Synthesis and Structure of Major Glycan Classes 1/24/05

Large O-linked Glycosaminoglycans and poly-lactosamine structures Glycoprotein N-linked and O-linked oligosaccharides Glycolipid oligosaccharides

Symbolic Representation Simplified Traditional The building blocks Symbolic Representation Simplified Traditional

Glycan synthesis in a cellular context

Overview From ER through Trans-Golgi and points inbetween

ER processing of N-linked glycans

Major Classes of N-Glycans “Hybrid” “Complex” “High-Mannose” (oligo-mannose)

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

GlcNAc Man Gal Sia Fuc Glc Biosynthesis of the N-Glycan Precursor on the Cytosolic Leaflet of the Endoplasmic Reticulum (ER) CDG = Congenital Disorder of Glycosylation in Humans Adapted from Marquardt T, Denecke J. Eur J Pediatr. 2003 Jun;162(6):359-79

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

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. 2003 Jun;162(6):359-79

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 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 GlcNAc Man Gal Sia Fuc Glc 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

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

ER glycolipid synthesis

Biosynthesis of Ceramide and Glucosylceramide

ER glycolipid synthesis

Basic Glycosylphosphatidylinositol (GPI) Anchor Phospholipid

Examples of GPI-Anchored Proteins Cell surface hydrolases alkaline phosphatase acetylcholinesterase 5’ nucleotidase Protozoal antigens trypanosome VSG leishmanial protease plasmodium antigens Adhesion molecules neural cell adhesion molecule heparan sulfate proteoglycan Mammalian antigens Thy-1 carcinoembryonic antigen Others decay accelerating factor scrapie prion protein folate receptor

Structure of the Basic GPI Anchor

Structural Analysis of the GPI Anchor Enzymatic and chemical cleavage sites are useful in identifying GPI anchored membrane proteins

Examples of C-Terminal Sequences Signaling the Addition of GPI-Anchors Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition

Golgi processing of N-linked glycans

Completion of Processing of N-Glycans in ER and Golgi GlcNAc Man Gal Sia Fuc Glc 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

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 Asn Yeast Slime Mold N Asn a3 6a b 2 Plants a3 “Pauci- mannose” a3 Insects 6a a6 Vertebrates a 3 4 N Asn

Golgi processing of O-linked glycans