1 tsri/novartis: 02/04 glycomics and bioinformatics what is glycomics? scope of the informatics...

TSRI/Novartis: 02/04 1

Glycomics and BioinformaticsGlycomics and Bioinformatics

• What is glycomics?• Scope of the informatics

problem.• Bioinformatics in the glycomics

space. • Public database resources.


GlycomicsGlycomics

• Glycomics: just another omics?- Genes :Genomics

- Proteins: Proteomics

- Glycans: Glycomics

• Glycomics: Study of the glycome

• Glycome : All the glycan structures present in an organism.


The Glycome : How should it be definedThe Glycome : How should it be defined

• Glycosylation is the most common post-translational modification of proteins, with 50-80% of all glycoproteins being glycosylated

• Glycan structure at a single site is often heterogenous, with single glycoproteins having 20-50 glycan structures.

• Glycosylation is cell type specific, so a single glycoprotein can have a different spectrum of glycan structures when expressed in different cells.

• Should the glycome be defined as the library of glycans expressed in an organism (1000s), or the glycans associated with every protein in every cell (1,000,000s)?


Challenges in GlycomicsChallenges in Glycomics

• Glycan structure analysis is low throughput. Technical hurdles for obtaining information is high.

• The goals of glycomics and proteo-glycomics are not clear.

• Will the information be useful if it is obtained?• The resources needed to do glycomics far

exceed the need for the information.• Solution: Develop a focused approach to

glycomics based on function and information content.


Functional Glycomics: Systems Biology Functional Glycomics: Systems Biology Focus on Glycan Binding Proteins Focus on Glycan Binding Proteins

>30,000 Proteins 50-80%

Glycosylated~100sGBP’s

1000s Glycan structures

~100sGBP

Ligands

Human and Murine Biology


Consortium for Functional GlycomicsConsortium for Functional Glycomics

Goal: Define paradigms by which

protein-carbohydrate interactions mediate cell communication

http://www.functionalglycomics.org

Define paradigms by which protein-carbohydrate interactions mediate

cell communication

• NIGMS sponsored 'glue grant' to TSRI (~$8 MM/yr).

• Over 110 participating investigators worldwide

• Focus in innate and acquired immunity


Glycan Binding Proteins in Cell Glycan Binding Proteins in Cell Communication : Three paradigmsCommunication : Three paradigms

One Cell

Other Cell

Glycan Ligand

Glycan Binding Protein

TRANSTRANS

Cell Adhesion

Selectins

CISCIS

ModulateReceptor Signaling

CD22(Siglec-2)

•Pathogen (HIV-DC-SIGN)

•Glycoprotein

Glycan Targeting /

Endocytosis


CBP Family Ligands Functions

C-type lectins Various• Cell Adhesion• Glycoprotein clearance• Inn ate Immunity

Siglecs( -I ty pe lectins) Sialosides

• Cell signaling• Cell adhesion

Galectins Galactosides • Extr -a cellula r matrixcros -s linking

CD1/TCRGlycolipids/

Glycopeptides• Inn ate immunity• Acquire d immunity

M-type lectins Hig h Man N-linked • Qualit y contro l inprotei n fold ing i n ER

L-typ e lectins Various • Protei n sorting inER

P-typ e lectins Man-6-phosphate • Lysosom al enzymesortin (g Post-Gol )gi

R-typ e lectins Various • Enzym e targeting• Hormon e turnover

Mammalian Glycan Binding Proteins (GBPs)Mammalian Glycan Binding Proteins (GBPs)

CBP Family Ligands Functions

C-type lectins Various• Cell Adhesion• Glycoprotein clearance• Inn ate Immunity

Siglecs( -I ty pe lectins) Sialosides

• Cell signaling• Cell adhesion

Galectins Galactosides • Extr -a cellula r matrixcros -s linking

CD1/TCRGlycolipids/

Glycopeptides• Inn ate immunity• Acquire d immunity

M-type lectins Hig h Man N-linked • Qualit y contro l inprotei n fold ing i n ER

L-typ e lectins Various • Protei n sorting inER

P-typ e lectins Man-6-phosphate • Lysosom al enzymesortin (g Post-Gol )gi

R-typ e lectins Various • Enzym e targeting• Hormon e turnover


Specific AimsSpecific Aims

For selected glycan binding proteins (GBPs):

• Define the specificity for binding sugar structures.

• Identify the macromolecular-ligands on biologically relevant cell types.

• Determine how GBP-ligand interactions mediate cell communication.

• Establish the extent to which regulation of glycosylation regulates their function.

• Determine the crystal structure.

