GLYCOMICS:

NOVEL PLATFORM FOR

DIAGNOCTICS AND THERAPY

Professor Jerka Dumić

University of Zagreb, Faculty of Pharmacy and
Biochemistry, Zagreb, Croatia

Nicolaus Copernicus
Nature 409:6822
February 15, 2001

What does genetics offer?

 number of genes genetic similarity

30.000 human 99.9% similarity between humans

30.000 chimpanzee 99% similarity to humans

30.000 mouse 70% similarity to humans

13.000 fly 60% similarity to humans

19.000 worm 20% similarity to humans

1
CH3COO‐ ?

?
pharnesylation

?
CH3
? ‐ OPO3–

Why posttranslational modifications?

• 30 000 genes vs. 200 000 proteins or more
• genomes of complex organisms contain much more genes coding
proteins participating in posttranslational modifications than
genomes of simple organisms at all
• wide‐spread (glycosylation, phosphorylation, ubiquitinylation, and methylation)
vs. uncommon posttranslational modifications (gluthationylation,
hydroxylation, sulphatation, transgluthaminylation, epimerisation)

• posttranslational modificatons – for cell “cheaper” and “faster”

than protein synthesis (“fine tuning”)

The cell membrane

WRONG

2
 Membranes of all cells are virtually covered
with complex carbohydrates

DID YOU KNOW ...

• > 50% of all today known proteins as well as
> 80% membrane proteins are gylcosylated
• glycans – the most abundant and diverse biopolymers found
in nature
• O‐N‐acetylglucosaminylation ‐ regulatory modification ‐ more
abundant than phosphorylation
• > 1% of the mammalian genome encodes enzymes that
produce and modify glycan structures
• CMP‐sialic acid hydroxylase – the only identified gene that
differs between humans and chimpanzees

KNOWLEDGE ABOUT GLYCAN STRUCTURES AND FUNCTIONS

IS INSUFFICIENT! ! !

3
 Glycans are one of four principal
components of a cell

GLYCOCONJUGATES
Proteoglycans

Glycoproteins

Glycolipids

Glycosylphosphatidyl
‐inositol anchors
Monosaccharide
Protein
Lipid
Ethanolamine phosphate
Inositol phosphate

Repertoire of monosaccharides found in

glycoconjugates is limited

OH
OH HO C H 2 HO C H 2
H O CH 2 HO CH 2 O O
O O
HO HO

HO HO HO
OH HO NH NH
OH C OH C OH
OH OH H3 C H 3C O
O

‐D‐Glucose ‐D‐Galactose N‐Acetyl‐‐D‐glucosamine N‐Acetyl‐‐D‐galactos‐

(Glc) (Gal) (GlcNAc) amine (GalNAc)
OH
OH
HO CH 2 H O H2 C C
O
H3 C O C O
HO OH O O C
HO HO
HO OH OH N OH
HO
OH OH O C OH
OH OH OH
CH 3
‐D‐Mannose (Man) ‐L‐Fucose (Fuc) ‐D‐Xylose (Xyl) N‐Acetylneuraminic acid (Neu5Ac)

D‐Glucuronic acid L‐Iduronic acid D‐Arabinose D‐ & L‐Rhamnose D‐Galacuronic acid

L‐Arabinofuranose D‐Galactofuranose N‐Acetylmannosamine 3‐N‐Acetyl‐D‐quinovosamine Neu5Gc
and a few more including their derivatives

4
 Potential diversity of glycan
structures is enormous
1. Monosaccharide sequence
2. Glycosidic bond position
OH
3. Anomeric configuration (α or β) HO C H 2
of glycosidic linkage O

4. Number of branching points HO

5. Position of branching C
NH

H 3C O O

OH 6
HO C H 2 C H2
O O
HO
HO
 O
OH OH
pentamer ABCDE nuber of isomers
1 3 OH
Oligosaccharides 2 144 640
oligopeptides 120

