Properties and Structure The mammalian immune system responds to the almost unlimited array of antigens by producing antibodies that react specifically with the molecules that induced their production. During the immune response, the structure of the inducing antigen is imprinted on the immune system, and subsequent challenges with the same or structurally related molecule(s) causes a more rapid rise in antibody levels to much greater concentrations than were achieved after the primary antigenic challenge. Thus the hallmarks of the humoral immune system include induction, specific protein interaction, and memory.

Antibodies belong to the family of proteins called the immunoglobulins. The basic structure of all immunoglobulins consists of a monomer that contains four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains covalently linked by disulfide bonds ( Fig. 1 ). The X-ray crystallographic structure of a monomeric immunoglobulin, specifically a mouse IgG2a mono clonal antibody (mAb), is shown depicted in both ribbon and space filling models in Fig. 2 . Depending on the angle between the constituent Fab (fragment antigen-binding) monomers, an immunoglobulin monomer consists of a Y- or T-like structure. The size of the Fab arms is 80 × 50 × 40 Å, and the size of base, called the Fc (fragment crystallizable) region, is approximately 70 × 45 × 40 Å according to the X-ray structure models. The Ig molecule exhibits considerable flexibility. In electron microscopic, low-angle X-ray scattering, transient electric birefringence, and resonance energy transfer studies, the angle between the Fab domains has been observed to vary from 0 to 180 degrees. All antibodies have two identical combining sites for antigen located at the ends of the Fab domains.

Fig1. DIAGRAMMATIC REPRESENTATION OF THE STRUCTURAL FEATURES OF AN IMMUNOGLOBULIN G (IGG) MOLECULE. NH 2 indicates the N-terminus and COOH the C-terminus. V h, C h1 , V l, and C l homology domains are shown as boxes. Only the disulfide linkages that join H and L chains are shown. Left, Approximate boundaries of the complementarity-determining region (CDR) regions in the V l and V h regions. Right, Sequences encoded by V h, D, J h, V l, and J l segments in the V h and V l regions.

Fig2. X-RAY CRYSTALLOGRAPHIC STRUCTURE OF AN INTACT IGG MOLECULE SHOWN AS A RIBBON DIAGRAM (A), OR A SPACE-FILLING MODEL (B). The structure is that of a mouse immunoglobulin G2a (IgG2a) monoclonal antibody (protein data base [PDB] file 1IGT) and it was the first intact IgG to have its structure determined. (A) The two-layer β -sandwich characteristic of the “immunoglobulin fold” is clearly visible within each of the constituent domains of the γ -heavy chains (blue and red) and κ -light chains (green and yellow), respectively. Black lines indicate the positions of inter-heavy chain disulfide bonds in the hinge region. (B) The constant domains of the heavy chains and light chains are in various shades of blue, and the glycan chain lining a region between apposing C H 2 domains is in white. The variable regions are colored according to the genetic segment encoding them. Dark green denotes the polypeptide region encoded by the V segment of V H and orange the DJ segment of V H . Light green denotes the polypeptide encoded by the V segment of V L and yellow that encoded by the J segment of V L . (A, Modified from http://proteopedia. org/wiki/index.php/Image:Opening_1igt.png ; B, From http://www.imgt.org/ IMGTeducation/Tutorials/IGandBcells/_UK/3Dstructure/Figure2.html .)

Fab and Fc represent functional domains in immunoglobulins. They were discovered by performing limited proteolytic digestion of the molecule. Both the H and L chains contribute amino acids that constitute the antigen-binding site in Fab. The monovalent Fab fragment will bind to, but will not precipitate, multivalent antigens, in contrast to native IgG. A fragment can be prepared, called F(ab ′ ) 2 , that is devoid of Fc but still precipitates antigen. This form of immunoglobulin consists of two Fabs disulfide bonded at a part of the molecule called the hinge region. The hinge region is the part of the Ig molecule that is responsible for the molecular flexibility exhibited by all immunoglobulins. The other major function of immunoglobulins, binding to specific receptors on cells and certain effector proteins such as C1q, is associated with binding sites also found in Fc. The Fc region of IgG, one of the classes of immunoglobulin, also inter acts with protein A, an immune evasion molecule on the cell walls of S. aureus. When bound to protein A, the binding of IgG to host effector molecules such as C1q is sterically interfered with.

