associate professor copenhagen center for glycomics ... · copenhagen center for glycomics,...

Hans H. Wandall, MD, PhDAssociate professor

Copenhagen Center for Glycomics, Department of Cellular and

Molecular Medicine

University of Copenhagen

<

Polypeptide GalNAc-Transferases (GalNAc-Ts) in biology and medicine

)

• Structure, enzymatic function, and complexity of the

large family of GalNAc-Ts

– Domain structure; kinetic activity; differential

expression

– A few words on model systems

• Search for the biological functions of GalNAc-Ts

– GlyMap

– Engineered cell models

• GalNAc-Ts – as a useful tool in translational research

– Discovery platforms for biomarkers, therapeutic

antibodies, and vaccines

– Development of novel production platforms for

human protein drugs

Polypeptide GalNAc-Transferases (GalNAc-Ts) in

biology and medicine

)

Essentials of Glycobiology, 2nd ed., P. Stanley 2011

• 10% of all proteins

• 50% of proteins going through Golgi

All cells are covered with glycoconjugates

Mucin-type O-glycans

Dias 4

)

Initiation of mammalian O-linked glycosylation

• Most O-linked genes non-redundant

• GalNAc-type genes partially redundant

)

Mucin (GalNAc)-type protein O-glycosylation

Formation of complex O-GalNAc glycans with different core structures

GalNAca1-O-

20 genes

T

UDP-GalNAc

GalNAc-transferase

NH2

)

mucin-type O-linked glycans

Distribution of unique glycan structures are differentially regulated

Tarp et al

)

GalNAc-Ts basics

• Family of 20 GalNAc-Ts highly conserved throughout evolution;

existing in pairs/clusters

• Individual GalNAc-Ts have distinct peptide specificities, although

considerable overlap exist

• Acceptors include both unmodified and modified (GalNAc

glycosylated) substrates

• A C-terminal GalNAcT lectin domain modulates the specificity

toward partially glycosylated GalNAc peptide substrates

• GalNAc-Ts are differentially expressed in tissues, during

differentiation, and changes are seen in malignancies

UDP-GalNAc

GalNAca1-O-

20 genes

T

GalNAc-transferase

NH2

)

T1 T2 T3

• Type II membrane structure (short N-terminal cytoplasmic tail,

hydrophobic membrane-spanning domain, stem region, catalytic

domain)

• Subcellular topology: Present throughout the Golgi with isoform-specific

differences

• Redistribution to ER during cellular activation (EGF-R, SrcKinase)

• Importance for glycosylation hierarchy inside cells?

Golgi localization of GalNAc-Ts

NH2

Further elongation

)

• Expression profiling has demonstrated differential

expression in tissues and organs

• Creation of specific monoclonal antibodies has

confirmed these finding with differential

expression in tissues

-ex. T11/T14 in kidney

• Differential expression changes during

differentiation

- skin as an exemplar

GalNAc-Ts change expression during

differentiation and development

Diffe

rentiation &

matu

ration

T1 T2 T3

)

Marked changes during cancer progression

• GalNAc-T2 (Liver: UP)

• GalNAc-T3 (Lung UP, Pancreas DOWN)

• GalNAc-T6 (Breast UP, Colon UP, Oral UP)

• GalNAc-T12 (Colon and gastric UP)

• GalNAc-T13 (High metastatic cell lines DOWN)

GalNAc-Ts change during malignant transformation

Illustration from Wandall et al 2007; Bennett et al. 2012

Normal

Oral Cancer

Normal

Oral Cancer

T3 T6

Normal Colon Cancer

T6 T6

Important information both for basic understanding as well as therapeutic and diagnostic applications?

Normal

Cancer

)

Domain structures

The catalytic domain

• The GalNAc-T catalytic domains contain a GT-A

structural motif characterized by two tightly interacting

folds

– A conserved DxH Mn2+ ion binding motif

interacts with the donor substrate UDP

– A Gal/GalNAc-T motif interacts with the GalNAc

moiety

• A pre-formed channel in the surface of the catalytic

domain interacts with the acceptor substrate

• Random peptide and glycopeptide libraries have

revealed acceptor substrate preferences for GalNAc-T

isoforms – still difficult to predict (P +3)!

