II.

ANTIBODY MOLECULES: THE IMMUNOGLOBULIN

SUPERFAMILY
Antibodies are produced in response to invasion by foreign compounds
(antigens) that can be proteins, carbohydrates, nucleic acids or microorganisms
(viruses, bacteria). Antibody binding to an antigen initiates a process that
eliminates the foreign substance.
Antigens may have several antigenic determinant regions in them; in proteins,
this can be as small as 6 or 7 amino acids.
Each human can produce about 10 million different antibody structures. All
antibodies have similar structures but differ in specificity.
In autoimmune diseases, the bodys own molecule is recognized by the immune
system.
Basic structure of immunoglobulins
Immunoglobulin molecules consist of four polypeptide chains, two identical
copies of two different chains, designated L and H.
Two light chains (L) of identical sequence combine with two heavy chains (H) of
identical sequence to form the structure (LH)2.
The four chains are covalently interconnected by disulfide bonds. One L binds to
one H, and the two heavy chains are also interconnected. The latter is called
the hinge region, which gives the molecule flexibility upon antigen binding.
Each H is associated with an L in such a way that the N termini are close to each
other.
In the most common immunoglobulin, IgG, the H chains have about 440 amino
acids (50 kDa) while the L has about 220 (25 kDa) amino acids.
In the other types of immunoglobulins, the H chains are different from those in
IgGs, and they can form dimers to pentamers.

Figure 9.1, Devlin. Linear Representation of four-chain IgG antibody molecule Two H chains
and two L chains in C to N terminal directions. Interchain disulfides link H and L chains together.
Domains of the constant C region of the H chain are CH1, CH2, and CH3. The constant region of
L is designated CL and variable, V, regions are VH and VL of H and L chains, respectively.

Constant and variable regions of the antibody structure

Sequences of the N terminal half of the L chains and the N terminal quarter of
the H chains are highly variable between different antibody molecules.
These N-terminal sequences are the variable (V) regions and are designated
VH and VL.
Within the V regions, certain segments are hypervariable.
These regions are termed the complementarity-determining regions (CDRs)
since they form the antigen binding sites.
Thus, variable regions give the antibody its specificity.

In contrast, the C-terminal three quarters of the H chains and the C terminal
half of the L chains are homologous in sequence with the other H or L chains
of the same class of antibodies.
These constant (C) regions with a homologous primary structure are
designated CH and CL in the H and L chains, respectively.
The CH regions determine the antibody class, provide binding site for
complement proteins and are the site necessary for antibodies to cross the
placental membrane.

Figure 9.2, Devlin. Diagrammatic of Structure for IgG. L chains are divided into domains VL and
CL. H chains are divided into domains VH and CH1, CH2, CH3. Antigen binding is in VH-VL.

Repeating amino acid sequences and homologous three-dimensional

domains occur within an antibody
Within each chain of an antibody molecule, there is a repeating pattern of
structure, called domains.
In the H chain, there are four domains. The H chain is divided into one VH
region and three CH regions.
The L chain is divided into 2 domains, one VL region and one CL region.
Each domain folds into a structure built by antiparallel stands which
generates a motif known as the all-beta immunoglobulin fold. These domains
are stabilized by disulfide bridges.

Figure 9.4, Devlin. Immunoglobulin Fold. (a) is scheme for folding of a CL domain, showing pleated sheet structure. Blue shows cystine bond. Light arrows are strands in plane above, dark
are below.

The H and L chains finally fold over each other and stabilized by disulfide bonds.
This gives the final 3D structure to the molecule.

Figure 9.6, Devlin. Model of an IgG antibody molecule. The two L are light gray spheres and the
H chains are lavender. Carbohydrates are green and orange. The CDR of the VH-VL are dark
red in the H chains and pink in the L. The heptapeptide hinge connecting the Fab and Fc are
dark red.