The chain structure of immunoglobulins explains neither antibody structural diversity nor antibody binding to antigen. The discovery of variable and constant regions of amino acid sequence formed the basis for understanding both phenomena. Thus in the L chain, the 100 or so amino acids in the amino-terminal half of the protein (variable region [V L ]) vary among antibody molecules, but in the second half (constant region [C L ]), there is virtual complete correspondence in amino acids, position for position, to the carboxy-terminus. The H chains exhibit a similar pattern and can be divided likewise into V H and C H 1, C H 2, and C H 3. Comparison of the amino acid sequence of many V L s has revealed that whereas certain parts of the variable region exhibit excess variability, others are less variable. The former regions are called hypervariable or complementarity-determining regions (CDRs). The latter framework regions function as a structural scaffold to support the CDRs. Antigen binding is mediated by six CDRs, three in each of the V H and V L domains. The combining site for antigen is a trough, cavity, or even flat surface composed of parts of the hypervariable regions of both the H and L chains. It is a small region, representing only 25% of the antibody V region. The region that interacts directly with the epitope on the antigen is even smaller and is formed by the association of the CDR regions, each of which consists of approximately 20 amino acids. Thus the variation in a few amino acids accounts for the specificity and diversity of antibodies with respect to antigen binding.

Immunoglobulins exhibit additional physical heterogeneity, which imparts to each immunoglobulin a special effector function that is reflected in unique biologic properties independent of antigen binding activity. In the pregenome era of immunochemical research, heterologous and autologous antisera raised against immunoglobulins were used to classify three types of physical heterogeneity. The first kind is based on the antigenic heterogeneity exhibited by immunoglobulin when it is used as an immunogen in other species. This is called class or isotypic variation. In humans, five isotypes can be distinguished based on unique antigenic (isotypic) determinants found on the H chain. These are designated by capital Roman letters as IgG, IgM, IgA, IgD, and IgE. The H chain of each class is designated by the lower-case Greek letter corresponding to the Roman letter of the class. Thus the H chain for IgG is γ , for IgM is μ , for IgA is α , for IgD is δ , and for IgE is ε . Some of the immunoglobulin classes are composed of polymers of the basic monomer. In humans, the two antigenic varieties of the L chain are kappa ( κ ) and lambda ( λ ). Each Ig has two identical L chains; the κ and λ are shared by all classes. The monomeric form of any immunoglobulin is described by its chain structure. The molecular mass of the immunoglobulins can vary from 150 to 1000 kDa. This variation is attributable to polymerization of the basic monomer form. None of the immunoglobulins are polymeric forms of another class. IgG is the most prevalent, constituting 75% of the total Ig in blood. It is present in normal adults at concentrations of 600 to 1500 mg/dL. IgG is designated γ 2 κ 2 or γ 2 λ 2. It is the only class of Ig that crosses the placenta ( Table1 ).

Table1. Human Immunoglobulins: Properties and Functions

The isotype IgM is predominantly a pentamer consisting of five monomeric units disulfide linked at the C-terminus of the H chain. Each monomer of IgM is 180 kDa because of the presence of an additional C H domain, specifically the C μ 2 domain, which replaces the hinge segment. The complete protein has a sedimentation coefficient of 19 S, which corresponds to a molecular mass of 850 kDa. IgM is designated ( μ 2 κ 2) 5 or ( μ 2 λ 2) 5 . IgM also contains a 15-kDa protein called the J chain. In the current structural model of IgM, the J chain forms a disulfide-bonded clasp at the C-terminus of two H chains (Fig.3 ).

