Download Antibody structure

Antibody structure
Overview
1. Antibodies belong to a class of proteins called
immunoglobulins
2. Antibody molecules belong to one of five classes
i.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. C’
activity or binding to cells
4. Ability of immunoglobulins to bind antigen determ.
by AA sequence in variable region
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, is
used 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
Serum and plasma
Serum
Blood
Plasma
Blood +
anticoagulant
Cells
Clot
Isolation & characterization
Chromatography
Molecular sieving
Ion exhange
Affinity
Salting out/ Dehydration
Ammonium sulfate
Alcohol precipitation
Ultracentrifugation
Immunochemical
Chromatography
Molecular sieving
+
+
-
+
+
+
+
+
Ion exchange
Affinity
Salting out
Ammonium sulfate (half saturated)
Ethanol (90%)
Density gradient
ultracentrifugation
Displacement = Svedberg
Kabat & Tiselius (1939)
+
Albumin
a
b
g
Globulins
-Discovered that hyperimmunizing rabbits resulted in increased g
-Purification revealed antibody activity resided in this serum portion
Electrophoretic analysis of
serum
Sample application
Separation by charge
-
+
Anode
-
+
Cathode
+
Albumin
a
b
Globulins
g
Protein concentration
Protein concentration
Characterizing
chains
Albumin
Broad peak of g = heterogenous proteins
a1 a2 b
+
g
normal
-
Need pure Ig to chemically analyze
Narrow peak of g = homogenous proteins
-
+
multiple myeloma
Bence-Jones proteins in urine of multiple
myeloma pts. = dimers of immunoglobulin
light chains, k or l
Porter (England)
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
Fab
SS
Fab specifically bind antigen, univalent (can’t ppt)
one binding site each, identical to each other
SS
SS
Fc
Fc crystallizable (thus homogenous) can’t bind ag
responsible for biological activity of molecule after
ag bound to Fab portions
Eddleman (USA)
Treatment with mercaptoethanol = 4 chains:
(mercaptoethanol breaks S-S bonds)
SH HS SH HS SH HS
2 chains = 53,000 daltons
2 chains = 22,000 daltons
All immunoglobulins basic unit consisting of 4 polypeptides i.e.
2 H, 2 L
Proteolytic enzyme digestion
reveals N- & C-terminal
N-terminus
Fab
Papain digestion results
in cleavage @ “N” terminus
in proline hinge region @
disulfide bridge
Fab
SS
SS
SS
Fc
Fab2
SS
Pepsin digestion results
in cleavage @ “C” terminal
portion, resulting in a divalent
fragment (Fab2) joins by S-S
& several Fc fragments
SS
SS
C-terminus
Cleavage of Ig
SH HS
SH HS
SH HS
Fab
Papain
4 polypeptide chains
Fab
SS
SS
SS
2 Fragment ag binding
1 Fragment crystallizable
1 Fab2
SS
SS
SS
Several small pieces
Fc
Myeloma cells
Plasma cell becomes tumorous = myeloma
produces homogenous (monoclonal) Ig
Myeloma Ig = myeloma “proteins”
Myeloma proteins may be purified & structurally analyzed
AA sequence in L & H chains
Normal, non immune
Normal, immune
Myeloid myeloma
Light chain sequences
N-terminus
Fab
N-terminus
Fab
SS
SS
214 AA in
two domains
SS
C terminus
Analysis from
several myelomas
C terminus
Different
sequences
in N-termin.
domain
Variable (VL)
Identical
sequences
in C-termin.
domain
Constant (CL)
Heavy-chain sequence
N-terminal domain varies
Heavy chain consists of 445 AA
in 4 domains
(HV)
Other 3 domains constant
(HC)
Flexible
Hinge
Region
Fab
N-terminus
Fab
SS
SS
SS
C terminus
%
var
Sequence variability not distributed
evenly in variable region
CDR 1
CDR 3
CDR 2
Variation restricted to 3 regions
Hypervariable regions (HV1,HV2,HV3)
Location of antigen binding site
AA sequence det. shape of ag binding site
(paratope)
Determine epitopes to which Ig binds =
complementary- determining regions (CDR)
CDR = 6-10 AA
CDR 1 = 24 - 34
CDR 2 = 50 - 56
CDR 3 = 89 -97
%
var
Framework regions = regions where AA seq.
is relatively constant (FR)
Heavy chain CDR
CDR 1 = 31-35
CDR 2 = 50-65
CDR 3 = 95-102
Wu & Kabat plot
Structure of variable &
constant domains (X-ray
crystallography)
Light chain
C domain
Two layers, linked by
disulfide bonds
Light chain
V domain
N terminus
CDR
Disulfide bonds
Each layer formed
several stretches
3 conformation = b strand
Layers = b sheet
CDR 1
CDR 2
Order of b strands
is characteristic for
each sheet
3 D structure = Ig fold
C terminus
B strands
B strands
Opening to reveal b strands
comprising each b sheet
Principal difference between C & V domains
V domain has 2 more b strains forming extra
loop
unique strands
C domain
V domain
Order & orientation
characteristic for each
domain
b strands lettered sequentially according to occurrence in AA sequence in dom.
