Antibody Struc
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Transcript of Antibody Struc
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Antibody structureOverview
1. Antibodies belong to a class of proteins called
immunoglobulins
2. Antibody molecules belong to one of five classesi.e. IgG, IgM, IgA, IgD & IgE
3. Immunoglobulins are Y shaped proteins. The
arms of the Y bind antigens. The tail of the
Y is responsible for biological activity eg. Cactivity or binding to cells
4. Ability of immunoglobulins to bind antigen determ.
by AA sequence in variable region
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General Characteristics of
antibody
Antigen-specific products of B cells
First of the molecules participating in immune response
to be characterized, best understood
Basic building block, immunoglobulin domain, isused in molecules of both immune system & other
biological recognition systems
Antibody molecule has 2 separable functions:1) specific binding to antigen eliciting response
2) biological activity - recruit cells & components
designed to destroy agent to which ab developed
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Serum and plasma
Blood
Blood +
anticoagulant Cells
Plasma
Serum
Clot
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Isolation & characterizationChromatography
Molecular sieving
Ion exhange
Affinity
Salting out/ Dehydration
Ammonium sulfate
Alcohol precipitation
Ultracentrifugation
Immunochemical
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Chromatography
Molecular sieving
+ +- ++
++ +
-Ion exchange
Affinity
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Salting outAmmonium sulfate (half saturated)
Ethanol (90%)
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Density gradient
ultracentrifugation
Centri
fuga
tion
60,000
-100,00X
G
Displacement = Svedberg
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Kabat & Tiselius (1939)
Albumin
+ -
Globulins
-Discovered that hyperimmunizing rabbits resulted in increased -Purification revealed antibody activity resided in this serum portion
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Electrophoretic analysis of
serum
+ -
Sample application
Anode Cathode
+ -
Separation by charge
Albumin
+ -
Globulins
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Characterizing chainsAlbumin
1 2 + -P
rotein
conce
ntra
tion
Broad peak of = heterogenous proteins
multiple myeloma
+ -Pro
tein
concentra
tion
normal Need pure Ig to chemically analyze
Narrow peak of = homogenous proteins
Bence-Jones proteins in urine of multiplemyeloma pts. = dimers of immunoglobulin
light chains, or
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Porter (England)
SSS S
SS
Fab Fab
Fc
Treatment with proteolytic enzyme,papain,
resulted in three approximately equal sized fragments:
2 capable of ag rx (fragment antigen binding)
1 could be crystallized (fragment- crystallizable)
Fab specifically bind antigen, univalent (cant ppt)
one binding site each, identical to each other
Fc crystallizable (thus homogenous) cant bind ag
responsible for biological activity of molecule after
ag bound to Fab portions
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Eddleman (USA)
SH HSSHHSHS SH
Treatment with mercaptoethanol = 4 chains:
(mercaptoethanol breaks S-S bonds)
2 chains = 53,000 daltons
2 chains = 22,000 daltons
All immunoglobulins basic unit consisting of 4 polypeptides i.e.
2 H, 2 L
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Proteolytic enzyme digestion
reveals N- & C-terminal
Fc
SSSS S S
Papain digestion results
in cleavage @ N terminusin proline hinge region @
disulfide bridge
SSS SSS
Fab FabN-terminus
Pepsin digestion results
in cleavage @ C terminal
portion, resulting in a divalent
fragment (Fab2) joins by S-S
& several Fc fragments
Fab2
C-terminus
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Cleavage of IgSH HSSH
HS
HS
SH
2 Fragment ag binding
1 Fragment crystallizable
1 Fab2
Several small pieces
Fc
Pepsin
Papain
Reduce
dwME
SSSS S S
SSS SSS
Fab Fab
4polypeptide chains
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Light chain sequences
SSS SSS
Fab FabN-terminus
C terminus
N-terminus
C terminus
214 AA in
two domains
Analysis from
several myelomas
Identical
sequences
in C-termin.
domain
Different
sequences
in N-termin.domain
Variable (VL
)
Constant (CL)
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Heavy-chain sequence
SSS SSS
Fab FabN-terminus
C terminus
Heavy chain consists of 445 AA
in 4 domains
Flexible
Hinge
Region
N-terminal domain varies
(HV)
Other 3 domains constant
(HC)
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Sequence variability not distributed
evenly in variable regionVariation restricted to 3 regions
Hypervariable regions (HV1,HV2,HV3)
Location of antigen binding siteAA sequence det. shape of ag binding site
(paratope)
Determine epitopes to which Ig binds =
complementary- determining regions (CDR)
Framework regions = regions where AA seq.
is relatively constant (FR)
CDR = 6-10 AACDR 1 = 24 - 34
CDR 2 = 50 - 56
CDR 3 = 89 -97
Wu & Kabat plot
Heavy chain CDR
CDR 1 = 31-35
CDR 2 = 50-65
CDR 3 = 95-102
CDR 1
CDR 2
CDR 3
%
var
%var
St t f i bl &
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Structure of variable &
constant domains (X-ray
crystallography)Two layers, linked by
disulfide bonds
Each layer formedseveral stretches
conformation = stranLayers = sheet
Order of strandsis characteristic for
each sheet
3 D structure = Ig fold
Light chain
V domain
Light chain
C domain
N terminus
C terminus
strands strands
Disulfide bonds
CDR 1
CDR 2
CDR 3
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Opening to reveal strandscomprising each sheet
V domainC domain
Principal difference between C & V domains
V domain has 2 more strains forming extraloop
unique strands
strands lettered sequentially according to occurrence in AA sequence in dom.
