An immunoglobulin molecule can be divided in various ways. It is composed of two identical heavy and two identical light chains. The heavy chains are approximately twice as "long" as the light chains because they contain about twice as many amino acids; 440 and 220, respectively.1 Each chain can be divided into variable and constant sections called domains (Figure 1).
The amino terminal quarter of each heavy chain consists of a variable sequence of amino acids with three or more hypervariable regions; the latter are intimately involved in the actual antigen binding. The amino acids in the V domain with its hypervariable segments are distributed in so many different patterns that one V domain will bind with one of the innumerable foreign molecules or antigens that the host encounters and lend the Ig molecule its unique specificity, called idiotype. The actual binding is done by VH plus VL. The remainder of the heavy-chain molecule is of a relatively constant composition; it consists of three constant (C) domains - CHI, CH2, and CH3 - constant in structure but not interchangeable in position.1
The variable and constant domains of the light (L) chain, VL and CL plus VH and the first constant domain, Cu1, of the heavy chain can be separated chemically from the remainder of the molecule at the "hinge" region; it contains the antigen binding domains and is called the Fab fragment while the rest of the molecule contains the CH2 and CH3domains, is crystallizable, and is called the Fc fragment (Figure I).1 The heavy chain fragment consisting of VH and CH1 is called the Fd fragment.
Immunoglobulins are recognized as belonging to five different classes: IgG, IgM, IgA, IgE and IgD. Their names are derived from the heavy chains in their molecule, ie, the y, µ, s,e and 5 chains, respectively. Moreover, IgG has four subclasses and IgA two subclasses; to date no subclasses have been identified for IgM, IgD, and IgE. IgG, IgA, and IgD heavy chains each have three constant domains while IgM and IgE have an additional C domain (Figure 2).1,2
The amino acid sequence of the C domains is consistent, although it differs from class to class and allows differentiation as well as identification of each class immunologically. Thus, an antibody to IgG usually raised in another species, most often goat or rabbit, will recognize the Fes of all other IgGs but will not recognize the Fes of Igs of other classes. The light chain has one V and one C domain; the latter allows the differentiation of one type of light chain called ? from the other called ?.
Figure 1. Structural diagram of Ig molecule.
AU immunoglobulins have interchain linkage by disulfide bonds. The interchain disulfide bonds that link the two heavy chains are located in the hinge region, the stretch of amino acids between CHi and CH2. This area contains the cysteine residues that make up the disulfide bonds. There are two such bonds in the IgGl. IgG4, and IgE molecules. There are four disulfide bonds between the two 7 chains of IgG2. Between five and 14 bonds have been reported to link the two heavy chains of IgG3. A single disulfide binds the two d chains of IgD.
In the case of the IgM molecule, one disulfide bond links the heavy chains of the monomer in the CH2 domain and another in the CH4 domain. An interchain disulfide appears to link the five monomers to each other in the area of CH2-CH3 while at the penultimate cysteine in CH4 they also link to a J chain which joins the monomers to form the pentamer.
The light chains are bound to heavy chains with a which usually links a cysteine amino acid found near the carboxy 1 end of the light chain with located between VH and CH, on the heavy chain. In the case of IgGl the link of the light chain with the ? chain occurs with a cysteine in CH1, and in the case of the allotype Ami of IgG A2, the two light chains are linked together at their carboxy terminals and not linked to the heavy chain.
The Ig chains have also been identified immunologically by their genetic markers. At least 25 Gm markers have been found for IgG molecules, whereas two Am markers are known for IgA and three so-called Km genetic markers have been found for the ? light chain, all immunologically.
The subclasses can also be identified immunologically. AU IgG subclasses fix complement and cross the placenta.2 All but IgG 2 can participate in passive cutaneous anaphylaxis. Antibodies against pure carbohydrate are predominantly of the IgG2 subclass. Thus, the antibodies against Streptococcus pneumoniae and H influenza B resulting from the vaccines against the polysaccharide capsules of these microorganisms are made poorly before lgG2 synthesis begins at about 24 months age.3,4
Figure 2. Schematic representation of heavy and light chains in the various classes and subclasses.
SEQUENCE OF SYNTHESIS
When the host defense system initially encounters an antigen, the B-lymphocyte is transformed into a plasma cell to make IgM antibody. IgM is usually found as a pentamer with the five different monomers linked by disulfide bonds and a J chain.2 After 7 to 10 days the defense system begins to make IgG, a simpler molecule that has a longer half-life (Figures 1 and 2), is smaller, and can cross the placenta. At sites lined with mucosal cells, such as the respiratory or digestive tracts, the patient manufactures secretory IgA, which consists of two molecules of IgA joined by a J chain and linked to a secretory component, manufactured locally by epithelial cells. Secretory IgA is found not only in respiratory and intestinal secretions, but also in the urogenital tract, mammary gland, saliva, and tears (Figure 2).1,2
IgE is present in serum in less than 10,000 times the amount of IgG; it plays an important role in both parasitic infections and allergic reactions.1 The function of IgD is not yet completely understood; it is found primarily on the surface of the immature B cell together with IgM. IgD is thought to play a role in the differentiation of the B cell and to assist in the switch from IgM to IgG or IgA production. Reports have described IgD as present in colostrum.1
Synthesis of IgG: Diversity
A B-lymphocyte precursor matures into the pre-B cell stage producing µ chains (but no light chains) and then into a B cell with both d and µ chains on its surface.5 This lymphocyte can be activated by mitogen or antigen or IL-2 or IL-I, the interleukin factors from T cells or mononuclear phagocytes (MNPs), respectively. Activated B cells differentiate into either Igbearing memory B cells or plasma cells that synthesize and secrete IgM, IgG, or immunoglobulins of other classes.6 (See article by Drs. Sherris arid Mayer on human B cell maturation and immune regulation in this issue.)
