email: [email protected]. what is mbl? endogenous binding ligands 1) the immunoglobulins...
TRANSCRIPT
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Email: [email protected]
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• What is MBL?
• Endogenous binding ligands
1) The Immunoglobulins
2) The Thiol-Ester Proteins
3) Plasma Fibronectin
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• 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
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Adapted from Petersen et al 2001
21 59 30 118 amino acids
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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
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MBL circulates bound to three serine proteases labelled MASP1-3
Glycans
Weis and Drickamer, 1994
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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
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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
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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
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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
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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
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• 5 classes of Ig• Glycosylation is important to both structure and
function
Roit, Brostoff and Male
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• 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
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• 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
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• 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
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Mattu et al, 1998
Roos et al, 2001
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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
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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
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0
0.05
0.1
0.15
0.2
0.25
0.3
BSA IgG IgD IgDunfolded
IgE IgEunfolded
OD492
Arnold et al., 2004
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Asn-445
Asn-496
Asn-354
Arnold et al., 2004
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Asn394Asn265
Asn383
Asn371
*
Constructed based upon crystal structure IgE
Fc Wan et al, 2002
FRONT VIEW TOP VIEW
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• 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
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• 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
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IgM Eluted from MBL Column
Pooled IgM
5 6 7 8 9 10 11
GU
2AB2AB 2AB2AB
2AB2AB
Arnold et al., 2005
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• 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
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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
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TOP VIEW
SIDE VIEW
MBL Target GlycansArnold et al., 2005
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• 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 )
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185
97
52
31
19
kDa
α-2Macroglobulin
IgM HC
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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
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Klodziej et al, 2002
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Klodziej et al, 2002
Thiol-Ester Intact
Thiol-Ester Cleaved
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YY Y Y Y Y
6 7 8 9 GU
YY
YYY
Man5 Man6
YY YY Y Y Y Y
Man7 FA1G1S1
Asn-846
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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
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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
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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
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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)
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Y
Microorganism Surface
C4
C4b
C4b
C3
C3b
C3bY
Y
Y
Y
Y
Y
A2M
Y
Y
Y
Y
Y YY
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Y
C4bC3bYY
YY
Microorganism Surface
YActivation/Amplifaction of Complement Activation
Opsonisation
A2M
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205
116978468
55
45
36
kDapFN V+
pFN V-
α2M
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• 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
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JBM(Mannosidase)
Undigested GlcNAc2Man6
GlcNAc2Man9GlcNAc2Man5
5 6 7 8 9 10 11 GU
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ClotCross-linked
Fibrin And
Fibronectin
Blood Vessel
C3
C3
C4
C2
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ClotCross-linked
Fibrin And
Fibronectin
Blood Vessel
MBL
C3bC4b C4
C2
ComplementActivation
Amplification
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• 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
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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