At least 2000 different glycan determinants are being

attached to polypeptide backbones

Glycoproteome is several orders

of magnitude more complex
than the proteome

Single IgG molecule is produced as a

mixture of over 100 glycoforms

5
Fine details of N‐glycans strongly affect
effector funcions of IgG

6
 antibody‐dependent cell‐mediated cytotoxicity

N‐Glycan branching affects membrane half‐life of

numerous receptors

Grigorian A et al. J. Biol. Chem. 2007

Notch signalling is regulated by glycosylation

Jafar-Nejad H et al. Glycobiology 2010;20:931-949

7
 Can we really understand the function of a
glycoprotein without knowing its glycan part?

Prion protein

HIV gp120

DNA does not contain templates for synthesis of

glycan parts of glycoproteins

DNA RNA proteins

glycoproteins

Glycans are being made by sequential

action of numerous enzymes

8
 Glycans are a result of an interplay of
hundreds of genes

1 gene  1‐10 proteins

>500 genes  1 glycoprotein

Nearly all proteins invented by multicellular

organisms are glycosylated

The majority of genes that affect protein glycosylation

are still not known

IC
TF

GO / TP
GTs

GDs

Out of 109 genes found to affect neural specific

glycosylation in Drosophila, only 14 were known glyco‐
genes

Yamamoto‐Hino et al, PLoS Genet 6(12): e1001254, 2010.

9
 Glycans are a result of an interplay of
thousands of genes

1 gene  1‐10 proteins

>2000 genes  protein glycosylation

With nearly 10% of the genome devoted to it, glycosylation is

the most complex process in humans.

Genome wide association studies initiated

revolution in genetics

Glycomics was set to join

large scale genomics after
high throughput HPLC
method was developed by
Pauline Rudd and
colleagues in 2008

10
Single IgG polypeptide is produced as a
mixture of over hundred glycoforms

HPLC is quantitative and groups glycans

according to structural similarities

Human N‐plasma glycome

(~ 4500 individuals)

 very high variability between individuals

 very stable regulation of glycan levels within an individual
– individual glycosylation profiles are very stable even after
prolonged times
– age and other parameters explain only up to 5% of variance
for most glycans
– heritability generally below 50%
 changes in specific glycans as a response to
environmental stimuli
– potential diagnostic applications
Knežević et al, J. Proteome Res, 2009
Gornik et al, Glycobiology 2009
Knežević et al, Glycobiology, 2010

11
 Some individuals have very different patterns of N‐
linked glycans in plasma (glyco‐phenotypes)
Normal N‐ Outlier A
glycan profile

Outlier Outlier C
B

Outlier Outlier E
D

Pučić et al, Glycobiology, 2010

HPLC glycan data can be used for GWAS studies

Lauc et al, Nature Precedings, 2009

The first GWAS study of human glycome –

HNF1A (hepatocyte nuclear factor 1A)

Lauc et al, PLOS Genetics, 6(12): e1001256, 2010

12
 Gene silencing experiments provided insight into
mechanisms of observed GWAS association

The first GWAS study of human glycome identified HNF1A as

a master regulator of plasma protein fucosylation

Lauc et al, PLOS Genetics, 6(12): e1001256

Diabetes diagnostics in the 21st century!

Severe Insulin
Glucokinase Mitochondrial
resistance
mutation mutation

Hepatocyte Other
Nuclear Factor-
1a mutation Type 2

Latent
Autoimmune
Diabetes in
Adults
Type 1

13
 Maturity‐onset diabetes of the young

• monogenic diabetes affecting ‐cells

• autosomal dominant inheritance
– classically at least 2 generations of DM
• early onset non‐insulin dependant DM
– classically one family member died <25 years
– evidence of endogenous insulin secretion

• ~90% of UK MODY cases undiagnosed

HNF1A/4A‐MODY respond better to

sulphonylurea drugs
1
Gliclazide Metformin
0 1 2

-1
Decrease in fpg mmol/l

-2

-3
p=0.45
-4
Type 2

-5 HNF1a MODY
-6

-7 p=0.002
(Pearson et al Lancet 2003)

Why Sulphonylureas sensitivity?

sulphonylureas
Ca2+ Voltage dependent
depolarisation Ca2+ Channel
KATP L type
Channel
Glucose