Part. Investigators•Explore Biology using tools

•Contribute data> 80 Institutions

Functional Glycomics : Systems Biology Perspective

Glycan Library Tools for specificity analysis and biology

TSRI

Mouse Phenotype UC San Diego

Mouse TransgenicsCreate & Distribute

KO mice

TSRI

Consortium DatabasesMIT

Steering Committee

AnalyticalGlycan Profiling

Imperial College

Glycan SpecificityGBP ligand specificity

U Oklahoma

Gene MicroarrayGBP and GT genes

TSRI


Consortium for Functional Glycomics : Consortium for Functional Glycomics : Glycobiology Resources Offered Glycobiology Resources Offered

Analysis of your RNA samples on a highly annotated custom gene microarray with 1800 human and mouse genes

Glyco-gene microarry

Glycan specificity of your carbohydrate binding protein on an array with 170 unique glycans representing glycoprotein and glycolipid glycans

Glycan array screening

Over 100 carbohydrate compounds represented on the glycan array are available with azido or biotin functional groups

Carbohydrate compounds

24 mouse strains with genes ablated for glycosyltransferase and glycan-binding proteins

Mouse knockout strains

http://www.functionalglycomics.org

http://www.functionalglycomics.org/



Part. Investigators•Explore Biology using tools

•Contribute data> 80 Institutions

Functional Glycomics : Systems Biology Perspective

Glycan Library Tools for specificity analysis and biology

TSRI

Mouse Phenotype UC San Diego

Mouse TransgenicsCreate & Distribute

KO mice

TSRI

Consortium DatabasesMIT

Steering Committee

AnalyticalGlycan Profiling

Imperial College

Glycan SpecificityGBP ligand specificity

U Oklahoma

Gene MicroarrayGBP and GT genes

TSRI


Glycan Profiling in Mouse and Human Tissues, Cells and Proteins

Imperial CollegeAnne DellStuart HaslemMark Sutton-Smith

TSRI/PARCDavid GoldbergRichard H. BrucePeter Kuhn

TSRIDan SalomonMouse Transgenic Core

Consortium Database

TSRI/Novartis: 02/04 14MALDI Screening of WT Kidney

KEY:

Fucose

NeuAc

GalNAc

Galactose

GlcNAc

Mannose

NeuGc


PNA Positive Lymphocytes in Germinal PNA Positive Lymphocytes in Germinal Centers of Spleen and Lymph NodeCenters of Spleen and Lymph Node

G. Cattoretti, Columbia Univ

Thorbecke, NY Univ. School of Med

Spleen Lymph Node

PNA(Gal1-3GalNAc) PNA

(Gal1-3GalNAc)


Protocol for Comparing Resting and Protocol for Comparing Resting and Activated LymphocytesActivated Lymphocytes

Prepare Mixed Splenocytes (B, CD4+, CD8+)

± Activation in vitro

Isolate B, CD4+ or CD8+ lymphocytes using

immuno-magnetic beads

Left over cellsDesired Resting or Activated Cells

For Glycan Profiling

Extract mRNA for Microarray

TSRI/Novartis: 02/04 172500 2700 2900 3100 3300 3500

Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1=>BC=>NF0.7[BP = 1133.6, 51713]

2653.2455

2655.2445

3306.5343

2601.2317

3308.52613102.44522519.1766 2928.37832723.2846 3104.42852603.2231

2898.33732696.2586 3310.46543134.44102521.2049 2926.37412827.29902625.2310 3027.4188 3379.59612720.2697 3221.53873123.51662541.1002 2789.7348

2500 2700 2900 3100 3300 3500

Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% Intensity

Voyager Spec #1=>BC=>NF0.7[BP = 2397.2, 48908]

3027.4080

2601.2399

3029.34772519.2553

2636.2781

2723.37092603.21972504.2313 2840.31312693.3151

2533.2539 2927.43973085.46952663.3107 2811.29532737.3507 2883.33932665.3168 3131.40092522.2347 2594.2482 3289.53742968.3952 3042.41492793.3733 2899.3482 3116.45522622.24982508.1122 3336.23762690.3760 3041.30142806.2946 2931.2588 3260.43083165.6332 3408.43442616.2495 3339.32332537.9715 2703.0636

CD8+ T-cells(IL12 Activated)

CD8+ T-cells (Fresh)


Gene Microarray: Few Changes in GT Gene Microarray: Few Changes in GT Expression After Activation of CD8+ T cellsExpression After Activation of CD8+ T cells

CD8+T Cells

Fold Change (Activated/Resting)

1.91

1.60

- 6.22

- 7.71

- 2.33

ST6GalNAc IV

1,3Gal GT

CMP-NeuAc Hydroxylase

ST6Gal I

ST8Sia IV

0

Relevant to Changes in N-

Linked Glycosylation


1-31-3

Altered Terminal Glycosylation of N-linked Altered Terminal Glycosylation of N-linked Glycans in Activated CD8+ T CellsGlycans in Activated CD8+ T Cells