Antibody diversity is not only due to the different classes of light and heavy chains but principally to the variable domains of both chains found in the Fab fragment.1 The variable domains with their hypervariable segments that differ from antigen to antigen represent the immunoglobulin 's idiotype for that particular antigen.7 The hypervariable segments of both VL and VH link with the antigen. It appears that the majority of amino acids of the hypervariable or complementarity-determining-regions play a role not only in the linkage but also in the recognition of antigen.8
Actually a microorganism such as the herpesvirus hominis or the EB virus of infectious mononucleosis has several antigenic determinants, each of which is called an epitope. Each epitope of the viral antigen reacts with a particular variable and hypervariable sequence of amino acids making that V domain specific for that epitope, and its idiotype. Rare exceptions that lead to cross-reactions may occur. Moreover antibodies against the idiotype develop spontaneously and thereby form part of a network that regulates antibody production.6-8
GENES CONTROLLING SYNTHESIS AND DIVERSITY
The immunoglobulin's molecular structure and the switch of production from one class of Ig to that of another class is controlled by genes. Antibody diversity in the heavy chain can be attributed, in part, to several types of genes regulating structure. An unknown number of V genes in the germline controlling the amino acid sequence of the variable and hypervariable sequences of the VH domain. One of these VH genes links to one of a still unknown number of diversity (D) genes. This V-D gene complex makes contact with one of the joining (J) genes, four of which have been recognized. The V-D-J gene complex finally binds one of the genes that control the synthesis of the five different constant (C) heavy-chain immunoglobulin classes. The gene to which the V-D-J complex binds is the one that is eventually synthesized. Each V-D-J gene complex 'can be "utilized" by the genes of all the heavy-chain classes.9,10 Antibody diversity is increased by the number of genes controlling the amino acid structure of the V domains of the ? and ? chains. There are four different sets of genes of VK light chain domains, and five different sets of Vx genes for the variable domains of the ? light chain. In addition to the VL genes there are J or joining genes that have been recognized as responsible for a host of different linkages of VK or Vx to CK or Cx, the constant segments of the light chains.9'1'
To enhance the myriad of structural alternatives produced by all these genes there is an almost incalculable number of random associations of DNA segment linkage possibilities of VH to D, D to JH, and JH to Cn.9"1 l At the same time the different VL and JL gene segment associations and subsequent linkages to one of the CL genes again present innumerable possibilities (until the exact number of gene segments for structure is known) leading to linkage diversity. The light chains that have been synthesized associate with a variety of VH - CH] combinations to form the antigen-binding sites of the Fab fragment, generating additional diversity that is further enhanced by somatic recombination of gene segments and point mutation nucleotide substitution throughout the V domain during B-lymphocyte differentiation." (See article by Drs. Sherris and Mayer on human B cell maturation and immune regulation in this issue. )
On the host's first encounter with an antigen, in an acute infection, each epitope links with a V domain; IgM is made first. Since the VH-D-JH gene repertoire is shared by all heavy-chain Ig classes and subclasses it probably links with ?µ first because C is the CH gene closest to the JH DNA segment.12 VH remains an unchanged part of the immunoglobulin molecule when a new linkage takes place between the VH-D-JH complex and the C gene of one of the other Ig classes. VL and the light chain also remain unchanged when the synthesis of another class of Ig occurs (Figure 3).
Figure 3. Schematic representation of genetic patterns controlling heavy and light chain structure.
Class switch is mediated by DNA rearrangement of two switch (S) regions found at the amino terminal of each CH gene except C0 called S-S recombination.12 Different CH genes are deleted depending on the CH gene expressed. The human CH gene band q32 is located on chromosome 14 with large duplicated sets of C7, Ce, and Cff genes as well as pseudogenes. The CH segments, beginning at the 5' terminal, are found in the following order: 5' - JH - ?µ - C8 - C73 - C7, - Ce2 (pseudogene) - Cal - C72 - C74 - Cel - Ce2 - 3' (Figure 3). B and pre- B- lymphocytes that bear both µ and d chains have the same VH domain.12·13 The DNA sequence for VK, JK, and CK is found on chromosome 2, with other VK gene DNA sequences, some of them pseudogenes, dispersed and located on chromosomes 1, 15, and 22. 14 The ? chain DNA sequence has been localized to chromosome 22. 13
Lymphocyte Ig and class switching can be considered to follow five steps: 1) Pre-B-lymphocyte with surface µ chain; 2) B-lymphocyte with surface IgM and IgD; 3) B cell with surface IgM and IgG, or IgM plus IgA, or IgM plus IgE; 4) B-lymphocyte with either IgG or IgA or IgE on its surface; 5) B-lymphocyte differentiates into plasma cell which secretes large amounts of IgG or IgA or IgE, respectively.10'13 The Ig class switch adds to an antibody's effector function role. Thus, each lymphocyte's specificity is determined by random associations of four DNA segments (VH, D, JH, and CH) in the case of the heavy chain and of three DNA segments (VL, JL, and CL) in the case of the light chain. The random associations plus somatic recombinations can thus be readily visualized to lead to the antibody diversity that gives each antigen's epitope its own idiotype.
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