email: james.arnold@bioch.ox.ac.uk. what is mbl? endogenous binding ligands 1) the immunoglobulins...

Email: James.arnold@bioch.ox.ac.uk

• What is MBL?

• Endogenous binding ligands

1) The Immunoglobulins

2) The Thiol-Ester Proteins

3) Plasma Fibronectin

• MBL is a member of the collectin family of proteins• Molecular Mass 450kDa • Produced in the liver• Resembles a ‘bunch of tulips’ in structure

x3 x6MBL

Adapted from Petersen et al 2001

21 59 30 118 amino acids

0

1

2

3

4

5

6

7

8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Pooled

Random Healthy UK Serum Sample Number

Average MBL concentration

Average MBL 1.21 ug/mlStandard deviation 1.27ug/ml

• A great difference of levels were recorded between individuals

MBL circulates bound to three serine proteases labelled MASP1-3

Glycans

Weis and Drickamer, 1994

C1q

Lectin Pathway

Classical Pathway

MASP Activation

C1r and C1s Activation

MBL

C2 and C4Cleavage

Alternative PathwayC3, Factors B, D, I and H C3bBb

C4b2a

C3 C3b

iC3b

Opsonisation

Factor ICo-Factors

C5 C5b+C9, C8, C7, C6

MACCell Lysis

• MBL acts as an opsonin

• MBL, via MASPs can activates complement

2a

• Starts with cleavage of C4

• Formation of C3 convertase

• Self surfaces protected by: DAF and CR1

C4

C2

C4b

C4b

C2C4b C4b

C2b

C3

C3b

C3b

2a

• C5 cleavage

• MAC : C5b + C6 + C7 + C8 + C9

• MASP-2 inhibited by C1-inhibitor and Alpha-2 macroglobulin

C4b

C5

C5b C9

C6 C7C8

O

OHH

OHH

H

H

HOH

OH

OH

O

OHOH

HH

OH

H

HOH

H

OH

Mannose

O

OHOH

HH

H

H

HOHOH

OH

Glucose

Galactose

3

4

3

3

4

4

di-equatorial) hydroxyls

di-equatorialhydroxyls

equatorial/axial hydroxyls

Dr Pauline Rudd

• MBL binds C-3 and C-4 hydroxyls in equatorial plane of pyranose ring

VirusesSalmonella montevideoEscherichia coliHaemophilus influenzaeListeria monocytogenesNeisseria meningitidisMycobacterium aviumChlamydia pneumoniaeBurkholderia cepaciaKlebsiella speciesStaphylococcus aureusActinomyces israeliiBifidobacterium bifidumFusobacterium (except F. mortiferum)Leptotrichia buccalisPropionibacterium acnesVeillonella dispar

BacteriaInfluenza AHerpes simplex 2HIV-1 and -2

FungiSaccharomyces cerevisiaeCandida albicansCryptococcus neoformansAspergillus fumigatus

ProtozoaLeishmania major and mexicanaTrypanosoma cruziCryptosporidium parvumPlasmodium falciparumTrichinella spiralis

• 5 classes of Ig• Glycosylation is important to both structure and

function

Roit, Brostoff and Male

• Antibody bound to antigen display “patterns” (collective motifs)

• Can be protein or glycan epitopes

• Glycosylation ‘patterns’ can be recognised by MBL

• Avidity of binding is much greater than the affinity

• Contains one conserved glycosylation site at Asn-297

• All biantennary complex glycans, predominantly:

FcA2 and FcA2B

Mark Wormald

Fab

Fc

Hinge

Light Chain

Heavy Chain

Glycan

Fc

A2

GlcNAc2

• IgG biantennary complex glycans are variable• Three predominant glycoforms:

IgG-G0- two exposed GlcNAc

IgG-G1-one exposed GlcNAc

IgG-G2-no exposed GlcNAc• IgG-G0 levels are elevated in

RA (Parekh et al, 1989)• MBL has been shown to bind

IgG-G0 (Malhotra et al, 1995) Dr Mark Wormald

Mattu et al, 1998

Roos et al, 2001

65.00 70.00 75.00 80.00 85.00 90.00 95.00 100.00 105.00 110.00 115.00 120.00 125.00Retention time (min)

5 6 7 8 9 10 11

ABS

undigested

GU

CH2

CH3

VH

CH1

IgD• Serum conc ~30ug/ml• 1972 discovered as part of BCR• 3 N-link glycosylation sites

N-linked Glycosylation siteO-linked Glycosylation site

Man9Man8

Arnold et al., 2004

N-linked glycosylation

CH3

CH4

VH

CH1

CH2

IgE

Man5

Asn394

Asn371

Constructed based upon crystal structure IgE

Fc Wan et al, 2002

• Serum conc <1ug/ml• IgE is directed towards allergens• 7 N-link glycosylation sites

5 6 7 8 9 10 11

Undigested

GU

Arnold et al., 2004

0

0.05

0.1

0.15

0.2

0.25

0.3

BSA IgG IgD IgDunfolded

IgE IgEunfolded

OD492

Arnold et al., 2004

Asn-445

Asn-496

Asn-354

Arnold et al., 2004

Asn394Asn265

Asn383

Asn371

*

Constructed based upon crystal structure IgE

Fc Wan et al, 2002

FRONT VIEW TOP VIEW

• IgM circulates as a pentamer or hexamer• Serum conc ~ 2.5mg/ml• Human IgM has 5 N-linked glycosylation sites: Asn-

171, Asn-332, Asn-395, Asn-402, Asn-563• Asn-402 and Asn-563 are occupied by

oligomannose glycans

IgM

N-Linked GlycanO-Linked GlycanInter-Domian Disulphide BridgeDomain

J Chain

IgM

N-Linked GlycanO-Linked GlycanInter-Domian Disulphide BridgeDomain

J Chain

• Pierce sell an purification Kit for mouse IgM

• Using column of rabbit MBL

• Evidence for binding human IgM

Based on paper by:

