Immunoglobulins (Ig)
Glycoprotein molecules that are produced by plasma cells in
response to an immunogen and which function as antibodies. The
immunoglobulins derive their name from the finding that they
migrate with globular proteins when antibody-containing serum is
placed in an electrical field (Figure 1).
II. GENERAL
FUNCTIONS OF IMMUNOGLOBULINS
A. Antigen
binding
Immunoglobulins bind specifically to one or a few closely
related antigens. Each immunoglobulin actually binds to a
specific antigenic determinant. Antigen binding by antibodies is
the primary function of antibodies and can result in protection
of the host. The valency of antibody refers to the number of
antigenic determinants that an individual antibody molecule can
bind. The valency of all antibodies is at least two and in some
instances more.
B.
Effector Functions
Frequently the binding of an antibody to an antigen has no
direct biological effect. Rather, the significant biological
effects are a consequence of secondary "effector functions" of
antibodies. The immunoglobulins mediate a variety of these
effector functions. Usually the ability to carry out a
particular effector function requires that the antibody bind to
its antigen. Not every immunoglobulin will mediate all effector
functions. Such effector functions include:
1.
Fixation of complement - This results in lysis of cells and
release of biologically active molecules
2.
Binding to various cell types - Phagocytic cells,
lymphocytes, platelets, mast cells, and basophils have
receptors that bind immunoglobulins. This binding can
activate the cells to perform some function. Some
immunoglobulins also bind to receptors on placental
trophoblasts, which results in transfer of the
immunoglobulin across the placenta. As a result, the
transferred maternal antibodies provide immunity to the
fetus and newborn
Figure 2A The basic structure of
immunoglobulins
Figure 2B
Click on the image at left for an animated tutorial on antibody
structure Requires Chime Plug-In. Get Chime
Figure 2C Ribbon drawing of the first intact antibody (IgG2A) every
crystallized.
Harris, L. J., Larson, S. B., Hasel, K. W.,
Day, J., Greenwood, A., McPherson, A. Nature 1992, 360, 369-372. ©
2000
Figure 2D
Rotating antibody Jose Saldanha,
Humanization by Design © 2000,
III. BASIC
STRUCTURE OF IMMUNOGLOBULINS
The basic
structure of the immunoglobulins is illustrated in the Figure 2.
Although different immunoglobulins can differ structurally they all
are built from the same basic units.
A. Heavy
and Light Chains
All immunoglobulins have a four chain structure as their basic
unit. They are composed of two identical light chains (23kD) and
two identical heavy chains (50-70kD)
B.
Disulfide bonds
1.
Inter-chain disulfide bonds - The heavy and light chains and
the two heavy chains are held together by inter-chain
disulfide bonds and by non-covalent interactions The number
of inter-chain disulfide bonds varies among different
immunoglobulin molecules.
2.
Intra-chain disulfide binds - Within each of the polypeptide
chains there are also intra-chain disulfide bonds.
C.
Variable (V) and Constant (C) Regions
After the amino acid sequences of many different heavy chains
and light chains were compared, it became clear that both the
heavy and light chain could be divided into two regions based on
variability in the amino acid sequences. These are the:
1. Light
Chain - VL (110 amino acids) and CL
(110 amino acids)
2. Heavy
Chain - VH (110 amino acids) and CH
(330-440 amino acids)
D. Hinge
Region
This is the region at which the arms of the antibody molecule
forms a Y. It is called the hinge region because there is some
flexibility in the molecule at this point.
E. Domains
Three dimensional images of the immunoglobulin molecule show
that it is not straight as depicted in Figure 2A. Rather, it is
folded into globular regions each of which contains an
intra-chain disulfide bond (figure 2B-D). These regions are
called domains.
1. Light
Chain Domains - VL and CL
2. Heavy
Chain Domains - VH, CH1 - CH3
(or CH4)
F.
Oligosaccharides
Carbohydrates are attached to the CH2 domain in most
immunoglobulins. However, in some cases carbohydrates may also
be attached at other locations.
IV. STRUCTURE
OF THE VARIABLE REGION
A.
Hypervariable (HVR) or complementarity determining regions (CDR)
Comparisons
of the amino acid sequences of the variable regions of
immunoglobulins show that most of the variability resides in
three regions called the hypervariable regions or the
complementarity determining regions as illustrated in Figure 3.
Antibodies with different specificities (i.e. different
combining sites) have different complementarity determining
regions while antibodies of the exact same specificity have
identical complementarity determining regions (i.e. CDR
is the antibody combining site). Complementarity determining
regions are found in both the H and the L chains.
B.
Framework regions
The regions
between the complementarity determining regions in the variable
region are called the framework regions (Figure 3). Based on
similarities and differences in the framework regions the
immunoglobulin heavy and light chain variable regions can be
divided into groups and subgroups. These represent the products
of different variable region genes.
