Transcript Immunoglobulins

Immunoglobulins
Structure
Definitions
Immunoglobulin is a generic term that refers to a
diverse group of molecules found in the blood and
tissue fluids
They are soluble globulin molecules and they
generally migrate in an electrophoretic field at or near
the gamma globulin fraction
An antibody is an immunoglobulin molecule capable of
binding specifically with a known substance (antigen)
Immunoglobulins are synthesized by B lymphocytes
and terminally differentiated B lymphocytes or plasma
cells
Introduction
The first to show that antibodies reside in the gamma
globulin fraction were Kabat and Tiselius in 1939 when
they performed serum electrophoresis and they called
them immunoglobulins
From the broad electrophoretic peak, it is clear that a
heterogeneous collection of immunoglobulin
molecules with slightly different charges is present
This heterogeneity was one of the early obstacles in
attempts to determine the structure of antibodies,
since analytical chemistry requires homogeneous
crystalizable compounds as starting proteins
Certain diseases (multiple myeloma and light chain
disease) provided the solution
Recently the hybridoma technology has made
available antibodies in pure form
Porter in 1959 treated antibody with papain and
produced three fragments of equal size
Two of the fragments were found to retain the
antibody’s ability to bind antigen specifically but could
no longer precipitate the antigen from solution
These two fragments were referred to as Fab
The third fragment could be crystallized out of solution
and called FC
ENZYME DIGESTION OF IMMUNOGLOBULINS
Fragment “antigenbinding”
Papain
Pepsin
Fragment
crystallisable
At about the same time, Edelman discovered
that when the immunoglobulin molecule was
extensively reduced of by mercaptoethanol, it
fell apart into four chains, two identical heavy
chains (53000 Daltons) and two identical light
chains (22000 Daltons)
Noble prize was awarded for Porter and
Edelman for revealing antibody structure
Antibody structure
LIGHT
HEAVY
H+L
Antibody
+
dimer
Structure
An immunoglobulin is composed of four polypeptide
(glycoprotein) chains; two light and two heavy chains
linked by disulfide bonds
Five different types of heavy chains; α, , µ, δ, and ε.
Two types of light chains; kappa (k) and lambda(λ)
with a ratio of 3:2
An individual B cell or immunoglobulin monomer
expresses either k or λ but not both
Within an immunoglobulin molecule, the two heavy
chains and light chains are identical
Schematic diagram of structure of Ig
Some immunoglobulin molecules are basic
monomers and others are composed of multiple
copies (dimers or pentamers) of identical
immunoglobulin monomers
Both heavy and light chains can be divided into
regions or domains, homologous portions of an
immunoglobulin chain, each composed of
approximately 110 amino acids and contains an
intradomain disulfide bridge
(b) The μ and Є heavy chains
contain an additional domain
that replaces the hinge region
(a) Heavy and light chains are
folded into domains, each containing
about 110 a.a. & an intrachain disulfide bond
that forms a loop of 60 amino acids.
Light chains contain two regions, a variable (VL) and a
constant ( CL) domain
Heavy chains contain a single variable (VH) and
multiple constant domains(CH1, CH2, CH3, CH4)
Variable regions in both heavy and light chains are so
named because of the extensive variation in the amino
acid sequence found in immunoglobulin molecules
made by different cells
The amino acid sequence determines the
conformational structure of VH and VL
The combination of a light variable and a heavy
variable region forms a pocket that constitute the
antigen-binding region of the immunoglobulin
molecule
Hinge Region
Made up predominantly of cysteine and proline
residues
It permits flexibility between the two Fab arms of
the Y shaped molecule
It allows Fab to open and close to accommodate
binding to two epitopes separated by a fixed
distance
Variable Domains
Three areas of hypervariability occur between
less variable stretches called Framework
regions
Because they bind specifically to epitopes, the
hypervariable regions are termed
complementarity -determining regions (CDRs)
of the L and H chains; CDR1, CDR2, and
CDR3
CDR1 CDR2
V
V
CDR1 CDR2
CDR3
D J
J
CDR3
Immunoglobulin Fragments
Fab: produced by papain cleavage (2 Fab)
FC: produced by papain cleavage
Fd: it is the heavy chain portion of an Fab fragment
cleaved by papain
F(ab)2: it is the dimeric molecule produced by pepsin
cleavage which fragments the FC
Fd─ : it is the heavy chain portion of an Fab fragment
cleaved by pepsin
Immunoglobulin Classes
Five different classes or isotypes depending
on heavy chain antigenicity
Monomers, dimers or pentamers
Monomers are divalent having two identical
antigen- binding sites
General structures of the five major classes
of antibody
IgM
First to be produced in an immune response. M for
macroglobulin, M. Wt. about 106 Daltons,
sedimentation coefficient 19S, Has an extra CH
domain
Cell surface bound monomer or secreted pentamer.
Five basic units (pentamer) joined by a J chain
(150.000 Daltons) synthesized by B cells or plasma
cells
Five antigen-binding sites instead of the expected
valence of 10 due to conformational constraints
imposed by polymerization
6-8% of serum immunoglobulins
IgG
Monomer, M. Wt 150.000, sedimentation
coefficient 7S. Least anodic of all serum
proteins
Four subclasses; IgG1, IgG2, IgG3, and IgG4
Predominant immunoglobulin in blood, lymph,
CSF, and peritoneal fluid
72-80% of serum immunoglobulins
IgA
The major immunoglobulin in external secretions such
as saliva, mucous, sweat, gastric fluid, and tears
Monomer (serum) or dimer (secretions), M.Wt 165000,
sedimentation coefficient 7S
Two subclasses; IgA1 and IgA2
13-19% of serum immunoglobulins
Serum IgA is predominantly monomeric and
monomeric IgA1 accounts for about 90% of serum IgA
Secretory Ig A
Secretory Ig A has a J chain and a secretory
piece (70.000 Daltons) which is synthesized by
epithelial cells to facilitate passage of secretory
IgA into mucous secretions and to protect it
from cleavage (secretory IgA is more resistant
than serum IgA)
Secretory IgA: - IgA1 90% in secretions above
the diaphragm
- IgA2 10% in lower GI
Structure and formation of secretory lgA
IgD
Monomer, M. Wt. 180.000, sedimentation
coefficient 7S
A major surface component of many B cells but
present in very low concentrations (< 1% of
serum immunoglobulins) in serum where it has
no function.
It is not secreted by plasma cells and it is
uniquely susceptible to proteolytic cleavage
IgE
Monomer, M. Wt 200.000, sedimentation
coefficient 8S
It has an extra CH domain
Less than 0.001% of serum immunoglobulins
Binds with high affinity to mast cells and
basophils (Homocytophilic)
Antigenic determinants of immunoglobulins
Allotypes
Allelic forms of the same protein as a result of the
presence of different forms of the same gene at a
given locus
Allotypic differences at known loci usually involve
changes in only one or two amino acids in the
constant region of a chain
Important genetic markers inherited as dominant traits;
Gm on  chain, Km (previously Inv) on kappa chains,
and Am on α chain
The genes encoding the markers are expressed
codominantly, so that an individual may be
homozygous or heterozygous for a given marker
Ideotypes
The antigenicity of the variable region of Fab
May or may not block binding of the antibody to the
antigen depending whether produced against CDRs or
Framework sequence. An ideotype represents an
“internal immage” of the epitope
Public or cross reacting epitopes are ideotypes on
different antibodies produced against the same
epitope. Private epitopes react with only a particular
antibody molecule
Regulation of an Immune response (Jerne’s Theory)