2-62-6

ST6Gal I

Key:

NeuGc

Gal1-

3GalT1-

3GalT

ST6Gal I

Major structure on resting cells

Major structure on activated

cells


Glycan Synthesis Core - TSRIGlycan Synthesis Core - TSRI

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Synthetic GlycansConstruct Library of All

Natural Terminal Glycan Sequences



Ola Blixt, Nahid Razi-TSRINicolai Bovin-Moscow

Printed Glycan

Microarray

Construct Library of All Natural Terminal

Glycan Sequences



Ola Blixt, Nahid Razi-TSRINicolai Bovin-Moscow

TSRI/Novartis: 02/04

Glycan-Array: Covalent printing of a glycan library on glass slides

Glycan-Array: Covalent printing of a glycan library on glass slides

Microarray DNA-core facility(Core-E) at Scripps

OO

NH2

OO

NH2

OO

NH2

N

O

O

O

O

OO

NH

O

O HN

NH2

O

CO2H

Glycoprotein Glycans

multistep chemoenzymatic synthesis

One-PotChemical Synthesis

PNGNase cleavageand purification

+

Stable amide bond

NHS-activated surface

Making a diverse carbohydrate library

Library with a selectivefunctional amino group

"High CarbChips"

O. Blixt: TSRI Glycan Synthesis CoreS. Head: TSRI Microarray-CoreC.-H. Wong, M. Bryan, F. Fazio: TSRIN. Bovin: MoscowR. Cummings and R. Alvarez : Univ.

Oklahoma


Screening Specificity of Human CD22 (Siglec-2)Screening Specificity of Human CD22 (Siglec-2)



hCD22

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

15000

16000

17000

18000

19000

20000

21000

22000

Neu5,9NAc2,6LacNAc

5Neu Gc2,6LacNAc

5Neu Gc2,6Tn5Neu Ac2,6-

Tn

5Neu Ac2,6 -LacNAc LacNAc

5Neu Ac2,6 2LacNAc

5Neu Ac2,6[6 ]Su LacNAc

5Neu Ac2,6 1LacNAc

Ligands

Neu5Ac2-6Gal(NAc)

• 4000 spots / slide (ng)• Low amount protein consumed

(0.5µg / slide)• No Blocking required, 1 hr

Analysis < 1 hr • Re-utilization of slides


CD22 is a Negative Regulator of theCD22 is a Negative Regulator of the B Cell Antigen Receptor Complex B Cell Antigen Receptor Complex

Antigen

(phosphatase-negative regulator)

CD22

SHP-1

IgM

IgIg IgIg

src kinase

(phosphorylase-positive regulator)

?

Does theLigand Binding

Domain ParticipateIn CD22 Function?


Public Databases : Glycosyltransferase and Public Databases : Glycosyltransferase and Glycan Binding ProteinGlycan Binding Protein

• Glycosyltransferase Database- CAZY (extensive genomic portal)

- Consortium for Functional Glycomics (Glycosylation Pathways: menu for CAZY)

- KEGG Glycosylation Pathways http://www.genome.ad.jp/kegg/pathway.html

• Glycan Binding Protein Database- Genomics Resource for Animal Lectins


Public Databases : Glycan StructurePublic Databases : Glycan Structure

• GlycoSuite DB (commercial license req.)

• Glycominds (commercial license req.)

• Sweet DB (first stop)

• Consortium for Functional Glycomics (glycan profiles)

• GlyCAM -3D (early stage)


Glycobiology ResourcesGlycobiology Resources

• Glycoforum : http://www.glycoforum.gr.jp/

• Consortium for Functional Glycomics http://functionalglycomics.org


URL Addresses:URL Addresses:

- CAZY (extensive genomic portal)http://afmb.cnrs-mrs.fr/CAZY/GT.html

- Consortium for Functional Glycomics http://www.functionalglycomics.org/

- KEGG Glycosylation Pathways http://www.genome.ad.jp/kegg/pathway.html

- Genomics Resource for Animal Lectinshttp://ctld.glycob.ox.ac.uk/

- GlycoSuite DB (commercial license req.)http://www.proteomesystems.com/glycosuite/

- Glycominds (commercial license req.)http://www.glycominds.com/GlycoInfo.asp?HighLight=LinearCode

- Sweet DB (first stop)http://www.dkfz-heidelberg.de/spec2/sweetdb/

- Consortium for Functional Glycomics (glycan profiles)http://www.functionalglycomics.org/

- GlyCAM -3D (early stage)http://glycam.ccrc.uga.edu/index.html


EndEnd

1 tsri/novartis: 02/04 glycomics and bioinformatics what is glycomics? scope of the informatics...

Documents

functional glycomics

glycan library

tsri glycan synthesis

sweet db

public databases

d early stagehttp

animal lectinshttp

hr analysis