ESSENTIALS OF GLYCOBIOLOGY LECTURE 13 OTHER TYPES OF GOLGI GLYCOSYLATION Hud Freeze

SUMMARY AND TAKE HOME MESSAGES “Rare” forms of glycosylation are not rare or insignificant Technical limitations slowed their identification O-linked Mannose-based glycans are abundant in brain These chains are prominent on  -dystroglycan Altered O-Mannose glycans cause Muscular Dystrophy O-Fucose, O-Glucose found in EGF domains Notch signaling pathways depend on glycosylation Other forms of glycosylation are likely to have functions

RARE FORMS OF GLYCOSYLATION Discovery  Minor component in an abundant source  Thorough analysis of a well known or important molecules  Antibody against a “glycan”  Dedicated analysis of mixtures of glycans, proteins or organs  Chance

RARE FORMS OF GLYCOSYLATION Roadblocks to Discovery  Degradation and analysis is difficult  Minor amounts  Biosynthetic route is unknown  Tools required are often state of the art level Significance  Ranges from Unknown-----> critically important  Primitive eukaryotic function - selection and adaptation to specific needs in mammals

Urinary Oligosaccharides and Glycosides Oligosaccharides Xyl  1,3Glc 5-10 *Fuc  1,2Glc 5-10 Glycopeptides Xyl  -Ser 1 Gal  -Hyl12 GlcNAc  -Asn 4 Glc  1,3Fuc  -Thr0.2 *Not reported in proteins

C-X-D/N-X-X-X-Y/F-C-X-C C-X-X-G-G-T/S-C C-X-S-X-P-C EGF MODULE AND ASSOCIATES

1--Glc  -Ser 2--Fuc  -Ser 3--  -hdroxy-Asp/Asn EGF Module of human Factor IX

What are the functions of these modifications? Little effect on half-life or activity of clotting proteins How about other proteins with EGF modules? Now it gets interesting

Notch - an EGF Signaling Protein Signaling molecule from C. elegans  Humans Signals induced in binding to Delta or Jagged (serrate) on adjacent cells Signaling defects in Notch cause abnormal development, leukemia and a complex disease of Cerebral Artiopathy and Infarcts Human Notch-1 (of 4) contains 12 O-Fucose and 17 O-glucose modification consensus sites Could these be involved in Signaling????

O-fucose O-glucose both Notch retains  Fucose and  Glucose glycosylation motifs

Are the Sugar Chains Important for Anything? Half of the glycosylation sites are conserved across species (3, 4, 10, 12-14, 13, and 14 all contain conserved sites). The EGF modules 11 and 12 are essential for binding to Delta. Modules 10, 12, 13, and 14 all contain conserved sites Abnormal wing vein mutations occur in modules Human disorders have been tracked to mutations in Notch 3. Fringe proteins differentially modify Notch binding to Delta and Serrate. Wonder what fringe is…....

Are the Sugar Chains Important for Anything? Mammalian fringe homologs exist--manic, lunatic, radical Fringe must be expressed in the same cell as Notch to exert its effect. Fringe is secreted.

Notch and its ligands both contain EGF domains Notch Delta Serrate SIGNALS FRINGE GENES

Jagged Notch + manic +lunatic Fringe proteins modify Notch/ligand binding and signaling Lunatic and manic modify different EGF modules

FRINGE IS A GLYCOSYL TRANSFERASE!!! ARE YOU REALLY SURPRISED? Fuc  Ser/Thr+UDP-GlcNAcGlcNAc ,1,3Fuc  Ser/Thr

Gal  1,4GlcNAc ,1,3Fuc  MINIMAL GLYCAN INVOLVED IN NOTCH SIGNALING That requires  1,4GalT, the usual one for N-linked chains DON’T FORGET Glc ,1,3Fuc  Thr FOUND IN HUMAN URINE AND CHO CELLS ADDITIONAL FUNCTIONS?

1--Glc  -Ser 2--Fuc  -Ser 3--  -hdroxy-Asp/Asn EGF Module of human Factor IX

BAH--  ASP/ASN HYDROXYLATION Mice have developmental Abnormalities and are also prone to intestinal polyps

O-Mannose: An Emerging Family of Glycans About 1/3 of brain O-linked chains are O-mannose based

Muscular Dystrophies *MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a  1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Encodes a glycosylt’ase--O-mannosyl transferase(?) Fukutin Related protein--(19q13.3) dystroglycan misglycosyaltion and laminin deficiency Overexpression of a specific  1,4GalNAc transferase reverses MD in a model of Duchenne MD (dystrophin) Common Features: Affects  -dystroglycan glycosylation and not  -dystroglycan Heriditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

O-Mannose: An Emerging Family of Glycans Mutated transferase

Muscular Dystrophies MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a  1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Encodes a glycosylt’ase--O-mannosyl transferase(?) *Fukutin Related protein--(19q13.3) dystroglycan misglycosyaltion and laminin deficiency Overexpression of a specific  1,4GalNAc transferase reverses MD in a model of Duchenne MD (dystrophin) Common Features: Affects  -dystroglycan glycosylation and not  -dystroglycan Heriditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

Dystroglycan in Myd Mouse

Muscular Dystrophies MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34) Encodes a  1,2GlcNAc transferase specific for O-Man Fukuyma-type CMD--fukutin (9q31) Encodes a glycosylt’ase--O-mannosyl transferase(?) Fukutin Related protein--(19q13.3) dystroglycan misglycosyaltion and laminin deficiency *Overexpression of a specific  1,4GalNAc transferase reverses MD in a model of Duchenne MD (dystrophin) Common Features: Affects  -dystroglycan glycosylation and not  -dystroglycan Heriditary Inclusion Body Myopathy II (9p12-13) UDP-GlcNAc epimerase/kinase used for CMP-Sia

O-Mannose: An Emerging Family of Glycans Over expressed

C-Mannosylation - Novel C-C bond Consensus sequence in 100’s of proteins and wide- spread in mammalian cells. Mannosyl transferase present in many cells. Antibody and structural determination are keys to finding this unusual form of protein modification WXXW--

Thrombospondin has Man(C)--Trp And Glc-Fuc-O-disaccharide in EGF domains Man(C)--Trp Glc-Fuc--O-Ser/Thr Residues implicated in binding to GAG chains

GLYCOSYLATION + AMINO ACID MODIFICATION A RECURRING THEME IN RECOGNITION? Sugar chain Tyr-SO 4 P-Selectin C- Type Lectin Domain CR-Repeat EGF- Domain Q - A - T - E - Y - E - Y - L - D - Y - D - F - L - P - E - T - E - P - P 33 11 66 44 33 33 33 O SO 3 - O - O -

VARIABLE TYPES OR AMOUNTS OF GLYCOSYLATION CAN STRONGLY AFFECT BIOLOGICAL READOUT AND PHYSIOLOGY-- WATCH OUT FOR THE NEWCOMERS