ESSENTIALS OF GLYCOBIOLOGY

LECTURE 33

GENETIC DISORDERS OF GLYCOSYLATION IN HUMANS

Hud Freeze

OVERVIEW AND SUMMARY

Many Human glycosylation disorders were discovered in the last 10 years, mostly in the N-linked pathway.

Disorders are underdiagnosed. Alert physicians.

Defects include monosaccharide activation, glycosyltransferases, transporters, biosynthetic glycosidasesGolgi trafficking proteins.

A few disorders can be treated with monosaccharides

Misglycosylation causes some types of muscular dystrophy

More glycosylation disorders will be found, phenotypesWill be broad and variable

GLYCOSYLATION DEFECTS IDENTIFIED GLYCOSYLATION DEFECTS IDENTIFIED

Year99989796959480-

930100

15

5

10

20

5

10

15

20N-linked DisordersMuscular Dystrophy

02-04

Nu

mb

er o

f d

efec

ts

-N-X-T/S

On Protein

Dol-P-P

LLO

TYPE I TYPE II

CONGENITAL DISORDERS OF GLYCOSYLATION

-N-X-T/S

CONGENITAL DISORDERS OF GLYCOSYLATION(CDG)

TYPE I---Defects in synthesis or transfer of oligosaccharides from Dol-PP carrier to proteins in the lumen of the endoplasmic reticulum

TYPE II--Defects in the processing of N-linked oligosaccharides OR in all other types of glycosylation

Types Number of Patients

Ia-IL-- >400 (Ia>300) IIa-IIe-- ~20 Ix or IIx-- >50

CDG--often (not always) recognized by glycosylation-dependentalterations in serum transferrin isoelectric focusing pattern

Isoelectric focusing of transferrinIsoelectric focusing of transferrin

4

2

0

control controlCDG patients

Sialic acidGalactose

N-acetylglucosamineMannose

normal missing chains altered processing

Sialic acids

30-40 GENES REQUIRED TO MAKE AND ADD AN ASN-LINKED CHAIN30-40 GENES REQUIRED TO MAKE AND ADD AN ASN-LINKED CHAIN30-40 GENES REQUIRED TO MAKE AND ADD AN ASN-LINKED CHAIN30-40 GENES REQUIRED TO MAKE AND ADD AN ASN-LINKED CHAIN

Man Fuc

GDP-Man

Man-l-P

GTP

NADP

Dol-P-Man

GDP-Fuc

Man-6-P

Dol-P

ATP

Fuc-1-P

GTP

ATP

ManNAc-6-P

PEP

Neu5AcCMP-Neu5Ac Neu5Ac-9-P

CTP

ManNAc

UDP- GlcNAcUDP-GalNAcGalNAc

ATP

GalNAc-1-P

UTP

GlcNAc-6-P

GlcNAc-l-P

UTP

Dol-P-Glc

GlcNAc

UDP-Xyl

-CO2

UDP-GlcA

UDP-Glc

Glycogen

NAD+

UDP-Gal

Gal

Gal-1-P

UTP UDP-Glc

Dol-P

ATP

Glc-1-P

Glc

Glc-6-P Fru-6-P

ATP

Glutamine

GlcN-6-P

-NH3

Pi

ATP

AcCoA

NAD

Glycolysis

CMP-Neu5Ac

NADP

OVERVIEW OF SUGAR METABOLISM IN CELLS

Man Fuc

GDP-Man

Man-l-P

GTP

NADP

Dol-P-Man

GDP-Fuc

Man-6-P

Dol-P

ATP

Fuc-1-P

GTP

ATP

ManNAc-6-P

PEP

Neu5AcCMP-Neu5Ac Neu5Ac-9-P

CTP

ManNAc

UDP- GlcNAcUDP-GalNAcGalNAc

ATP

GalNAc-1-P

UTP

GlcNAc-6-P

GlcNAc-l-P

UTP

Dol-P-Glc

GlcNAc

UDP-Xyl

-CO2

UDP-GlcA

UDP-Glc

Glycogen

NAD+

UDP-Gal

Gal

Gal-1-P

UTP UDP-Glc

Dol-P

ATP

Glc-1-P

Glc

Glc-6-P Fru-6-P

ATP

Glutamine

GlcN-6-P

-NH3

Pi

ATP

AcCoA

NAD

Glycolysis

CMP-Neu5Ac

NADP

Biosynthesis and Interconversion of Monosaccharides. The relative contributions of

each under physiological conditions are unknown. and - donors;