The biological function of the antibodies

The function of the antibodies is the recognition and binding of foreign molecules
and the initiation of their removal.
When antibodies bind to antigens, small changes in conformation of the CDRs
occur that is transferred to the other domains of the molecule. Change in
conformation at the C-terminal region exposes the complement-binding site and
activates the complement system.
At least 11 different complement proteins exist in plasma. They are activated by
IgG or IgM antibodies binding to antigens on the outer cell membrane of invading
bacterial cells, protozoa or tumor cells. The 11 proteins are sequentially activated
and associate with the cell membrane to cause lysis.
Many complement proteins are precursors of proteolytic enzymes. Similarly to
the blood-clotting cascade, one protein will activate another protein in the
complement cascade.
Upon association of the antibody with the antigen, the antibody changes
conformation and exposes the complement-binding site at the Fc region. This will
allow the binding of the C1 complex, which consists of C1q, C1r and C1s. C1r
and C1s undergo a conformational change and become active enzymes on the
cell surface. The activated C1 complex (C1a) hydrolyzes a bond in C2 and C4
that now become active and associate on the cell surface. The now active C2C4 complex then hydrolyzes a bond in C3. Activated C3 then binds to the cell
surface and activated C2-C4-C3 activates C5. Activated C5 then associates with
C6, C7, C8 and six molecules of C9. This complex binds to the cell surface and
lyses the membrane. (You do not need to know the members of the complement
system and the order they are activated.)

Amplification of the triggering signal occurs in this cascade.

The major role of the complement system is to generate opsonins an old term
for proteins that stimulate phagocytosis by neutrophils and macrophages. The
major opsonin is C3b, which can bind to specific receptors on the macrophage
cell surface.
Patients with inherited deficiency of C3 are subject to repeated bacterial
infections.

Antibody classes
Immunoglobulins in a single class contain common homologous regions.
Characteristics of each class are due to differences in the heavy chains.
The different H chains are designated as , , , , and occur in IgG,
IgA, IgD, IgE and IgM classes, respectively.
Two types of L chains are made, lambda () and kappa (), either of which
are found combined with the five classes of H chains.

IgDs, IgEs and IgGs are monomers (LH) 2 , containing delta, epsylon and gamma
heavy chains, respectively.
IgAs in serum are mostly monomeric (LH) 2 (alpha heavy chains), however, in
seromucosal secretions are covalently linked dimeric structures ((LH)2)2 kept together
by the so-called J-chain. These secretory IgA molecules also contain an additional
secretory component (a short polypeptidet), which facilitates transport of the secretory
IgA across the mucosal epithelium and protects the secretory IgA from proteolysis.
IgMs are pentamers ((LH)2)5 kept together by the so-called J-chain (same as in
IgA).
IgG is the major immunoglobulin in plasma, it is monomeric (LH) 2.
Biosynthesis of a specific IgG in significant levels takes about 10 days
after exposure to a new antigen.
IgMs are made first and faster and serve as the first line of defense until IgGs are
made.

Figure 9.3 Devlin. Time course of specific antibody IgM and IgG response to antigen

FUNCTIONS OF THE DIFFFERENT ANTIBODY CLASSES

The IgA class of immunoglobulins is found in serum (15% serum antibodies) and
is the predominant antibody in seromucous secretions (bronchial, nasal and
intestinal secretions and in tears, milk and colostrum). They are the initial
defense against viral and bacterial pathogens prior to entry into plasma or
internal space. IgAs neutralize viruses, toxins and block bacterial adherence on
mucosal surfaces.
The IgM class is found mostly in plasma (10% of serum antibodies). They are the
first antibodies elicited in significant quantities on exposure to foreign antigen.
They are also found in many external secretions but at lower levels than IgA. IgM
promotes
phagocytosis
of
microorganisms
by
macrophages
and
polymorphonuclear leukocytes, and is a potent activator of the complement
system.
IgG class of antigens occurs in high concentrations in plasma (75% of serum
antibodies); it promotes phagocytosis of antigens by macrophages and activates
complement. Their response to foreign antigens is slower than that of the IgMs
but build up to a much higher concentration in plasma. This is the only antibody
that is able to cross the placenta. It also can secrete to breast milk, thus IgG
provides immunity to newborns.
IgD is the predominant immunoglobulin, with IgM, on the surface of Blymphocytes (<1% of serum antibodies). It serves as antigen receptor on
lymphocytes and participates in their activation.
IgE (< 0.004% of serum antibodies) binds strongly to receptors for IgE on
basophils and mast cells. IgE mediates immediate hypersensitivity reactions,
thus plays a role in allergic responses such as anaphylactic shock, hay fever and
asthma. It is also considered and anti-parasite antibody.
Immunoglobulin deficiency results in increased susceptibility to infection. The Xlinked agammaglobulinemias and common variable immunodeficiencies are
known. The commonest disorder is selective IgA deficiency that results in
recurrent infection of sinus and the respiratory tract.