• Peptide versus Glycopeptide acceptors

Gerken et al. ; Bourne and Henrissat; Fritz et al.; Kubota et al. 2006; Wandall et al.2007; Pedersen et al. 2011; 2011, Bennett et al; Jill et al 2011

NH2

　�

Domain structures

The Lectin domain

• GalNAc-TFs contain a C-terminal lectin domain with a β-trefoil structure (three

repeats: α, β and γ)

• Lectin domain modulate and improve the catalytic efficiency of GalNAc-Ts

with incompleted GalNAc-substrates (mucins).

Gerken et al. ; Bourne and Henrissat; Fritz et al.; Kubota et al. 2006; Wandall et al.2007; Pedersen et al. 2011; 2011, Bennett et al; Jill et al 2011

NH2

)

Single site and high density glycosylation

• Synthesis of mucins (multiple sites) covered by multiple GalNAc-Ts (redundant)

• Synthesis of single site covered by individual GalNAc-Ts (non-redundant) – how

do we identify their functions?

Further elongation

)

Mouse models• Mice deficient in galnt4, t5, t4&t5, t8, or t14 have no overt phenotype

confirming the predicted redundancy.

• Loss of a single galnt gene may thus produce more discrete phenotypes in

select organs:

– Mice deficient in galnt1 exhibit a bleeding disorder and have deficiency

in B-cell maturation.

– Mice deficient in galnt13 exhibit decreased expression of Tn

carbohydrate in brain tissues, but no apparent phenotype.

– Mice deficient in galnt3 develop the classical familiar Tumor Calcinosis

(a heridetary disease with calcified masses).

• More detailed analysis required to decipher the subtle phenotypes

(Marth 1996; Lowe and Marth 2003; Tenno et al. 2007; Ten Hagen et al. 2002; Zhang et al. 2003; Tabak 2010; Manzi et al. 2000;

)

Fly models

• Drosophila Melanogaster useful model system to identify

essential biological functions.

• Fly GalNAc-Ts are highly homologues enzymes with

preserved domain structure and enzymatic functions

• Studies in Drosophila melanogaster provided the first

demonstration that one member of this gene family

(pgant35A; GalNAc-T11 homologue) is essential for

viability

• pgant35A is important for the diffusion barrier formation

in the developing respiratory system

• Another member, pgant3, affect integrin-mediated cell

adhesion during Drosophila development by influencing

the secretion of an extracellular matrix protein

)

Essentials of Glycobiology. 2nd edition.Varki A, Cummings RD, Esko JD, et al. 2009

How does O-linked glycans impinge on cellular function and

communication?

•Assembly of signaling molecules

•Modulation of cell-adhesion

•Protein trafficking

The complex nature of vertebrate GalNAc-TFs makes it difficult to dissect the molecular

mechanisms

)

GalNAc-Ts; Teaching objectives




expression

– A few words on model systems


– GlyMap






human protein drugs

)

GalNAc-Ts basics

• Family of 20 GalNAc-Ts highly conserved throughout evolution;

existing in pairs/clusters

• Individual GalNAc-Ts have distinct peptide specificities, although

considerable overlap exist

• Acceptors include both unmodified and modified (GalNAc

glycosylated) substrates

• GalNAc-Ts are differentially expressed in tissues, during

differentiation, and changes are seen during malignant

development

UDP-GalNAc

GalNAca1-O-

20 genes

T

GalNAc-transferase

NH2

)

Specific non-redundant functions of GalNAc-Ts

Dias 20

How does O-linked glycans impinge on cellular function and communication?




Mucin domains and clustered O-glycans

•Many biological functions

•Synthesis covered by multiple GalNAc-Ts (redundant)

Single or isolated O-glycans

•Functions?

•Synthesis covered by individual GalNAc-Ts (non/less-redundant)

How to identify isoform-specific non-redundant functions

Murine model, fly model, and human model?