Location of CDR & FR in Ig L
& H chains
CDR
1
FR1
2
FR2
FR1
3
FR3
FR2
1
FR4
FR4 CH1
FR3
2
CDR
CL
3
H
L & H chain folding to yield 3
CDR in each chain to form
walls of ag binding groove
Immunoglobulin consists of 4
polypeptide chains
N terminus
Ag binding site
Ag binding site
Variable regions
VH
VL
CH1
CL
CH2
Constant
region
CH3
C terminus
Disulfide
bonds
EM Rabbit Ig
X 2,042,500
Light chain
Heavy chain
Immunologic analysis of
immunoglobulins
Ig (like most other proteins) stimulate ab in other
animal species
All species have two major classes of L chains i.e. k,l
individual of species produces both types; ratio of
k : l varies by species (e.g. mouse 95%k ; human
60% k)
in any Ig molecule, both L chains = either k or l,
never one of each
Ig of all species consist of 5 classes (isotypes) differ in
structure of H chains
H chains among isotypes differ serologically, CHO
content, size & biological function
H chains
H chain confers unique biologic properties of molecule e.g.
1/2 life, receptor binding , enzyme-, C’ activation with ag
Immunoglobulin isotype
IgM
IgG
IgA
IgD
IgE
Heavy chain
m
g
a
d
e
Individual of species produces all H chains, in proportion
characteristic for species, but ab molecule H chains are
identical ( i.e., 2e, 2d etc.)
Isotypic (Class) structure of Ig
L H
L H
L H
L H
IgG isotype = k2g2 or l2g2, but not k1l1g1a1
IgE isotype = k2e2 or l2e2
….etc…...
H chains
H chain confers unique biologic properties of molecule e.g.
1/2 life, receptor binding , enzyme-, C’ activation with ag
Immunoglobulin isotype
IgM
IgG
IgA
IgD
IgE
Heavy chain
m
g
a
d
e
Individual of species produces all H chains, in proportion
characteristic for species, but ab molecule H chains are
identical ( i.e., 2e, 2d etc.)
Immunoglobulin consists of 4
polypeptide chains
N terminus
Ag binding site
Ag binding site
Variable regions
VH
VL
CH1
CL
CH2
Constant
region
CH3
C terminus
Disulfide
bonds
EM Rabbit Ig
X 2,042,500
Light chain
Heavy chain
Isotypic (Class) structure of Ig
L H
L H
L H
L H
IgG isotype = k2g2 or l2g2, but not k1l1g1a1
IgE isotype = k2e2 or l2e2
….etc…...
Hinge region
Consists of approx 12 AA between CH1 & CH2
No homology between hinge & other Heavy chain domains
Angle = 900
0
AA
sequence
unique
for
each
class
Angle = 60
& subclass
Angle = 0o
AA sequence in hinge region
Hinge region
Light Chain
Cys
-Arg-Val-Glu-Pro-Lys-Ser- Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-Glu-Arg-Val-Glu-Pro-Lys-Ser- Cys-Asp-Lys-Thr-His-Thr-Cys-Pro-Pro-Cys -Pro-Ala-Pro-GluCys
Light Chain
Papain
Fab region
Fc region
Characteristics of hinge region
Immunoglobulins (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 and proline 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
IgG
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 ag
Only activates classical C’ p’way
IgG1
Subclasses show close overall relation
Heavy chains = g1,g2,g3,g4
IgG2
All normal indiv. have all
IgG1>IgG2>IgG3>IgG4
IgG3 has shortest 1/2 life,
highest catabolic rate,
highest # S-S
IgG4 = monoval. no aggl
ppt rx., autoab to clot fac
Autoab to DNA = IgG1,IgG3
IgG3
IgG4
Structural features of IgG
Two g H chains, Two L chains
(either k or l but not both)
Each H chain = 50 kD
Each L chain = 25 kD
HV
Variable
150 kD, 7S, “g globulin”
Least anodic of all serum proteins
Constant
-
+
Anode
Cathode
Structural features of IgM
SS
SS
SS
Pentamer (5)
First Ig produced following immun.
Macroglobulin (M)
900 kD, 19S
J chain
Doesn’t have hinge region
has additional H domain
Has a J chain (one of 2 Ig isotypes)
15 kD
Structural features of IgA
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 g
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 piece
In serum, primarily a monomer, no
secretory piece
Secretory component protects IgA from
proteolytic digestion
Structural features of IgD
Primarily a B cell antigen receptor
Long exposed hinge region
170kD, 7S, migrates as fast g
No interchain S-S bridges in H chains
Readily denatured
Structural features of IgE
Sometimes called reaginic ag
Mediates allergies (Type I
hypersensitivies)
190 kDa, 8S, migrates as fast g
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
Immunoglobulin variations
Isotype = class of Ig, all people have
Allotype = genetic difference, depends on existence of
allelic forms of the Ig , result of different forms of
the same gene, involve changes of 1-3 AA in constant
region; known allotypes IgG:
Gm (Ig Markers)
Km (markers on k chain
Idiotype = one of several thousand Ig, each of which directed
toward specific epitope