PROTEOMICS IN VIRAL DISEASEPROTEOMICS IN VIRAL DISEASE

BEVIN GANGADHARAN

Third Year Graduate Student

BEVIN GANGADHARAN

Third Year Graduate Student

Supervisor : Nicole Zitzmann

Oxford GlycoProteomics GlycoBiology Institute Trinity Term 2005

Supervisor : Nicole Zitzmann

Oxford GlycoProteomics GlycoBiology Institute Trinity Term 2005

What is proteomics?What is proteomics?

The term ‘proteome’ was first established by Wasinger et al (1995) and means the protein complement of a genome

The systematic separation, identification and characterisation of the proteins present in a tissue or other biological sample is termed as ‘proteomics’

Two key analytical techniques used in proteomics are two dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry

The term ‘proteome’ was first established by Wasinger et al (1995) and means the protein complement of a genome

The systematic separation, identification and characterisation of the proteins present in a tissue or other biological sample is termed as ‘proteomics’

Two key analytical techniques used in proteomics are two dimensional polyacrylamide gel electrophoresis (2D PAGE) and mass spectrometry

Proteomics at OGBIProteomics at OGBI

Spot/band excision & Trypsin digestionSpot/band excision & Trypsin digestion

Database searchingDatabase searching

Comparative Image analysis

Comparative Image analysis

2D-PAGE

Protein samplePseudomonas, Serum, Platelets, Jurkat, Uveal melanoma, Skin

Protein samplePseudomonas, Serum, Platelets, Jurkat, Uveal melanoma, Skin

Staining & scanningStaining & scanning

Nanospray MS/MSNanospray MS/MS

SDS-PAGE

In-solution IEF

Direct LC-MSDirect LC-MS

Peristaltic pump driven gradient

gel casting

Peristaltic pump driven gradient

gel casting

m/z

Ab

un

dan

ce

m/z

Ab

un

dan

ce

MS MS/MS

1. Determine fibrosis marker(s) in serum by using proteomics and glycan analysis

2. Identify serum proteins and glycans that have changed post treatment with 231B in HCV infected patients

3. Identification of proteins changing in Jurkat lipid rafts in the presence and absence of HIV Nef

1. Determine fibrosis marker(s) in serum by using proteomics and glycan analysis

2. Identify serum proteins and glycans that have changed post treatment with 231B in HCV infected patients

3. Identification of proteins changing in Jurkat lipid rafts in the presence and absence of HIV Nef

List of ProjectsList of Projects

Hepatitis C Virus (HCV)Hepatitis C Virus (HCV)

HCV enveloped RNA virus

No vaccine

Member of the flaviviridae family

Main target organ is the liver

Estimated 200 million (3% popn) are infected of which 170 million (85%) are at risk of developing liver cirrhosis and/or liver cancer

HCV enveloped RNA virus

No vaccine

Member of the flaviviridae family

Main target organ is the liver

Estimated 200 million (3% popn) are infected of which 170 million (85%) are at risk of developing liver cirrhosis and/or liver cancer

HCV Liver fibrosis

Activation of hepatic stellate cells

Accumulation of extracellular matrix

Liver biopsies - most reliable analysis

HCV Liver fibrosis

Activation of hepatic stellate cells

Accumulation of extracellular matrix

Liver biopsies - most reliable analysis

HCV induced liver fibrosisHCV induced liver fibrosis

CirrhosisNormal

HCV Serum : Project ObjectiveHCV Serum : Project Objective

Determine fibrosis marker(s) in

serum by using proteomics and

glycan analysis to compare

normal and HCV infected

fibrotic serum samples

Determine fibrosis marker(s) in

serum by using proteomics and

glycan analysis to compare

normal and HCV infected

fibrotic serum samples

Methods – Fibrosis marker Methods – Fibrosis marker Normal along with mild, moderate and cirrhotic serum samples were provided by Paul Klenerman

Normal along with mild, moderate and cirrhotic serum samples were provided by Paul Klenerman

2D-PAGE gels were run for all samples2D-PAGE gels were run for all samples

Glycan analysis done by Yun-Gon Kim, Louise Royle

and Pauline Rudd

Glycan analysis done by Yun-Gon Kim, Louise Royle

and Pauline Rudd

Normal

Mild fibrosis

Moderate fibrosis

Cirrhosis

Normal

Mild fibrosis

Moderate fibrosis

Cirrhosis

2D-PAGE proteomics2D-PAGE proteomics Glycan analysisGlycan analysis

N-glycans were released from serum proteins, then labelled with 2-AB followed by HPLC

A range of exoglycosidases were used

N-glycans were released from serum proteins, then labelled with 2-AB followed by HPLC

A range of exoglycosidases were used

Results - Fibrosis markerResults - Fibrosis marker2D-PAGE proteomics2D-PAGE proteomics Glycan analysisGlycan analysis