Figure 3
Structure of the variable region framework
regions
Click on image at
left to see a rotable molecular structure of an Fab fragment bound
to an influenza hemagglutinin peptide.
Requires Chime plug-in. Get Chime
here)
Click on image at left to see details of the
interaction of a mouse monoclonal antibody interacting with hen egg
white lysozyme. Requires Chime plug-in. Get
Chime here)
V. IMMUNOGLOBULIN FRAGMENTS:
STRUCTURE/FUNCTION RELATIONSHIPS
Immunoglobulin
fragments produced by proteolytic digestion have proven very useful
in elucidating structure/function relationships in immunoglobulins.
A. Fab
Digestion with papain breaks the immunoglobulin molecule in the
hinge region before the H-H inter-chain disulfide bond Figure 4.
This results in the formation of two identical fragments that
contain the light chain and the VH and CH1
domains of the heavy chain.
Antigen
binding - These fragments were called the Fab fragments
because they contained the antigen binding sites of the
antibody. Each Fab fragment is monovalent whereas the
original molecule was divalent. The combining site of the
antibody is created by both VH and VL.
An antibody is able to bind a particular antigenic
determinant because it has a particular combination of VH
and VL. Different combinations of a VH
and VL result in antibodies that can bind a
different antigenic determinants.
B. Fc
Digestion with papain also produces a fragment that contains the
remainder of the two heavy chains each containing a CH2
and CH3 domain. This fragment was called Fc because
it was easily crystallized.
Figure 4 Immunoglobulin fragments:
Structure/function relationships
Effector functions -
The effector functions of immunoglobulins are mediated by
this part of the molecule. Different functions are mediated
by the different domains in this fragment (Figure 5).
Normally the ability of an antibody to carry out an effector
function requires the prior binding of an antigen; however,
there are exceptions to this rule.
Figure 5 Immunoglobulin
fragments: Structure function relationships
C. F(ab')2
Treatment of immunoglobulins with pepsin results in cleavage of
the heavy chain after the H-H inter-chain disulfide bonds
resulting in a fragment that contains both antigen binding sites
(Figure 6). This fragment was called F(ab')2 because
it was divalent. The Fc region of the molecule is digested into
small peptides by pepsin. The F(ab')2 binds antigen
but it does not mediate the effector functions of antibodies.
Figure 6 Immunoglobulin fragments:
Structure/function relationships
VI. HUMAN
IMMUNOGLOBULIN CLASSES, SUBCLASSES, TYPES AND SUBTYPES
A.
Immunoglobulin classes
The immunoglobulins can be divided into five different classes,
based on differences in the amino acid sequences in the constant
region of the heavy chains. All immunoglobulins within a given
class will have very similar heavy chain constant regions. These
differences can be detected by sequence studies or more commonly
by serological means (i.e. by the use of antibodies
directed to these differences).
1. IgG -
Gamma heavy chains
2. IgM -
Mu heavy chains
3. IgA -
Alpha heavy chains
4. IgD -
Delta heavy chains
5. IgE -
Epsilon heavy chains
B.
Immunoglobulin Subclasses
The classes of immunoglobulins can de divided into subclasses
based on small differences in the amino acid sequences in the
constant region of the heavy chains. All immunoglobulins within
a subclass will have very similar heavy chain constant region
amino acid sequences. Again these differences are most commonly
detected by serological means.
1. IgG
Subclasses
a)
IgG1 - Gamma 1 heavy chains
b)
IgG2 - Gamma 2 heavy chains
c)
IgG3 - Gamma 3 heavy chains
d)
IgG4 - Gamma 4 heavy chains
2. IgA
Subclasses
a)
IgA1 - Alpha 1 heavy chains
b)
IgA2 - Alpha 2 heavy chains
C.
Immunoglobulin Types
Immunoglobulins can also be classified by the type of light
chain that they have. Light chain types are based on differences
in the amino acid sequence in the constant region of the light
chain. These differences are detected by serological means.
1. Kappa
light chains
2. Lambda
light chains
D.
Immunoglobulin Subtypes
The light chains can also be divided into subtypes based on
differences in the amino acid sequences in the constant region
of the light chain.
1. Lambda
subtypes
a)
Lambda 1
b)
Lambda 2
c)
Lambda 3
d)
Lambda 4
E.
Nomenclature
Immunoglobulins are named based on the class, or subclass of
the heavy chain and type or subtype of light chain. Unless it is
stated precisely you are to assume that all subclass, types and
subtypes are present. IgG means that all subclasses and types
are present.
F.
Heterogeneity
Immunoglobulins considered as a population of molecules are
normally very heterogeneous because they are composed of
different classes and subclasses each of which has different
types and subtypes of light chains. In addition, different
immunoglobulin molecules can have different antigen binding
properties because of different VH and VL
regions.