)

GALNT1

GALNT2

GALNT3

GALNT4

GALNT5

GALNT6

GALNT7

GALNT8

GALNT9

GALNT10

GALNT11

GALNT12

GALNT13

GALNT14

GALNT15

GALNT16

GALNT17

GALNT18

GALNT19

GALNT20

Familial Tumoral Calcinosis

HDL/TG dyslipidemia?

Newborn heart heterotaxy?

Trail drug sensitivity?

Colon cancer susceptibility

Sickle cell pulmonary complication?

Male infertility (immobile sperm)

B-cell immunity?

Elevated BMI?

GalNAca1-O-

20 genes

T

UDP-GalNAc

GalNAc-transferase

NH2

Disease/phenotype association of the 20

polypeptide GalNAc-Ts

㜰�

Backtrack

Backtrack

㜰�

Discovery of diseases of the glycogenome

• Diseases caused by non-global defects in glycosylation missed

• Strategies: GlyMAP and SimpleCells ~ Targeted Glycomics

GLYMAP

Map of mutations

with functional

consequences

SIMPLE CELLS

Cell lines

engineered to

analyze enzyme

function

Targeted Glycomics

(Copenhagen Center for Glycomics)

)

The GlyMAP Strategy – Functional Mutational MAP

Exome sequence information of glycogenes from >2,000 persons

GlyMAPMap of functional mutations in population

Glycogene

Deleterious mutations

Discovery by genotyping select disease populations

閐ʹ

Structural information is key: ZFN Engineered Isogenic Cell Lines

Steentoft et al. Nat. Methods 2011

Backtrack

Tn

STn

Core 1

Core 2

ST

C1GalT1

cosmc

Core 3

Core 4

a O-glycosylation pathway

GalNAc Galactose GlcNAc Sialic acid

ZFN targeting

COSMC–/–

+Neuraminidase

+Trypsin

VVA chromatography

Colo205

K562

Capan-1

SimpleCell Digest

nLCHCD-MS2

b and y ions

ETD-MS2

c and z ions

FT-MS1 and precursor selection

Time (min)

50 150100

Tota

l io

n c

urr

ent100

0

m/z

1,200800400

100

0

100

0

m/z

500 1,000 1,500 2,000

204.086

44.64

506 508

z = 4

Rela

tive a

bu

nd

an

ce

閐ʹ

GALNT1

GALNT2

GALNT3

GALNT4

GALNT5

GALNT6

GALNT7

GALNT8

GALNT9

GALNT10

GALNT11

GALNT12

GALNT13

GALNT14

GALNT15

GALNT16

GALNT17

GALNT18

GALNT19

GALNT20

GWAS: HDL/Triglyceride dyslipidemia?

Example: Disease/phenotype association between T2

and changes in lipid metabolism

GalNAc-T2 regulates HDL-Cholsetrol

• Overexpression of GalNAc-T2 ⇒ HDL-Cholesterol↓

• Knock-down of GalNAc-T2 ⇒ HDL-Cholesterol↑

)

Complex regulation of lipid metabolism

Modified from Willer Nat Gen 2008, News & Views

閐ʹ

Example: GALNT2 and Dyslipidemia?

Candidate Disease Gene

GWASGALNT2 in lipid metabolism

+-.

......

Differential glycoproteome

m/z

nLC-MS/MS

-+SimpleCells +/- GALNT2

Biomarker

Wild type

GalNAc-T2 Tn

SimpleCells

+T2

-T2

+T2

-T2

HepG2 liver cells

Schjoldager et al. PNAS 2012

잠;

Example: GALNT2 and Dyslipidemia?



+-.

......


m/z

nLC-MS/MS


Biomarker/Function

Probing isoform-specific functions of GalNAc-Ts

VVA

chromatography

nLC-MS/MS


잠;

Complex regulation of lipid metabolism

Modified from Willer Nat Gen 2008, News & Views

APO CIII; protein

component of VLDL

(very low density

lipoprotein).