- monosaccharides; -control points

Mannose supplements can treat CDG-Ib, Fru-6-P-->Man-6-P defect

0

40

80

120

160

200

-15 -13 -11 -9 -7 -5 -3 -1 1 3 5 7 9 11 13 15 17

0

1

2

3

4

5

6

7

-15 -13 -11 -9 -7 -5 -3 -1 1 3 5 7 9 11 13 15 17

0

2

4

6

8

10

-15 -13 -11 -9 -7 -5 -3 -1 1 3 5 7 9 11 13 15 17

anticoagulation (Marcumar R )

i. v. albumin substitution

albumin (g/dl)

fecal alpha1 AT (mg/g)

AT III

time in months

BEFORE MANNOSE DURING MANNOSE

CDG defect sugar transporter Man GlcNAc

-6-P

-1-P GDP-

Dol-P-

Fru-6-PPMI

PMM

Therapy for CDG-Ib

Man Fuc

GDP-Man

Man-l-P

GTP

NADP

Dol-P-Man

GDP-Fuc

Man-6-P

Dol-P

ATP

Fuc-1-P

GTP

ATP

ManNAc-6-P

PEP

Neu5AcCMP-Neu5Ac Neu5Ac-9-P

CTP

ManNAc

UDP- GlcNAcUDP-GalNAcGalNAc

ATP

GalNAc-1-P

UTP

GlcNAc-6-P

GlcNAc-l-P

UTP

Dol-P-Glc

GlcNAc

UDP-Xyl

-CO2

UDP-GlcA

UDP-Glc

Glycogen

NAD+

UDP-Gal

Gal

Gal-1-P

UTP UDP-Glc

Dol-P

ATP

Glc-1-P

Glc

Glc-6-P Fru-6-P

ATP

Glutamine

GlcN-6-P

-NH3

Pi

ATP

AcCoA

NAD

Glycolysis

CMP-Neu5Ac

NADP

Oral fucose supplements used to Treat CDG-IIc-- Defective in Golgi GDP-Fuc Transporter

Patient is deficient in fucosylationWhat symptoms would you expect?Cause?What therapy?

FUCOSE THERAPY NORMALIZES NEUTROPHILCOUNTS OF ONE CDG-IIc PATIENT

Serum Fucose(µM)

Normal Range

QuickTime™ and aBMP decompressor

are needed to see this picture.

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

CLINICAL FEATURES OF CDG PATIENTS

Used with permission T. Marquardt.

European J. Ped (2003)162 359-379.

Kindly provided by Dr. Thorsten Marquardt

Dysmorphic features of a baby with CDG-Ia (PMM-deficiency)

-6P

GDP-

Ia-6P -1P

DEFECT

-6P -1P

GDP-

GDP-

-N-X-T/S

Golgi

CytosolIIa

IIb

IIcUDP

CMPUDP

UDP

-N-X-T/S -N-X-T/S

-N-X-T/S -N-X-T/S

-N-X-T/S -N-X-T/S

-N-X-T/S -N-X-T/S

-N-X-T/S

IId

Dol-P-P

-6P Dol-P-

GDP-

Dol-P-Ib Ia

Ic

Id

Ie If

Dol-P-P

IgDol-P-P

Dol-P-P

Dol-P-P

Dol-P-P

Dol-P-P

Dol-P-P

LLO

On Protein

-N-X-T/S -N-X-T/S

T/ST/S

Possible Defect in Sialic Acid Synthesis or Transport?

Defect is Shared by N-Linked and O-Linked Pathways

Defects in Multiple Sialyl Transferases?

Core1GalT and ST3Gal-I activities in control and patient fibroblasts(pmol/mg protein/min)

Enzyme Control H.D.