GLUT 1,3 K+
Glucokinase ↑ [Ca2+]
↑ATP
Glycolysis ↓ MgADP Insulin

Mitochondria
HNF-1a
acts

Leads to regulated but reset glycaemia

14
 HNF1A as a master regulator of plasma protein
fucosylation

Plasma protein glycosylation can be indicative for a

function of a protein in and inacessible tissue

Mutations in HNF1 (HNF1A‐MODY) result in

decreased fucosylation of plasma proteins
DG9
12

10
% of the plasma glycome

0
HNF1A Type 2 Type 1 GCK general
MODY diabetes diabetes MODY population

Glycan ratio DG9/(DG8+DG9) is a robust index which

can be analyzed in both plasma and serum

DG8 DG9

DG9/(DG8+DG9) quantifies the amount of antennary

fucose on triantennary glycans

15
 GLYCANS ARE INVOLVED IN MANY
MEDICAL PROBLEMS

• tumor development and metastasis

• some storage diseases and mucopolysaccharidoses
• congenital disorders of glycosylation (CDGs)
• microbial infections
• allergies, autoimmune diseases and other immune disorders
• rejection of xenotransplants
• disruption of homeostasis
• individual (non) response to therapy
– and many others

Synthesis of N‐linked glycans occurs in cytoplasm & ER (formation of

the lipid‐linked oligosaccharide (LLO) precursor of N‐linked glycans) and GA
(processing of the protein‐bound sugar chains)

Congenital disorders of glycosylation

• a group of mainly autosomal recessive diseases

(metabolic syndromes)
• the primary defects either impair the formation of
the lipid‐linked oligosaccharide (LLO) precursor of N‐
linked glycosylation (CDGs type I) or affect
processing of the protein‐bound sugar chains (CDGs
type II)
• 45 types
• 1000 CDG patients diagnosed so far, but the true
incidence is estimated to be much higher (based on
frequency analysis for certain common mutations)

16
 PMM2- MPI-
Man-1-P Man-6-P Fru-6-P Glycolysi
Cytoplasm CDG
Ia CDG
Ib s
b
GDP-Man GDP
UDP-

CTP CDP RFT1-CDG

P P P P P P P
P P P P P P P P (In)

P P
DPAGT1- ALG1- ALG2- ALG11- ALG11- ALG3-
CDG (Ij) CDG (Ik)CDG (Ii) CDG (Ip) CDG (Ip) P CDG (Id) P

ALG9-
CDG (IL)
Endoplasmic reticulum

PP

GCS1- ALG8- ALG6- ALG9- ALG12-

CDG (IIb) OST P CDG (Ih) P CDG (Ic) P CDG (IL) P CDG (Ig)

P P P P

GA MGAT1-CDG
P
MGAT2-CDG (IIa) N-acetylglucosamine Mannose Glucose Dolichol-phosphate

TUSC3-CDG
Supraha et al, 2012

Congenital disorders of glycosylation

• 1980 the first N‐glycosylation defect was described and characterized
• all known CDGs have a recessive inheritance except
– EXT1/EXT2‐CDG ‐ dominantly inherited
– MGAT1‐CDG ‐ X‐linked
• 4 categories: defects of
– protein N‐glycosylation (16 types of CDGs)
– protein O‐glycosylation (8 types of CDGs)
– lipid glycosylation and glycosylphosphatidylinositol anchor glycosylation
(3 types of CDGs)
– defects in multiple glycosylation pathways and in other pathways (17
types of CDGs)
– several CDGs of so far unknown etiology (CDG‐x) have been recognized

CDGs are followed with wide spectrum of

symptoms and degree of severity
 dysmorphic features
 psychomotor retardation
 stroke‐like episodes
 coagulation disorders
 cerebellar ataxia
 floppiness
 feeding problems
 hypotonia

 often CDGs are under‐ or

misdiagnosed (neurological
disorders)

17
 Laboratory diagnostics
• the simplest and still wildly used methods for CDG screening are
isoelectric focusing (IEF) of transferrin (Tf) and apolipoprotein C‐III
(ApoC‐III) for N‐ and O‐glycan synthesis defects, respectively

Ctrl CDG - I CDG- II

+ pentasialo-Tf

tetrasialo-Tf

trisialo-Tf

disialo-Tf

monosialo-Tf
-
asialo-Tf
1 2 3

Laboratory diagnostics

• Glycan analysis
• Measurements of enzyme activities and metabolites
• Biochemical analysis (PMM2‐CDG ‐ low plasma cholesterol concentration
and cholinesterase activity with proteinuria PMM2‐CDG; ‐ high aminotransferases
activities; CDGs previously classified as type II – increased AST and normal ALT
activity but CDGs type‐I are not; PIGV ‐ hyperposphatasia.... transferrin
concentration and thrombocytopenia in various cases)
• Molecular diagnostics
• Prenatal diagnosis