Angiopoietin-

like 3 (ANGPTL3)

is a determinant

factor of HDL

level

ᢐҘ

Biomarker for GALNT2 Deficiency – ApoC-III

10 Glycosylated

ApoC-IIIStd

HepG2

WT -T2 +T2 -T1

Non-Glycosylated

Human serum (1 uL)

#1 #2 #3

ApoC-III SDS-PAGE WB


Loss of GalNAc-T2 glycosylation of ApoC-III could be used as biomarkers and

may point to the molecular explanation of GalNAc-T2 involvement in HDL

metabolism

߰ͻ

ANGPTL3 is specifically glycosylated by GalNAc-T2

Proprotein convertase

(Furin)

(Activates)

GalNAc-T2

Angiopoietin-like 3 (ANGPTL3)

•Secreted factor produced in the liver

•In humans, ANGPTL3 is positively correlated with

plasma HDL cholesterol

Loss of GalNAc-T2 glycosylation of ANGPTL3 would increase cleavage and

release of ANGPTL3 positively correlated with plasma HDL levels

��

Perspectives – An Example (GALNT2)



Exome sequence variants

Functional mutations in population

Genotyping

PatientMolecular mechanism


+-.

......


APO-CIII

Disease mechanism/Biomarker

m/z

nLC-MS/MS

߰ͻ

GALNT1

GALNT2

GALNT3

GALNT4

GALNT5

GALNT6

GALNT7

GALNT8

GALNT9

GALNT10

GALNT11

GALNT12

GALNT13

GALNT14

GALNT15

GALNT16

GALNT17

GALNT18

GALNT19

GALNT20

HDL/TriGlyceride dyslipidemia?

Example: Disease/phenotype association of the 20

polypeptide GalNAc-Ts for O-glycosylation

Familiar Calcinosis and T3

߰ͻ

GALNT3 and FTC – another example of essential

co-regulation of PC processing

• Familial tumoral calcinosis (FTC)

• Hyperphosphatemia

• Calcium deposits and ossifications

• Caused by loss of FGF23

• FGF23 is responsible for phosphate metabolism

• PC processing inactivates FGF23

• Lack of GalNAc-T3 glycosylation cause inactivation

of FGF23

Topaz et al., Nat. Genet., 2004. Kato et al., JBC, 2006. Benét-Pages et al., Hum. Mol. Genet., 2005

Proprotein convertase (furin)

T3-/-

߰ͻ

GalNAc O-glycosylation modifies PC processing




•Differentiation

•Pro-Protein processing

ꃠͺ

GalNAc-Ts; Teaching objectives




expression

– Model systems


– Specific and redundant functions






human protein drugs

ꃠͺ

GalNAc-Ts – as a useful tool in translational research

• Discovery platforms for biomarkers:

Autoantibodies and tumor specific

products

• Cancer specific antibodies and chemo-

enzymatic creation of antigens for cancer

vaccines

• Enzymatic modification of protein drugs for

improvement of pharmacokinetic properties

)

Glyco-challenges in cancer detection and treatment

Healthy

Premalignant

Stage I

Stage II

Stage III

Stage IV

• Surgical

• Chemotherapy

• Biological treatment

• Immunotherapy (?)

Active and passive

Early detection Treatment

Clinical diagnosisS

urv

ival

Breast cancer

ꃠͺ

Glycan changes during cancer development

Healthy

Premalignant

Stage I

Stage II

Stage III

Stage IV

Surface carbohydrates

ꃠͺ

Cancer associated changes:

• Truncated glycans

• Increased sialyation

• Altered glycosylation densitiy

• Molecular background:

- Disruption of the secretory pathway, pH change

- Down regulation or mutations in glycosyltransferases

and chaperones

Cancer associated mucin-type O-linked glycans

ꃠͺ

Fig: Sørensen et al., 2012

• Current serological cancer markers are tumor products:

proteins (PSA, CA125)

Limitations:

• Vanishing small amounts produced with short serum half

life

• Proteins carrying cancer associated glycans selectively

cleared by carbohydrate receptors

• Remaining circulating proteins carry glycans resembling

glycans on proteins from non-malignant cells

Diagnostic implications:

How does cancer associated glycans impact biomarker discovery?

Stage I

Stage II

Stage III

Stage IV

Su

rviva

l

Pre-

malignant

Wahrenbrock MG, Varki A, Canc. Res., 2006; Wandall et al., Canc.Res. 2010

ꃠͺ

Selective clearance of cancer associated glycans

– autoantibodies as an alternative?