Figure 7
IgG Structure
VII. STRUCTURE AND SOME
PROPERTIES OF IG CLASSES AND SUBCLASSES
A. IgG
1.
Structure
The structures of the IgG subclasses are presented in Figure
7. All IgG's are monomers (7S immunoglobulin). The
subclasses differ in the number of disulfide bonds and
length of the hinge region.
2.
Properties
Most versatile immunoglobulin because it is capable of
carrying out all of the functions of immunoglobulin
molecules.
a)
IgG is the major Ig in serum - 75% of serum Ig is IgG
b)
IgG is the major Ig in extra vascular spaces
c)
Placental transfer - IgG is the only class of Ig that
crosses the placenta. Transfer is mediated by receptor
on placental cells for the Fc region of IgG. Not all
subclasses cross equally; IgG2 does not cross well.
d)
Fixes complement - Not all subclasses fix equally well;
IgG4 does not fix complement
e)
Binding to cells - Macrophages, monocytes, PMN's and
some lymphocytes have Fc receptors for the Fc region of
IgG. Not all subclasses bind equally well; IgG2 and IgG4
do not bind to Fc receptors. A consequence of binding to
the Fc receptors on PMN's, monocytes and macrophages is
that the cell can now internalize the antigen better.
The antibody has prepared the antigen for eating by the
phagocytic cells. The term opsonin is used to
describe substances that enhance phagocytosis. IgG is a
good opsonin. Binding of IgG to Fc receptors on other
types of cells results in the activation of other
functions.
Figure 8
Pentameric serum IgM structure
Figure 9
Cell surface IgM structure
Figure 10 B cell antigen
receptor (BcR)
B. IgM
1.
Structure
The structure of IgM is presented in Figure 8. IgM normally
exists as a pentamer (19S immunoglobulin) but it can also
exist as a monomer. In the pentameric form all heavy chains
are identical and all light chains are identical. Thus, the
valence is theoretically 10. IgM has an extra domain on the
mu chain (CH4) and it has another protein
covalently bound via a S-S bond called the J chain. This
chain functions in polymerization of the molecule into a
pentamer.
2.
Properties
a)
IgM is the third most common serum Ig.
b)
IgM is the first Ig to be made by the fetus and the
first Ig to be made by a virgin B cells when it is
stimulated by antigen.
c) As
a consequence of its pentameric structure, IgM is a good
complement fixing Ig. Thus, IgM antibodies are very
efficient in leading to the lysis of microorganisms.
d) As
a consequence of its structure, IgM is also a good
agglutinating Ig . Thus, IgM antibodies are very good in
clumping microorganisms for eventual elimination from
the body.
e)
IgM binds to some cells via Fc receptors.
f) B
cell surface Ig
Surface IgM exists as a monomer and lacks J chain but it
has an extra 20 amino acids at the C-terminus to anchor
it into the membrane (Figure 9). Cell surface IgM
functions as a receptor for antigen on B cells. Surface
IgM is noncovalently associated with two additional
proteins in the membrane of the B cell called Ig-alpha
and Ig-beta as indicated in Figure 10. These additional
proteins act as signal transducing molecules since the
cytoplasmic tail of the Ig molecule itself is too short
to transduce a signal. Contact between surface
immunoglobulin and an antigen is required before a
signal can be transduced by the Ig-alpha and Ig-beta
chains. In the case of T-independent antigens, contact
between the antigen and surface immunoglobulin is
sufficient to activate B cells to differentiate into
antibody secreting plasma cells. However, for
T-dependent antigens, a second signal provided by helper
T cells is required before B cells are activated.
Figure 11 IgA Structure
Figure 12 Origin of soluble IgA
C. IgA
1.
Structure
Serum IgA is a monomer but IgA found in secretions is a
dimer as presented in Figure 11. When IgA exits as a dimer,
a J chain is associated with it.
When IgA is found in
secretions is also has another protein associated with it
called the secretory piece or T piece; sIgA is sometimes
referred to as 11S immunoglobulin. Unlike the remainder of
the IgA which is made in the plasma cell, the secretory
piece is made in epithelial cells and is added to the IgA as
it passes into the secretions (Figure 12). The secretory
piece helps IgA to be transported across mucosa and also
protects it from degradation in the secretions.
2.
Properties
a)
IgA is the 2nd most common serum Ig.
b)
IgA is the major class of Ig in secretions - tears,
saliva, colostrum, mucus. Since it is found in
secretions secretory IgA is important in local (mucosal)
immunity.
c)
Normally IgA does not fix complement, unless aggregated.
d)
IgA can binding to some cells - PMN's and some
lymphocytes.
