Antibody Structure And Function - pediagenosis
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Monday, July 30, 2018

Antibody Structure And Function

Antibody Structure And Function
Considering that the antibody in serum is a mixture of perhaps 100 million slightly different types of molecule, the unravelling of its structure was no mean feat. Early work depended on separation into fragments by chemical treatment (top left in figure); the fine details have come from amino acid sequencing and X-ray crystallography, both of which require the use of completely homogeneous (monoclonal) antibody. This was originally available only in the form of myeloma proteins, the product of malignant B lymphocytes, but is produced nowadays by the hybridoma method (see Fig. 15) or by genetic engineering in bacteria, yeast or a variety of mammalian cell types.

Antibody Structure And Function

A typical antibody molecule (IgG, centre) has 12 domains, arranged in two heavy and two light (H and L) chains, linked through cysteine residues by disulphide bonds so that the domains lie together in pairs, the whole molecule having the shape of a flexible Y. In each chain the N-terminal domain is the most variable, the rest being relatively constant. Within the variable (V) regions, the maximum variation in amino acid sequence is seen in the six hypervariable regions (three per chain) which come together to form the antigen-binding site (bottom left in figure). The constant (C) regions vary mainly in those portions that interact with complement or various cell-surface receptors; the right-hand part of the figure shows the different features of the C region in the five classes of antibody: M, G, A, E and D. The result is a huge variety of molecules able to bring any antigen into contact with any one of several effective disposal mechanisms. The basic structure (MW about 160 000) can form dimers (IgA, MW 400 000) or pentamers (IgM, MW 900 000) (see right-hand side of figure).
There are species differences, especially in the heavy chain sub- classes, which have evolved comparatively recently; the examples shown here illustrate human antibodies. Interestingly, camels and llamas also have antibodies with only heavy chains. These antibodies may be able to attach to some targets not accessible to conventional antibodies, and examples are being tested as possible new ways of preventing infection by viruses such as HIV (see Fig. 28). The carbohydrate side-chains (shown here in black) may constitute up to 12% of the whole molecule.
Note: The illustration shows an IgG molecule with its 12 domains stylized. The actual three-dimensional structure is more like the molecule shown binding to antigen in Fig. 13 (extreme right).
Fragments produced by chemical treatment: these fragments were of great importance in elucidating the chemical structure of antibody. Fab and F(ab)2 fragments allow binding to specific antigen in the absence of secondary interactions with other cells mediated via the constant region.
1            H, L: heavy and light chains which, being only disulphide-linked, separate under reducing conditions.
2               Fab: antigen-binding fragment (papain digestion).
3               Fc: crystallizable (because relatively homogeneous) fragment (papain digestion).
4               F(ab)2: two Fab fragments united by disulphide bonds (pepsin digestion).
Affinity and avidity The strength of binding between one V domain and an antigen is called the affinity of the antibody (see Fig. 20). Typi- cally, it is of the order of 108 L/mol or above. However, antibody molecules have two, or in the case of IgM 10, identical binding domains. If the antigen recognized also has repeated units, such as the surface of many bacteria or viruses, one antibody molecule can make multiple attachments to the same target antigen. The strength of this overall binding is known as the antibody’s avidity. It can be much greater than the affinity, typically 100 times more for a divalent anti- body and up to 100 000 times more for IgM.
Chains The heavy and two types of light (κ, λ) chains are coded for by genes on different chromosomes, but sequence homologies suggest that all Ig domains originated from a common ‘precursor’ molecule about 110 amino acids long (see Fig. 10). A proportion of antibodies consisting only of heavy chains are found in some species (e.g. camels and llamas). These antibodies are narrower and can sometimes fit into sites conventional antibodies cannot reach, which may make them useful for some types of therapy.
Classes Physical, antigenic and functional variations between constant regions define the five main classes of heavy chain: M, G, A, E and D. These are different molecules, all of which are present in all members of most higher species. Points of interest are listed below.