Order & orientation
characteristic for each
domain
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Location of CDR & FR in Ig L
& H chains
FR1 FR2 FR3 FR4
FR1 FR2 FR3 FR4
CL
CH1 H
1 2 3
1 2 3
CDR
CDR
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L & H chain folding to yield 3
CDR in each chain to form
walls of ag binding groove
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Immunoglobulin consists of 4
polypeptide chains
C terminus
Constant
region
N terminus
Variable regions
Disulfide
bonds
Ag binding site Ag binding site
VHVL
CH1
CL
CH2
CH3 Light chain
Heavy chain
EM Rabbit Ig
X 2,042,500
I l i l i f
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Immunologic analysis of
immunoglobulins
Ig (like most other proteins) stimulate ab in otheranimal species
ll species have two major classes of L chains i.e. ,individual of species produces both types; ratio of
: varies by species (e.g. mouse 95% ; human60% )
in any Ig molecule, both L chains = either or ,never one of each
Ig of all species consist of 5 classes (isotypes) differ instructure of H chains
H chains among isotypes differ serologically, CHO
content, size & biological function
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H chains
Immunoglobulin isotype Heavy chain
IgM IgG IgA IgD IgE
Individual of species produces all H chains, in proportion
characteristic for species, but ab molecule H chains are
identical ( i.e., 2 , 2 etc.)
H chain confers unique biologic properties of molecule e.g.
1/2 life, receptor binding , enzyme-, C activation with ag
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Isotypic (Class) structure of Ig
gG isotype = 2 2 or 2 2 , but not 1 1 1 1IgE isotype = 2 2 or 2 2
.etc...
L H L H
L HL H
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H chains
Immunoglobulin isotype Heavy chain
IgM IgG IgA IgD IgE
Individual of species produces all H chains, in proportion
characteristic for species, but ab molecule H chains are
identical ( i.e., 2 , 2 etc.)
H chain confers unique biologic properties of molecule e.g.
1/2 life, receptor binding , enzyme-, C activation with ag
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Immunoglobulin consists of 4
polypeptide chains
C terminus
Constant
region
N terminus
Variable regions
Disulfide
bonds
Ag binding site Ag binding site
VH VL
CH1
CL
CH2
CH3 Light chain
Heavy chain
EM Rabbit Ig
X 2,042,500
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Isotypic (Class) structure of Ig
gG isotype = 2 2 or 2 2 , but not 1 1 1 1IgE isotype = 2 2 or 2 2
.etc...
L H L H
L HL H
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Hinge region
Angle = 0o
Angle = 600Angle = 900
Consists of approx 12 AA between CH1 & CH2
No homology between hinge & other Heavy chain domains
AA sequence unique for each class& subclass
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AA sequence in hinge region
Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu--Arg-Val-Glu-Pro-Lys-Ser-
Light Chain
Cys
Light Chain
Cys
Papain
Fab region Fc region
Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu--Arg-Val-Glu-Pro-Lys-Ser-
Hinge region
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Characteristics of hinge regionImmunoglobulins (with possible exception of IgM & IgE) contain hinge between
CH1 & CH2
No homology between AA sequence of hinge & heavy chains
AA sequence differs with different classes
Comprised of many cysteine andproline residues
Cysteine involved in formation of interchain disulfide bonds
Proline prevents folding in a globular structure, allowing flexibility between
two Fab arms of the Y-shaped antibody; allows open & close to accommodate
binding to two epitopes; because it is open, it can be cleaved by proteases
(e.g. papain) to generate the Fab & Fc fragments
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IgG
IgG1 IgG2
I G3 I G4
Subclasses show close overall relation
Heavy chains = 1, 2, 3,
Highest concentration in serum
Plays major role in immune dfns.
MW approx. 150 kDa
Small size ppt in surfaces
(e.g. cross placental barrier)
Opsinize, aggl. & ppt
agOnly activates classical C pway
All normal indiv. have all
IgG1>IgG2>IgG3>IgG4
IgG3 has shortest 1/2 life,
highest catabolic rate,
highest # S-SIgG4 = monoval. no aggl
ppt rx., autoab to clot fac
Autoab to DNA = IgG1,IgG
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Structural features of IgG
HV
Constant
Variable
Two H chains, Two L chains(either or but not both)Each H chain = 50 kD
Each L chain = 25 kD
150 kD, 7S, globulinLeast anodic of all serum proteins
+ -
Anode Cathode
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Structural features of IgMPentamer (5)
First Ig produced following immun.
Macroglobulin (M)
900 kD, 19S
SSS S
SS
SS
SS
SS
SS
SS
S
S
SS
S
S
S
S
SS
SS
SS
J chain Doesnt have hinge region
has additional H domain
Has a J chain (one of 2 Ig isotypes)
15 kD
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Structural features of IgA
S
S
SS
S
S
SS
SS
S
S
Secretory
piece
J chain
Principal Ig in external secretions e.g.saliva, mucus,sweat, gastric fluid & tears
Major Ig of colostrum & milk, provides
neonate with major source of intestinal
protection against pathogens
165 kD, 7S, migrates as fast Plasma cell forms basic IgA molecule with
J chain which form dimers (second Ig to
contain J chain)
When released from plasma cell, bind to
basal membranes of epithelia via
secretory pieceIn serum, primarily a monomer, no
secretory piece
Secretory component protects IgA from
proteolytic digestion
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Structural features of IgESometimes called reaginic ag
Mediates allergies (Type Ihypersensitivies)
190 kDa, 8S, migrates as fast Contains an extra domain (CH4)
which binds to mast cells
& basophils
May remain attached for long time
when ag reappears, cross links
IgE on mast cell surface, release
mast-cell granules & signs of
anaphylaxis
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