Wandall et al., Canc.Res., 2010

• Auto-antibodies could be used as amplified signals for the

presence of cancer

• Auto-antibodies appears years prior clinical appearance (p53).

• Mostly nuclear and cytoplasmic proteins identified to date with

High throughput methods: phage-display, recombinant cDNA

expression cloning (SEREX), peptide and protein arrays, and

self-assembling protein arrays.

• Change in PTMs induces presentation of specific targets to the

immune system

Stage I

Stage II

Stage III

Stage IV

Su

rviva

l

Pre-

malignant

Clinical

diagnosis

p53-Auto

antibodies

ꃠͺ

Autologous immuneresponse to proteins carrying

cancer associated glycoforms

Tolerance

Surface/SecretedGolgiER

Glycopeptide antibodies specific for cancer cells?

Clearance/Dendritic cell uptake via MGL et al.

Napoletano et al., 2007; Madsen et al, 2012, Van Kooyk

ꃠͺ

Creation of the discovery platform: Mucin glycopeptide microarray

Serum ab

Anti-IgG

Glycopeptide

antigenCore 3 synthase

ST6GalNAc-I

Core 1 synthase

Tn ppGalNAc-Ts

T

Core 3

STn

Pedersen et al. Int J. Cancer, 2010

ꃠͺ

• >100 Tandem repeats carrying O-linked glycans

• Over-expressed by common cancers

MUC1 carry aberrant glycans in cancer

Core 3 synthase

ST6GalNAc-I

Core 1 synthase

Tn ppGalNAc-Ts

T

Core 3

STn

Serum ab

Anti-IgG

Glycopeptide

antigen

ꃠͺ

MUC1 STn

Con

trols

CRC

0

20000

40000

60000

80000

2SD3SD

RFU

MUC1 Core3

Con

trols

CRC

0

20000

40000

60000

80000

2SD3SD

RFU

MUC1

Con

trols

CRC

0

10000

20000

30000

40000

RFU

MUC1 Tn

Con

trols

CRC

0

20000

40000

60000

RFU

VTSAPDTRPAPGSTAPPAHG x 3MUC1 VTSAPDTRPAPGSTAPPAHG x 3MUC1 15TnMUC1 15Core3MUC1 15STn

6.9% 30% 33%57%

IgG auto-antibodies to MUC1 glycopeptides

• ~ 50% of colorectal cancer patients have auto-antibodies to MUC1 Glycopeptides.

Pedersen et al. Int J. Cancer, 2010

隠ҕ

Autoantibodies in cancer:

Diagnostic and Therapeutic perspectives?

Autoantibodies

Selection of targets

Targets used for

production of selective

mAbs

Targets potential

candidates for active

vaccines

• Change in PTMs present specific SURFACE targets to the immune system

隠ҕ

• Specific targeting of cancer cells

• Breaking tolerance - providing danger signal to APC cells that

leads to enhanced T-cell activation

• Presentation to CD8+ AND CD4+ T-cells

Challenges in immunotherapy

What about antibodies?

ꃠͺ

Louis M Weiner, Madhav V Dhodapkar, Soldano Ferrone. Lancet, 2009

Antibody responses

Antibodies can stimulate antigen specific immune responses trough:

1.Antibody-dependent cellular cytotoxicity

2.Promotion of antibody-targeted cross-presentation of tumor antigens

)

Could carbohydrate antigens be targeted – challenges and

possibilities?

• Carbohydrates are often poorly immunogenic.

• Carbohydrate-specific antibodies have low affinity compared with protein-specific antibodies

• Heterogeneous display of glycans on target cells dilute the effect

• NEED FOR MORE THAN JUST SUGARS – Glycopeptides?