Therapy
• MPI‐CDG (CDG‐Ib)
– defect of mannosephosphate isomerase which blocks the
endogenous mannose formation from glucose
– oral administration of mannose
• SLC35C1‐CDG (CDG‐IIc)
– defect of GDP‐fucose transporter ‐ immunodeficiency caused by
the absence of carbohydrate‐based selectin ligands on the
surface of neutrophils resulting in recurrent infections with
hyperleukocytosis as well as severe psychomotor and mental
retardation
– in some patients, increased level of fucose achieved by oral
supplementation might overcome low affinity of the fucose
transporters

18
How different
we really are?
at the gene level ~1%

5‐N‐acetylneuramine acid (Neu5Ac)

Sia Sia • one of the sialic acids

(26) (26)
• the only negatively charged sugars
Gal Gal
β(14) β(14)
• usually present at the termini of the
GlcNAc GlcNAc
glycan structures
β(12) β(12)
GlcNAc
Man Man
β(14)
(13)
Man
(16)
OH
β(14) HOH 2C C
GlcNAc C O
HO O C
β(14)
N OH
GlcNAc Fuc
(16)
O C OH
OH
Asn (297)
CH 3

N‐acetyl‐neuramine acid

19
 Enzyme CMP‐Neu5Ac hydroxylase

• CMP‐Neu5Ac hydroxylase, and consequently Neu5Gc

are normally present in all mammals, including
primates, except in humans
• in primates Neu5Gc is not present in brain
• gene for CMP‐Neu5Ac hydroxylase in humans is not
functional because of deletion of 92 kb
• humans can not synthesize N‐glycolylneuramine acid
• humans produce antibodies against N‐
glycolylneuramine acid

It was estimated that deletion in

gene encoding CMP-Neu5Ac
hydroxylase has occured ~2.9
milllion years ago – followed by
tremendous development of the
inteligence of Homo sp.

When did we step

on our own way?

Numbers mark million years of the

existence of the mutual ancestor

Loss of Neu5Gc –
a selective advantage?

• Plasmodium falciparum – the main cause of malaria

in the world
• in chimps it causes a mild fever
• Plasmodium reichenowi causes malaria in chimps,
but not in humans
• P. falciparum selectively binds Neu5Ac at the human
erythrocytes while P. reichenowi selectively binds
Neu5Gc at the chipms erythrocytes

20
 Neu5Ac can be attached to galactose
through 2,3 or 2,6 linkage

Neu5Ac Neu5Ac
(23) (26)

Gal Gal
β(14) β(14)

GlcNAc GlcNAc
β(12) β(12)
GlcNAc
Man Man
β(14)
(13) (16)
Man
β(14)

GlcNAc
β(14)

GlcNAc Fuc
(16)

Asn

Influenza – caused by
H5N1 virus

Photo: Matko Biljak/Reuters

Influenza

• influenza – 1918 virus H1N1 (50 mil. people died)

– 1957 virus H2N2
– 1968 virus H3N2
• at the virus surface there is HEMAGLUTININ, a lectin,
responsible for the attachment of virus on the surface of the
host cell
• of 16 avian and human hemaglutinins H1, H2 and H3 could be
bound on the surface of human cells
• human lungs cells are mainly covered by 2,6‐Neu5Ac

21
 Hemaglutinin H5N1
(Viet04)

• hemaglutin of H1, H2 i H3
types binds 2,6‐Neu5Ac
• although H5N1 mainly binds
2,3‐Neu5Ac, as a
consequence of mutations it
can also bind 2,6‐Neu5Ac
and become pathogenic for
humans

Hemaglutinin H5N1
Science, April 21, 2006

To take home message

• the majority of proteins are glycosylated
• glycans perform numerous structural, functional and
regulatory roles
• glycans are promissing diagnostic and prognostic
markers
• there are no direct genetic templates and genetics of
glycosylation is very complex
• knowledge on glycan structure is of utmost
importance for understanding biological processes
• glycobiology is one of the biggest challenges of the
molecular life sciences of the 21st century

22