IgM is usually the first class of antibody made in a response and is also thought to have been the first to appear during evolution (see Fig. 46). Because its pentameric structure gives it up to 10 antigen- combining sites, it can show high avidity, even though it may have a relatively low specific affinity. It is therefore extremely efficient at binding and agglutinating microorganisms early in the response. However, it is also very efficient at making larger immune complexes (see Fig. 20), which can activate unwanted inflammation and disease (see Fig 36). Its production is therefore downregulated as soon as sufficient IgG has been generated.
IgG is a later development that owes its value to the ability of its Fc portion to bind avidly to C1q (see Fig. 6) and to receptors on phagocytic cells (see Fig. 9). It also gains access to the extravascular spaces and (via the placenta) to the fetus. In most species, IgG has become further diversified into subclasses (see below).
IgA is the major antibody of secretions such as tears, sweat and the contents of lungs, gut, urine, etc., where, thanks to its secretory piece (see below), it avoids digestion. It blocks the entry of microorganisms from these external surfaces to the tissues themselves.
IgE is a curious molecule whose main property is to bind to mast cells and promote their degranulation. The desirable and undesirable consequences of this are discussed in Fig. 35.
IgD  appears to function only on the surface of B cells, where it may have some regulatory role. In the mouse it is unusual in having two instead of three constant regions in the heavy chain.
Subclasses, subtypes Within classes, smaller variations between constant regions define the subclasses found in different molecules of all members of an individual species. The IgG subclasses are generally the most varied. All these variants found in all individuals of a species are called ‘isotypic’.
Allotypes In contrast, ‘allotypic’ variations (not shown in the figure) distinguish the Ig molecules of some individuals from others (compare blood groups). They are genetically determined and occur mainly in the C regions. No biological function has yet been discovered. Unlike blood groups, Ig allotypes are expressed singly on individual B cells, a process known as ‘allelic exclusion’, which shows that only one of the cell’s two sets of chromosomes are used for making antibody – presumably the first one to rearrange its Ig genes successfully.
Hypervariable regions Three parts of each of the variable regions of heavy and light chains, spaced roughly equally apart in the amino acid sequence (see lower left of figure) but brought close together as the chain folds into a β-pleated sheet, form the antigen-combining site. It is because of the enormous degree of variation in the DNA coding for these regions that the total number of combinations is so high. The hypervariable regions are also unusually susceptible to further somatic mutation, which occurs during B-cell proliferation within the germinal centre of lymph nodes or spleen. This further increases the range of available combining sites.
Idiotypes In many cases, antibody molecules with different antigen- combining sites can in turn be distinguished by other antibodies made against them. The latter are known as ‘anti-idiotypic’, implying that each combining site is associated with a different shape, although this is not always the antigen-binding site itself. Anti-idiotypic antibodies are thought to be formed normally and may possibly help to regulate immune responses.
Hinge region Both flexibility and proteolytic digestion are facilitated by the repeated proline residues in this part of the molecule. In IgM, the hinge region is as large as a normal domain, and is called CH2, so that the other two constant region domains are called CH3 and CH4.
J chain A glycopeptide molecule that aids polymerization of IgA and IgM.
Secretory piece A polypeptide derived from the poly-Ig receptor (see Fig. 10) and added to IgA dimers in epithelial cells to enable them to be transported across the epithelium and secreted into the gut, tears, milk, etc. where IgA predominates.
Clq The first component of the classic complement sequence, a hexavalent glycoprotein activated by binding to CH2 domains of IgM and some IgG subclasses (in the human, IgG1 and IgG3; see Fig. 6).
Antibodies for therapy Pure preparations of monoclonal antibodies are increasingly in clinical use, notably for autoimmune diseases and cancer (see Figs 38 and 42). Various strategies are available to avoid the recipient mounting an immune response against these antibodies, including the use of ‘humanized’ mouse monoclonals or of human cells  genetically  engineered  to  produce  antibodies  of  the  desired specificity.

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