52

黰ҕ

• >100 Tandem repeats carrying O-linked glycans

• Over-expressed by common cancers

MUC1 carry aberrant glycans in cancer

Autoantibodies

Selection of targets

Targets used for

production of

selective mAbs

Targets potential

candidates for

active vaccines

MUC1 HMFG2

IgG auto-antibodies to MUC1 glycopeptides

Tn-MUC1 5E5

Burchel et al.; Sørensen et al,2006

)

Breast cancer express the 5E5 epitope

Breast cancer Normal Breast Lactating breast

Tn-MUC1

MUC1 HMFG2

5E5

Lavrsen et al. 2012

• Tn-MUC1 epitope is selectively expressed in

breast cancer tissue

• 5E5 has high affinity to Tn-MUC1 (Kd 1.7 nM)

• 5E5 mediate ADCC of breast cancer cell lines

齀ҕ

-KLH

• Pilot Phase I study: Subcutanous delivery Tn-106merMUC1-KLH (20 high-risk

breast cancer patients immunized x 3)

• 19 patients developed significant anti Tn-MUC1 IgG antibody titers

• Tn-MUC1 induced immune response mimics reactivity of mAb 5E5

• Increase in ADCC in 3 out of 17 patients associated with “no evidence of

disease” (3/5)

0

1

2

3

Pre-vaccination serum samples

0

1

2

3

Post-vaccination serum samples

Pre-Immunization Post-Immunization

5E5 epitope

Wandall et al, Cancer Research, 2009

Active immunotherapy: Tn-MUC1 overrides tolerance in humans

5E5 like response

)

• GalNAc conjugated to larger molecules demonstrated to increase uptake and

promote both CD4 and CD8 responses

• Position of GalNAc(s) is critical for processing and hence CD8 responses

• GalNAc-MUC1 with 1 (one) GalNAc in SAPDT(GalNAc)RPAPG as fusion molecules

with TLR9 helper sequences demonstrates both CD4 and CD8 GalNAc-peptide

specific response

Glycans help uptake and induce cross presentation

Boons and Gendler et al., Kooyk et al.; Madsen et al.

龐ҕ

Fig: Sørensen et al., 2012

• Protein drugs often have short serum half life

• Proteins carrying truncated glycans are selectively cleared by carbohydrate receptors

• Human Granulocyte Colony Stimulating Factor (hGCSF) for the treatment of leukemia

as an example.

How to improve circulation time of protein drugs?

Wahrenbrock MG, Varki A, Canc. Res., 2006; Wandall et al., Canc.Res. 2010

)

58

• Covalent attachment of PEG prolongs the half-life and enhance the

pharmacodynamics of therapeutic proteins

• Methods for PEGylation largely rely on chemical conjugation which often

interferes with bioactivity

• GalNAc-TFs presents a novel strategy for site-directed PEGylation attaching

PEG to O-glycans – example human Granulocyte Colony Stimulating Factor

(hGCSF) for the treatment of leukemia

• Enzymatic GalNAc glycosylation at specific serine and threonine residues in

proteins expressed in Escherichia coli, followed by enzymatic transfer of sialic

acid conjugated with PEG to the introduced GalNAc residues

The use of GalNAc-TFs for site-directed modification of human

protein drugs

�ҕ

Teaching objectives




expression

– Model systems


– GlyMap






human protein drugs

)

Financial support U. of Copenhagen

Danish Research Councils

The Carlsberg Foundation

The Alfred Benzons Foundation

The AP Møller Foundation

The Lundbeck Foundation

EU FP7, EU Marie Curie

NIH-NCI

Yun Kong

Christoffer Goth

Stjepan Kracun

Kowa Chen

Catharina Steentoft

Kirstine Lavrsen

Sarah King Smith

Caroline Benedicte Madsen

Catharina Steentoft

Lara Da Silva

Diana Campos

Rikke Svava

Henrik Clausen

Eric Bennett

Ulla Mandel

Hans Wandall

Ola Blixt

Sergey Vakhrushev

Steven Levery

Malene Vester-Christensen

Nis Borbye Pedersen

Zhang Yang

Yoshiki Narimatsu

Hiren Josh

Katrine Schjoldager

Johannes W. Pedersen

Thank you!

Special thx to Katrine Schjoldager& Eric P.

Bennettfor slide support

associate professor copenhagen center for glycomics ... · copenhagen center for glycomics,...

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