Core1GalT 3.1 ± 0.6 1.9 ± 0.2

ST3 -Gal I 0.97 ± 0.08 0.37 ± 0.05

pm

ol/

mg

pro

tein

Endogenous acceptor

pm

ol/

mg

pro

tein

0 4 6 8 10 122

Exogenous acceptor (GAP)

D

Vmax 182

Lec20

50

100

150

200Control CHO

D

E

F

ControlVmax 43

Control

Vmax 306

HD Vmax 18

HDVmax 92

0

50

150

250300

200

100

01020

304050

CMP-Sia (uM)

UDP-Gal (uM)

CMP-Sia (uM)

60Time (min)

0

50

100

150

200

250

CMP-[3H]Sia

0

50

100

150

200

250[3H]CMP-Sia

0

50

100

150

200

250

UDP-[3H]Gal

A

B

C

3

Control Control+TX-100 HD

Nucleotide Sugar Transport is reduced

Vmax is reduced, Km remains the same

H

DC

ontr

ol

F

D Pre-bleach bleach 10min 30min 60 min

Recovery

ST-GFP Trafficking is reduced in HD and FD cells

T/SST3Gal-IST3Gal-I

ldlB=Cog1 ldlC=Cog2

ldlB and ldlC deficient cells are defective in glycosylation

Cog7 mutation decreases mRNA and protein

Destabilizes COG complex, mislocalizing some COG subunits

Decreases trafficking of glycosyltransferases and transporters

Decreases enzyme/transporter stability and normal localization

Decreases terminal modifications on glycans

Mutation Function

SUMMARY• Physicians must know many faces of glycosylation • Defective Glycosylation causes disease • Only a few of the potential types of CDG are known• Simple therapy may help a few CDG patients• CDG may be the cause of unexplained cases of developmental

delay, coagulopathy, and hypoglycemia• CDG presentations can be very mild and broad

WHENWHEN TO CONSIDER CDG?TO CONSIDER CDG?

””EVERY TIME YOU SUSPECT IT….EVERY TIME YOU SUSPECT IT….AND EVERY TIME YOU DON’T”AND EVERY TIME YOU DON’T”

-J. Jaeken-J. Jaeken

Man Fuc

GDP-Man

Man-l-P

GTP

NADP

Dol-P-Man

GDP-Fuc

Man-6-P

Dol-P

ATP

Fuc-1-P

GTP

ATP

ManNAc-6-P

PEP

Neu5AcCMP-Neu5Ac Neu5Ac-9-P

CTP

ManNAc

UDP- GlcNAcUDP-GalNAcGalNAc

ATP

GalNAc-1-P

UTP

GlcNAc-6-P

GlcNAc-l-P

UTP

Dol-P-Glc

GlcNAc

UDP-Xyl

-CO2

UDP-GlcA

UDP-Glc

Glycogen

NAD+

UDP-Gal

Gal

Gal-1-P

UTP UDP-Glc

Dol-P

ATP

Glc-1-P

Glc

Glc-6-P Fru-6-P

ATP

Glutamine

GlcN-6-P

-NH3

Pi

ATP

AcCoA

NAD

Glycolysis

CMP-Neu5Ac

NADP

UDP-GlcNAc epimerase/kinase Defective in two human diseases

Sialuria and Hereditary Inclusion Body Myopathy-II

Sialuria--hypotonia, cerebellar ataxia, and mental retardation

Muscular Dystrophies

Walker Warburg Syndrome--POMT1 mutations cause 30%MEB-Muscle Eye Brain Disease--POMGnT1(1p32-34)

Encodes a 1,2GlcNAc transferase specific for O-ManFukuyma-type CMD--fukutin (9q31)

Putative transferase in golgi (?)Fukutin Related protein--(19q13.3)

Putative transferase (?)LARGE- cause of myd mouse, tandem glycosyltransfrases(?)

Common Features:Affects -dystroglycan glycosylation and not -dystroglycan

Hereditary Inclusion Body Myopathy II (9p12-13)UDP-GlcNAc epimerase/kinase used for CMP-Sia

O-Mannose: An Emerging Family of Glycans

WWS-Mutated transferase

O-Mannose: An Emerging Family of Glycans

MEB- Mutated transferase

MACULAR CORNEAL DYSTROPHY

Autosomal recessiveProgressive punctate corneal opacity, requires transplantCaused by mutation in GlcNAc-6-Sulfotransferase (CHST6)

used for sulfation of keratan sulfate

http://www.burnham.org/presentations/human_glycosylation_disorders/index.htm