ESSENTIALS OF GLYCOBIOLOGY

LECTURE 13OTHER 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

OligosaccharidesXyl1,3Glc 5-10*Fuc1,2Glc 5-10

GlycopeptidesXyl-Ser 1Gal-Hyl 12GlcNAc-Asn 4Glc1,3Fuc-Thr 0.2

*Not reported in proteins

C-X-D/N-X-X-X-Y/F-C-X-CC-X-X-G-G-T/S-C

C-X-S-X-P-C

EGF MODULE AND ASSOCIATES

1--Glc-Ser2--Fuc-Ser3---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-fucoseO-glucoseboth

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 24-29.

• 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

SIGNALSSIGNALS

SIGNALSSIGNALS

SIGNALSSIGNALS

SIGNALS

FRINGE GENES

Jagged Jagged Jagged

Notch Notch 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?

FucSer/Thr+UDP-GlcNAc GlcNAc,1,3FucSer/Thr

Gal1,4GlcNAc,1,3Fuc

MINIMAL GLYCAN INVOLVED IN NOTCH SIGNALINGThat requires 1,4GalT, the usual one for N-linked chains

DON’T FORGET

Glc,1,3FucThr

FOUND IN HUMAN URINE AND CHO CELLSADDITIONAL FUNCTIONS?

1--Glc-Ser2--Fuc-Ser3---hdroxy-Asp/Asn

EGF Module of human Factor IX

BAH-- ASP/ASN HYDROXYLATION

Mice have developmentalAbnormalities andare also prone tointestinal 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 reversesMD 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 reversesMD 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 reversesMD 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)--TrpAnd Glc-Fuc-O-disaccharide in EGF domains

Man(C)--Trp Glc-Fuc--O-Ser/Thr

368 377420 432423 ---480 489

Residues implicated in binding to GAG chains

GLYCOSYLATION + AMINO ACID MODIFICATION

A RECURRING THEME IN RECOGNITION?

Sugar chain

Tyr-SO4

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 - 42 46 48 51 57

3

16

4

3

3

3

O

SO3

-O

SO3

-O

SO3

-

VARIABLE TYPES OR AMOUNTS OF GLYCOSYLATION CANSTRONGLY AFFECT BIOLOGICAL READOUT AND

PHYSIOLOGY--WATCH OUT FOR THE NEWCOMERS