Normal

Mild

Moderate

Cirrhosis

Normal

Mild

Moderate

Cirrhosis

Decrease in haptoglobinDecrease in haptoglobin

Increase in 2 macroglobulinIncrease in 2 macroglobulin

Glycan analysis done by Yun-Gon Kim, Louise Royle

and Pauline Rudd

Glycan analysis done by Yun-Gon Kim, Louise Royle

and Pauline Rudd

core fucosylation seen in cirrhotic serum compared to

normal healthy controls

core fucosylation seen in cirrhotic serum compared to

normal healthy controls

0

10

20

30

%a

ge

co

refu

co

sy

lati

on

Hea

lth

yco

ntr

ols

Cir

rho

tic

Rx

Cir

rho

tic

w/o

Rx

Changes with liver fibrosisChanges with liver fibrosis2 macroglobulin (increased)2 macroglobulin (increased)

Secreted from hepatocytes and stellate cells in fibrosisSecreted from hepatocytes and stellate cells in fibrosis

Binds to and modulates cytokines and growth factors

TGF- main mediator of hepatic stellate cell activationhepatic stellate cell activation

Protease inhibitor inhibition of ECM catabolism

Haptoglobin (decreased)

HGF decreases haptoglobin synthesis

Associated with TGF1 – a profibrogenic cytokine

Binds to haemoglobin preventing loss of iron

Core fucosylation (increased)

up-regulation of core--1,6-fucosyltransferase*

2 macroglobulin (increased)2 macroglobulin (increased)

Secreted from hepatocytes and stellate cells in fibrosisSecreted from hepatocytes and stellate cells in fibrosis

Binds to and modulates cytokines and growth factors

TGF- main mediator of hepatic stellate cell activationhepatic stellate cell activation

Protease inhibitor inhibition of ECM catabolism

Haptoglobin (decreased)

HGF decreases haptoglobin synthesis

Associated with TGF1 – a profibrogenic cytokine

Binds to haemoglobin preventing loss of iron

Core fucosylation (increased)

up-regulation of core--1,6-fucosyltransferase*

* Noda et al, (1998), Hepatology, 28, 944-52

231B – An antiviral iminosugar231B – An antiviral iminosugar

N

O HOH

CH3

O

Long alkyl chain DGJ compound

Plaque reducing activity against the cytopathic

strain of Bovine Viral Diarrhoea Virus (BVDV) *

Potential therapeutic antiviral iminosugar for HCV

Long alkyl chain DGJ compound

Plaque reducing activity against the cytopathic

strain of Bovine Viral Diarrhoea Virus (BVDV) *

Potential therapeutic antiviral iminosugar for HCV

O H

* Durantel et al, (2001), J Virol, 75, 8987-98* Durantel et al, (2001), J Virol, 75, 8987-98

231B (N7-oxanonyl-6deoxy-DGJ)231B (N7-oxanonyl-6deoxy-DGJ)

231B Project Objectives231B Project Objectives

Identify serum proteins and glycans

that have changed due to the

toxicological or pharmacological

influences of the drug 231B in HCV

infected patients

Identify serum proteins and glycans

that have changed due to the

toxicological or pharmacological

influences of the drug 231B in HCV

infected patients

Patient matched samples for

baseline and after 12 weeks of 231B

(United Therapeutics)

All had zero to moderate fibrosis

All failed other forms of treatment

Samples for weeks 10 and 24 of one

responder were also provided

(treatment stopped at Week 8)

32 samples covering 15 patients

(including the responder)

2D-PAGE and glycan analysis

Patient matched samples for

baseline and after 12 weeks of 231B

(United Therapeutics)

All had zero to moderate fibrosis

All failed other forms of treatment

Samples for weeks 10 and 24 of one

responder were also provided

(treatment stopped at Week 8)

32 samples covering 15 patients

(including the responder)

2D-PAGE and glycan analysis

Baseline vs. 12 weeksBaseline vs. 12 weeks

231B Project Methods231B Project Methods2

D-P

AG

E2

D-P

AG

EG

lyca

nG

lyca

n

Glycan analysis done by Yun-Gon Kim, Louise Royle

and Pauline Rudd

Glycan analysis done by Yun-Gon Kim, Louise Royle

and Pauline Rudd

Proteomics dataProteomics data

50

60

70

80

90

100

110

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Number of weeks post start of 231B treatment

To

tal

pro

tein

co

nc

en

tra

tio

n (

mg

/ml)

Total protein concentration for serum from responder

231B terminated

Concentrations higher than normal indicate inflammation (hepatitis)

Normal total protein reference range for serum (60-83 mg/ml)

Base-line

114

97

86

99

231B : 2D-PAGE results231B : 2D-PAGE results No changes observed so far between baseline and 12 weeks of 231B treatment

One change observed for responder and one other patient

The validity of this change is presently being investigated by re-running several gels

Few changes observed between healthy controls and baseline samples

No changes observed so far between baseline and 12 weeks of 231B treatment

One change observed for responder and one other patient

The validity of this change is presently being investigated by re-running several gels