Nevens et al, 1992

IgM Eluted from MBL Column

Pooled IgM

5 6 7 8 9 10 11

GU

2AB2AB 2AB2AB

2AB2AB

Arnold et al., 2005

• MBL is binding to GlcNAc terminating structures • Must only potentially occupy three exposed (non

oligomannose sites): Asn-171, Asn-332, Asn-395

• 30 N-glycosylation sites over pentamer structure• IgM from MBL column has statistically 3 of

GlcNAc terminating structures per pentamer• The MBL binding glycoform accounts for ~20%

of pooled serum IgM

020406080

100120140160180200

AntigenBound Anti-

TNP BSA IgM

Anti-TNPBSA IgM

BSA Serum IgM MBL ResinEluted IgM

200

100

Antigen Bound Anti-TNP-BSA IgM

Anti-TNP-BSA IgM BSA Serum IgM MBL-Resin

Eluted IgM

ABS +BTG Treated IgM

Arnold et al., 2005

TOP VIEW

SIDE VIEW

MBL Target GlycansArnold et al., 2005

• MBL previously shown to bind IgG-G0 and polymeric IgA

• MBL does not to bind IgD and IgE• MBL can bind a glycoform of IgM (approximately

20% of total serum IgM )

185

97

52

31

19

kDa

α-2Macroglobulin

IgM HC

Bait region

180kDa

85kDa

120kDa60kDa

95kDa

Thiol ester

Protease cleavage

Autolytic cleavage

Y

Y

Y

Y

Y

Y

Y

Y

Y

Y Y Y Y Y

Y Y Y Y Y

Y Y Y Y Y

Asn

-32

Asn

-47

Asn

-22

4

Asn

-37

3A

sn-3

87

Asn

-84

6

Asn

-96

8

Asn

-14

01

• Thiol-ester proteins family: includes A2M, C3, C4, C5 and CD109• A2M Inhibits foreign and host proteases via a ‘trap mechanism’• Conformational change upon cleavage of the ‘bait region’ or

cleavage of thiol-ester group which traps protease in cage

Klodziej et al, 2002

Klodziej et al, 2002

Thiol-Ester Intact

Thiol-Ester Cleaved

YY Y Y Y Y

6 7 8 9 GU

YY

YYY

Man5 Man6

YY YY Y Y Y Y

Man7 FA1G1S1

Asn-846

Thiol-ester Intact

Elution

Nucleophile-Treated Thiol ester-cleaved

Flowthrough

1 2 1 2

Thiol-ester Intact + 10mM Mannose

Thiol-ester Intact + 10mM Galactose

Thrombin Cleaved

α2MAutolytic cleavage

α2M

Autolytic cleavage

α2M

0

0.02

0.04

0.06

0.08

0.1

Live Alpha-2Macroglobulin

Dead Alpha 2Macroglobulin

Mannan BSA

OD

492

(off scale: 0.608)0.1

Live A2M Dead A2M Mannan BSA

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

0 500 1,000 1,500 2,000

A2M

NONE

BSA

1 MBL: X α2M or BSA

20001000

1.5

1.0

0

1hour

OD

405

2mins

Human

185

97

52

31

19

kDa

C3 α chainC4 α chain

C4 β chain

C3 β chain

C4 γ chain

• MBL also bound plasma C3 and C4

• C3 and C4 are also occupied by oligomannose glycans (Ritchie et al., 2002)

Y

Microorganism Surface

C4

C4b

C4b

C3

C3b

C3bY

Y

Y

Y

Y

Y

A2M

Y

Y

Y

Y

Y YY

Y

C4bC3bYY

YY

Microorganism Surface

YActivation/Amplifaction of Complement Activation

Opsonisation

A2M

205

116978468

55

45

36

kDapFN V+

pFN V-

α2M

• 8 N-linked glycosylation sites

• Several isoforms• Cell adhesion• pFN is Incorporated

into blood clots

1 2 3 4 5 6 7 8 9 10 11 12 12 13 14 15NH2 COOH

SS

14 15 V120

V95

V89

V64

V0

SH SH

Type I

Type II

Type III

EII-B EII-A

Y

Y YY Y YY Y

Y

Y

Y

Y

Asn-430Asn-528

Asn-542 Asn-877 Asn-1007

Asn-1244

Asn-2108

N-linked Glycosylation

V+

Variable Domain

JBM(Mannosidase)

Undigested GlcNAc2Man6

GlcNAc2Man9GlcNAc2Man5

5 6 7 8 9 10 11 GU

ClotCross-linked

Fibrin And

Fibronectin

Blood Vessel

C3

C3

C4

C2

ClotCross-linked

Fibrin And

Fibronectin

Blood Vessel

MBL

C3bC4b C4

C2

ComplementActivation

Amplification

• MBL binds to glycans on the surface of endogenous ligands

• IgG-G0, polymeric IgA, glycoforms of IgM• A2M, C3 and C4• pFN• The interactions occur in the serum however they

are relatively weak and short lived• When these ligands are multiply presented they

present ‘arrays’ of glycans to which MBL will bind with high avidity

Supervisor: Robert Sim Howard ClarkRussell WallisYu Hoi KangJulia PresanisStefanos TsiftsoglouTony WillisAlister DoddsDan MitchellJackie ShawDirector: Professor Kenneth Reid

Pauline Rudd Professor Raymond DwekCatherine Radcliffe Mark WormaldTony Merry Louise Royle David HarveyMax CrispinDavid Suter