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

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

Synthesis and Structure of Major Glycan Classes 1/24/05 Large O-linked Glycosaminoglycans and polylactosamine structures Glycoprotein N-linked and Olinked oligosaccharides Glycolipid oligosaccharides The building blocks = Glucose (Glc) Hexose,

= Fucose (Fuc) = Mannose (Man) unspecified (Hex) = Xylose (Xyl) = Galactose (Gal) = Sialic acid, unspecified (Sia) = N-acetylglucosamine (GlcNAc) = Glucuronic acid (GlcA)

= N-acetylgalactosamine (GalNAc) = Iduronic acid (IdoA) = N-acetylhexosamine, unspecified (HexNAc) = Uronic acid, unspecified (HexA) Fuc 3 Simplified Traditional

3Gl4GlcNAc2Mn Si Fuc 6 4GlcNAc4GlcNAc Mn 3 6 9Ac-SiaSi6Gl4GlcNAc2Mn Symbolic

Representation 3 9Ac 6 4 3 4 3

2 2 6 3 4 6 4 3

9Ac 4 2 6 3 3 4 2

4 4 6 9Ac Glycan synthesis in a cellular context Overview From ER through

Trans-Golgi and points ER processing of N-linked glycans Major Classes of N-Glycans Hybrid High-Mannose (oligo-mannose) Complex GlcNAc Man Glc

Gal Sia Fuc 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 Glc Gal Sia Fuc 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 GlcNAc Man Glc Gal Sia Fuc 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 Glc Gal Sia Fuc 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 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 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 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 asic Glycosylphosphatidylinositol (GPI) Ancho Protein Glycan Phospholipid Examples of GPI-Anchored Proteins Cell surface hydrolases alkaline phosphatase Protozoal antigens trypanosome VSG

acetylcholinesterase leishmanial protease 5 nucleotidase plasmodium antigens Adhesion molecules neural cell adhesion molecule heparan sulfate proteoglycan Mammalian antigens carcinoembryonic antigen

Thy-1 Others scrapie prion protein folate receptor decay accelerating factor Structure of the Basic GPI Anchor GPI-Linked Protein = Mannose (Man) NH2 = Glucosamine Etn = Ethanolamine P

Etn P = Phosphate PNH Defect Pig-A NH2 INOSITOL P Cell Surface Membrane

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 Protein GPI-Signal Sequence Acetylcholinesterase (Torpedo)

NQFLPKLLNATAC DGELSSSGTSSSKGIIFYVLFSILYLIFY Alkaline Phosphatase (placenta) TACDLAPPAGTTD AAHPGRSVVPALLPLLAGTLLLLETATAP Decay Accelerating Factor HETTPNKGSGTTS GTTRLLSGHTCFTLTGLLGTLVTMGLLT PARP (T. Brucei) EPEPEPEPEPEPG AATLKSVALPFAIAAAALVAAF

Prion Protein (hamster) QKESQAYYDGRRS SAVLFSSPPVILLISFLIFLMVG Thy-1 (rat) Variant Surface Glycoprotein (T. Brucei) KTINVIRDKLVKC GGISLLVQNTSWLLLLLLSLSFLQATDFISI ESNCKWENNACKD SSILVTKKFALTVVSAAFVALLF Bold AA is site of GPI attachment Sequence to right is cleaved by the transpeptidase upon Anchor addition Golgi processing of N-linked glycans

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

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 Slime Mold Yeast Pauci- Plants mannose

Insects 3 3 N Asn 6 3 6 4

N Asn 4 2 4 3 6 N Asn

N Asn Vertebrates Golgi processing of O-linked glycans

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