Figure 13 IgD Structure
D. IgD
1. Structure
The structure of IgD is presented in the Figure 13. IgD exists
only as a monomer.
2. Properties
a) IgD is
found in low levels in serum; its role in serum uncertain.
b) IgD is
primarily found on B cell surfaces where it functions as a
receptor for antigen. IgD on the surface of B cells has
extra amino acids at C-terminal end for anchoring to the
membrane. It also associates with the Ig-alpha and Ig-beta
chains.
c) IgD
does not bind complement.
Figure 14 IgE Structure
E. IgE
1. Structure
The structure of IgE is presented in Figure 14. IgE exists as a
monomer and has an extra domain in the constant region.
2. Properties
a) IgE is
the least common serum Ig since it binds very tightly to Fc
receptors on basophils and mast cells even before
interacting with antigen.
b)
Involved in allergic reactions - As a consequence of its
binding to basophils an mast cells, IgE is involved in
allergic reactions. Binding of the allergen to the IgE on
the cells results in the release of various pharmacological
mediators that result in allergic symptoms.
c) IgE
also plays a role in parasitic helminth diseases. Since
serum IgE levels rise in parasitic diseases, measuring IgE
levels is helpful in diagnosing parasitic infections.
Eosinophils have Fc receptors for IgE and binding of
eosinophils to IgE-coated helminths results in killing of
the parasite.
d) IgE
does not fix complement.
Figure 15 Rotating antibody
© 2000Clinical
Implications of Human Immunoglobulin Classes
Adapted
from:F.T. Fischbach in "A Manual of Laboratory Diagnostic Tests,"
2nd Ed., J.B. Lippincott Co., Philadelphia, PA, 1984.
IgG
1.
Increases in:
a)
Chronic granulomatous infections
b) Infections of all types
c) Hyperimmunization
d) Liver disease
e) Malnutrition (severe)
f) Dysproteinemia
g) Disease associated with hypersensitivity granulomas,
dermatologic disorders, and IgG myeloma
h) Rheumatoid arthritis
2.
Decreases in:
a)
Agammaglobulinemia
b) Lymphoid aplasia
c) Selective IgG, IgA deficiency
d) IgA myeloma
e) Bence Jones proteinemia
f) Chronic lymphoblastic leukemia
IgM
1.
Increases (in adults) in:
a)
Waldenström's macroglobulinemia
b) Trypanosomiasis
c) Actinomycosis
d) Carrión's disease (bartonellosis)
e) Malaria
f) Infectious mononucleosis
g) Lupus erythematosus
h) Rheumatoid arthritis
I) Dysgammaglobulinemia (certain cases)
Note:
In the newborn, a level of IgM above 20 ng./dl is an
indication of in utero stimulation of the immune
system and stimulation by the rubella virus, the
cytomegalovirus, syphilis, or toxoplasmosis.
2.
Decreases in:
a)
Agammaglobulinemia
b) Lymphoproliferative disorders (certain cases)
c) Lymphoid aplasia
d) IgG and IgA myeloma
e) Dysgammaglobulinemia
f) Chronic lymphoblastic leukemia
IgA
1.
Increases in:
a)
Wiskott-Aldrich syndrome
b) Cirrhosis of the liver (most cases)
c) Certain stages of collagen and other autoimmune disorders
such as rheumatoid arthritis and lupus erythematosus
d) Chronic infections not based on immunologic deficiencies
e) IgA myeloma
2.
Decreases in:
a)
Hereditary ataxia telangiectasia
b) Immunologic deficiency states (e.g.,
dysgammaglobulinemia, congenital and acquired
agammaglobulinemia, and hypogammaglobulinemia)
c) Malabsorption syndromes
d) Lymphoid aplasia
e) IgG myeloma
f) Acute lymphoblastic leukemia
g) Chronic lymphoblastic leukemia
IgD
1.
Increases in:
a)
Chronic infections
b) IgD myelomas
IgE
1.
Increases in:
a)
Atopic skin diseases such as eczema
b) Hay fever
c) Asthma
d) Anaphylactic shock
e) IgE-myeloma
2.
Decreases in:
a)
Congenital agammaglobulinemia
b) Hypogammaglobulinemia due to faulty metabolism or
synthesis of immunoglobulins
|
Figure 3
Structure of the variable region framework
regions
Figure 4 Immunoglobulin fragments:
Structure/function relationships
Figure 5 Immunoglobulin
fragments: Structure function relationships
Figure 6 Immunoglobulin fragments:
Structure/function relationships
Figure 7
IgG Structure
VII. STRUCTURE AND SOME
PROPERTIES OF IG CLASSES AND SUBCLASSES
Figure 8
Pentameric serum IgM structure
Figure 13 IgD Structure
Figure 14 IgE Structure