IMMUNIZATION
An immunizing vaccine can consist of killed bacterial cells, inactivated viruses, killed
parasites, a nonvirulent form of live bacterium related to a virulent bacterium or
denatured bacterial toxin or recombinant protein.
Antigens not only cause differentiation of lymphoid cells so they produce antibody but
also cause differentiation of some of the cells into memory cells. Memory cells do not
secrete antibody but place antibody on the outer surface and serve as radar. Later,
when infection occurs, the binding of antigen to memory cells stimulates memory cells to
divide into antibody producing cells and new memory cells. This reduces the time
needed for defense and increases the concentration of antigen specific antibody. Newer
vaccines for adults include pneumococcal vaccine, hepatitis B and influenza vaccine.

SUMMARY:
(1) The heavy and light chains fold into multiple domains each; these domains
are called the immunoglobulin fold;
(2) The 4 chains associate non-covalently to form the 3D structure of the
molecule and then stabilized by disulfide bonds;
(3) The antigen binding sites (CDRs) contain multiple loops at the N-terminal
hypervariable regions of VL and HL to bind antigen;
(4) The interactions between antigen and antibody are non-covalent;
(5) Small conformational changes occur in the VL-VH domains upon binding to
antigen;
(6) This antigen binding induces conformational changes in distant regions such
as the Fc region and exposes the complement or C1q binding domain.
(7) Conformational change in the antibody structure is crucial to trigger the
elimination of the antigen.

________________________________________________________________

MINICASE, p. 91 (Mongomery, Conway and Spector) Multiple

myeloma
F.R., a 66-year-old man, had fallen out of a tree 4 years previously without any
apparent fractures. Since that time he complained of pain over the ribs. The pain
worsened by coughing or straining and became more severe in the 2 months
before he sought medical help. This patient had had a firm, marble-sized mass in
his neck for 10 to 15 years; the size had remained constant until the previous 6
months. At that point, the mass approximately doubled in size. The patient
denied pain associated with the mass, hoarseness, dysphagia, or respiratory
obstruction as well as easy bruising or bleeding, anemia, shortness of breath on
exertion, palpitation, or tachycardia.
An x-ray film obtained 1 year previously by the patient's local physician revealed
multiple rib fractures, and he was referred to a university hospital. A complete
workup was done, the significant finding was that serum and urine
immunoelectrophoresis reveal the presence of monoclonal L (light) chains.
Multiple myeloma, a neoplasm of plasma cells, belongs to the a group of related
disorders known as plasma dyscrasias and characterized by an uncontrolled
proliferation of plasma cells and the synthesis of a homogenous gamma globulin,
called M-protein (also called monoclonal gammopathies). Only one type of
immunoglobulin is produced with one type of light chain. In most cases, one type
of light chain is overproduced, showing elevated levels in the plasma and
secreted by the urine. Plasma and urine electrophoreses show typical single
peak in the gamma globulin fraction. This light chain secreted in the urine is
called Bence-Jones protein, and named after H. Bence-Jones who first detected
these proteins in urines of patients with multiple myeloma in 1847. Bence- Jones
protein is a doublet of a kappa or lambda light chain. (Note: the presence of a
single light chain in the urine made possible to work out the sequence and the
structure of immunoglobulins.)
Because of the excessive proliferation of plasma cells and the abnormal
secretion of the M-protein, the clinical manifestations of multiple myeloma are
variable. Bone fractures are common because of the erosion of the bone by the
plasma cells. When plasma cells invade the bone marrow, anemia, later
granulocytopenia and thrombocytopenia may develop. Bleeding can occur since
monoclonal immunoglobulins may interfere with the platelet function. Chronic
renal failure is a common symptom because the overloading of the renal tubules
with protein causing tubular cellular degeneration.
References Cohen HJ: Monoclonal gammopathies and aging, Hosp Pract 23:75, 1988; Durie
BGM: Staging and kinetics of multiple myeloma, Semin Oncol 13:300, 1986; Tonegawa S:
The molecules of the immune system, Sci Am 253(8):122, 1985.