Few changes observed between healthy controls and baseline samples

ControlControl BaselineBaseline Week 12Week 12

Normal vs. HCV+ serum changesNormal vs. HCV+ serum changes

Increase in:

1. 2 macroglobulin

Decrease in:

2. Haptoglobin

3. Complement C3

4. Transthyretin

5. Retinol-binding protein

Increase in:

1. 2 macroglobulin

Decrease in:

2. Haptoglobin

3. Complement C3

4. Transthyretin

5. Retinol-binding protein

2

2

2

1

1

4

4

3

5

50

10

200

Mas

s (k

Da)

3 10pI

Decrease in Complement C3Decrease in Complement C3

Decreased complement activity already observed in hepatitis and cirrhosis*

Reported to be not consistent

Compromised hepatic synthetic function results in decreased complement synthesis

Decreased complement activity already observed in hepatitis and cirrhosis*

Reported to be not consistent

Compromised hepatic synthetic function results in decreased complement synthesis

* Steel et al, (2003), Proteomics, 3, 601-9

controlscontrols HCV+ serumHCV+ serum

controlscontrols HCV+ serumHCV+ serum

Retinol BP and transthyretinRetinol BP and transthyretin

Retinol BPRetinol BP

TransthyretinTransthyretin

Vitamin A and fibrosisVitamin A and fibrosis

Hepatic stellate cells (HSC) deposit collagen fibrosis

HSCs are the major site for Vitamin A storage

HSCs have receptors for retinol-binding protein

In fibrosis, HSCs lose their ability to store Vitamin A

Decrease in retinol-binding protein

Binds to retinol (vitamin A alcohol)

Delivers retinol from the liver to peripheral tissues

Decrease in transthyretin

Complexes with retinol-binding protein preventing loss through kidneys

Hepatic stellate cells (HSC) deposit collagen fibrosis

HSCs are the major site for Vitamin A storage

HSCs have receptors for retinol-binding protein

In fibrosis, HSCs lose their ability to store Vitamin A

Decrease in retinol-binding protein

Binds to retinol (vitamin A alcohol)

Delivers retinol from the liver to peripheral tissues

Decrease in transthyretin

Complexes with retinol-binding protein preventing loss through kidneys

Glycan dataGlycan data

0

5

10

15

20

25

30

35

40

Patient Initials

% o

f co

re f

uco

syla

tio

n

Before treatment

Treatment(after 12 weeks)

The % of core fucosylated glycansThe % of core fucosylated glycans in whole serum from HCV patients

After digestion with sialidase, galactosidase & N-acetylhexosaminidase

Data from Yun-Gon Kim, Louise Royle, Pauline Rudd

Baseline

12 weeks 231B

*

* 231B stopped Week 8

Average control

0

5

10

15

20

25

30

Average of 4individualcontrolsamples

Beforetreatment

10 weeks 12 weeks 24 weeks

% o

f co

re f

uco

syla

tio

n

Data from Yun-Gon Kim, Louise Royle, Pauline Rudd

The % core fucosylation of samples from 231B responder

Baseline

(231B was stopped at Week 8)

Further WorkFurther Work

Start image analysis on serum gels for 231B project

Re-run more gels for the 231B serum study to check the validity of changes

More serum samples needed (controls, fibrotic / cirrhotic and HCC) for both 2D-PAGE and glycan analysis

Start image analysis on serum gels for 231B project

Re-run more gels for the 231B serum study to check the validity of changes

More serum samples needed (controls, fibrotic / cirrhotic and HCC) for both 2D-PAGE and glycan analysis

AcknowledgementsAcknowledgements

Dr. Nicole ZitzmannProfessor Raymond Dwek

Dr. Pauline RuddDr. Louise RoyleYun-Gon Kim

Dr. Paul KlenermanDr. Paul KlenermanDr. Alison SimmonsDr. Alison Simmons

Dr. Nicole ZitzmannProfessor Raymond Dwek

Dr. Pauline RuddDr. Louise RoyleYun-Gon Kim

Dr. Paul KlenermanDr. Paul KlenermanDr. Alison SimmonsDr. Alison Simmons

Medical Research

Council

Medical Research

CouncilOxford GlycoSciences

Oxford GlycoSciences

Proteomics GroupProteomics GroupDr. Robin AntrobusDr. Robin AntrobusDr. Sripadi PrabhakarDr. Sripadi PrabhakarDr. Ángel García-AlonsoDr. Ángel García-AlonsoDr. María Pardo-PérezDr. María Pardo-PérezDave ChittendenDavid GilesDavid Giles

Virus GroupVirus Group

Proteomics GroupProteomics GroupDr. Robin AntrobusDr. Robin AntrobusDr. Sripadi PrabhakarDr. Sripadi PrabhakarDr. Ángel García-AlonsoDr. Ángel García-AlonsoDr. María Pardo-PérezDr. María Pardo-PérezDave ChittendenDavid GilesDavid Giles

Virus GroupVirus Group

United Therapeutics

United Therapeutics Oxford

GlycobiologyInstitute

Oxford Glycobiology

Institute