IMMUNOGLOBULINS - STRUCTURE AND FUNCTION

DEFINITION:

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

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

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BASIC STRUCTURE OF IMMUNOGLOBULINS

A. Heavy and Light Chains

All immunoglobulins have a four-chain structure as their basic unit. They are
composed of two identical light chains and two identical heavy chains

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 form 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. Rather, it is
folded into globular regions each of which contains an intra-chain disulfide bond 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.

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. This results in the formation
of two identical fragments that contain the light chain and the V H 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. Both VH and VL
create the combining site of the antibody. An antibody is able to bind a
particular antigenic determinant because it has a particular combination of V H and VL. Different
combinations of a VH and VL result in antibodies that can bind different antigenic determinant

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 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.

Effector functions - The effector functions of immunoglobulins are mediated

by this part of the molecule. The different domains in this fragment mediate
different functions. 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

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. 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.
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

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 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.

STRUCTURE AND SOME PROPERTIES OF IG CLASSES AND SUBCLASSES

A. IgG

1. Structure

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

B. IgM

1. Structure

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

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 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 extra 20 amino acids at
the C-terminus to anchor it into the membrane

☺ 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.

☺ 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 the Ig-alpha and Ig-beta chains can transduce a signal

☺ 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.

C. IgA

1. Structure
 Serum IgA is a monomer but IgA found in secretions is a
dimmer.

☺ 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. The Secretory piece helps
IgA to be transported across mucosa and also protects it from degradation in the
secretions.

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 2. Properties

a) IgA is the 2nd most common serum Ig.

b) IgA is the major class of Ig in secretions - tears, saliva,

colostrum, and 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 bind to some cells - PMN's and some

lymphocytes.

D. IgD

1. Structure
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.

E. IgE

1. Structure
.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.

Clinical Implications of Human Immunoglobulin Classes

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 IgG
Increased in Decreased In
› Chronic granulomatous infections
Infections of all types › Agammaglobulinemia
› Hyperimmunization › Lymphoid aplasia
› Liver disease › Selective IgG, IgA deficiency
› Malnutrition (severe) › IgA myeloma
› Dysproteinemia › Bence Jones proteinemia
› Disease associated with hypersensitivity › Chronic lymphoblastic leukemia
granulomas, dermatologic disorders, and IgG
myeloma
› Rheumatoid arthritis

IgM
› Waldenström's macroglobulinemia
› Trypanosomiasis
› Actinomycosis
› Carrión's disease (bartonellosis)
› Malaria
› Agammaglobulinemia
› Infectious mononucleosis
› Lymphoproliferative disorders (certain cases)
› Lupus erythematosus
› Lymphoid aplasia
› Rheumatoid arthritis
› IgG and IgA myeloma
Dysgammaglobulinemia (certain cases)
› Dysgammaglobulinemia
› Chronic lymphoblastic leukemia
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

IgA
› Hereditary ataxia telangiectasia
› Wiskott-Aldrich syndrome › Immunologic deficiency states (e.g.,
› Cirrhosis of the liver (most cases) dysgammaglobulinemia, congenital and
› Certain stages of collagen and other acquired agammaglobulinemia, and
autoimmune disorders such as rheumatoid hypogammaglobulinemia)
arthritis and lupus erythematosus › Malabsorption syndromes
› Chronic infections not based on immunologic › Lymphoid aplasia
deficiencies › IgG myeloma
› IgA myeloma › Acute lymphoblastic leukemia
› Chronic lymphoblastic leukemia

IgD
› Chronic infections
› IgD myelomas

IgE
› Atopic skin diseases such as eczema
› Hay fever › Congenital agammaglobulinemia
› Asthma › Hypogammaglobulinemia due to faulty
› Anaphylactic shock metabolism or synthesis of immunoglobulins
› IgE-myeloma
Kinds of Antibodies:

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Natural antibodies:
☺ Inherited antibodies, they are not acquired as a result of stimulation by antigen

Acquired Antibodies:
☺ Includes, agglutinins, precipitins, lysins, opsonins, antitoxins, virus neutralizing and allergenic
antibodies.

☺ Agglutinins: These are antibodies responsible for the immobilization of motile organisms and for
clumping of cells. Agglutinins can only be demonstrated against particulate antigen. Agglutinins can
only be demonstrated against a particulate antigen.

› Warm agglutinins: Can act best at body temperature.

› Cold Agglutinins: Act at 0-8C
› Hemagglutinin: Causes agglutination of red cells
› Isohemagglutinins: May act against red cells or other members of the same specie.
› Heterophile agglutinins: Act against red cells of different species.

☺ Agglutinoids: Are agglutinins modified by heat in such a manner that they can no longer bring
about agglutination but which are still capable of combining with the specific agglutinogens.

☺ Precipitins: These are antibodies that can cause precipitate formation by forming complex with
antigen molecules in the solution. Precipitin can only be demonstrated with soluble antigens.

☺ Lysins: These are antibodies that cause the dissolution (lysis) of antigenic cells. The presence of
complement is required to complete the reaction.

› Bacteriolysin: It is a lysin, which acts upon bacterial cells.

› Hemolysin: It is a lysin, which acts uponred cells.

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 › Luekocidins: Will kill leukocytes with or without lysis. Certain pathogenic bacteria produce
them.

☺ Opsonins: These are antibodies which act upon bacterial invaders, weakening their resistance to
the phagocytozing action of leukocytes

☺ Neutralizing (protective) antibodies. These are antibodies, which render their antigenic
microorganism (usually viruses) harmless.

☺ Antitoxins. These are antibodies, which neutralizes (or flocculates) with specific toxins.

☺ Allergic Antibodies. These are antibodies, which react with allergies in allergic reactions.

Classification of Antibodies:

1. According to the temperature at which they react:

a. Cold Antibodies: react at 4C to room temperature
b. Warm Antibodies: react at 37C

2. According to Occurrence:
a. Natural antibodies: appear without apparent stimulus
b. Immune antibodies: appear following the introduction of an antigen

3. According to the specie that produce antibodies:

a. Iso-antibodies: antibodies which came from the same specie
b. Heterophile antibodies: antibodies from one specie and produce antibodies in another specie

4. According to their In vitro behavior:

a. Complete or bivalent: (saline antibody) Capable of agglutinating antigens in saline
b. Incomplete (univalent or blocking). Able to coat antigens but to not agglutinate them, unless another
reagent is added. Like albumin, enzymes or antihuman globulins.