates  3  4  4  4 N Asn Yeast  Insects   2 Plants  Slime Mold N Asn  “Pauci- mannose”

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

-6P-1P GDP- -N-X-T/S Golgi Cytosol IIa IIb IIc UDP CMP UDP -N-X-T/S IId Dol-P- P LLO On ProteinCongenital Disorders of Glycosylation H.Freeze

Mouse Mutants in N-Glycosylation J.Marth

FUNCTIONAL EFFECTS OF MODIFYING OR ELIMINATING N-LINKED CHAINS ON GLYCOPROTEINS 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

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