Fig3. (A) Structure of the four subclasses of human immunoglobulin G (IgG). Constant region domains are indicated by C n N, where n is the subclass and N is the domain. (B) Structure of human IgM. The J chain is shown in the model as disulfide linked to two μ -chains. Other models have been proposed. Filled circles indicate carbohydrate. (C) Structure of human secretory IgA. This model shows the possible arrangement of the two IgA monomers in relation to the secretory component and J chain. As the IgA molecule passes through the epithelial cells, the secretory components are synthesized and attached covalently to the Fc domain of the α -chains that have previously been joined to the J chain with disulfide links. Light chains are shown in blue, heavy chains in purple, disulfide bonds as gray lines, and carbohydrates as red circles. (From Turner M. Molecules which recognize antigens. In: Roitt DK, ed. Immunology. London: Gower; 1989:51.)

The structure of the other isotypes of immunoglobulins are summarized as follows. The isotype IgA has a variable number of monomeric units and is designated ( α 2 κ 2) n or ( α 2 λ 2) n , where n = 1–5. Serum IgA constitutes 20% of the total serum immunoglobulin, and 80% of this is monomeric. The remainder exists as polymers, where n = 2–5. The other form of IgA is found in external secretions such as saliva, tracheobronchial secretions, colostrum, milk, and genitourinary secretions. Secretory IgA consists of four components: a dimer of two monomeric molecules, a 70-kDa secretory component that binds noncovalently to the IgA dimer, and the 15-kDa J chain that is believed to form a disulfide-bonded clasp at the C-terminus of the H chains (see Fig. 3 ). The isotype IgD has a molecular mass of 180 kDa. Its serum concentration is very low, approximately 3 mg/dL. IgD apparently functions as a membrane molecule, being associated on mature but unstimulated B cells in association with IgM. IgE is the homocytotropic or reaginic Ig and mediates immediate hypersensitivity. It has a molecular mass of 180 kDa and, similar to IgM, has four C domains. The Fc portion of IgE binds strongly to a receptor on mast cells, Fc ε R, and this is how this immunoglobulin exerts its particular activity. The overall properties of the immunoglobulins are summarized in Table1.

Subclasses of isotypes IgG, IgA, and IgM have been identified. The structural basis for this antigenic heterogeneity is variation in amino acid sequence in the Fc portion of the H chain of a given class. The subclasses of human IgG, called IgG1, IgG2, IgG3, and IgG4, are the best characterized. Each has a slightly different structure, with the most notable differences being in the length of the hinge and in the number of interchain disulfide bonds (see Fig. 3 and Table1). IgG1 constitutes 70% of the total IgG and IgG2 20%. IgG3 and IgG4 constitute 8% and 2%, respectively, of the total IgG. The sub classes of IgG exhibit different catabolic rates and bind differentially to cell-associated Fc receptors (Fc γ R) and to C1q. Specifically, IgG2 does not bind to the Fc γ Rs and IgG4 binds about 10-fold less well than do IgG1 and IgG3. For C1q binding, the rank order of affinities is IgG3 > IgG1 > IgG2 ≫ IgG4. Despite the most obvious sequence differences among the human IgG isotypes being in their hinge regions, studies using engineered domain-swapped chimeric molecules have demonstrated that it is the more subtle amino acid sequence differences within the respective C γ 2 domains that account for the differences in binding to C1q and to the Fc γ Rs. Transport across the placenta is mediated by the Fc-neonatal receptor (FcRn) and for this functional activity IgG2 crosses the placenta slightly more slowly than the other three subclasses. The other known subclasses of Ig isotypes are associated with IgM (IgM1 and IgM2) and IgA (IgA1 and IgA2). The properties and function of these subclasses are less well known.

The second type of variation is called allotypic variation. It is attributable to genetically controlled antigenic determinants found on both the H and L chains. Although each human has all immunoglobulin isotypes, an individual has only one form of each allotype on his or her immunoglobulin molecules. Allotypes are codominantly expressed, but an individual B lymphocyte secretes only one of the parental forms. This phenomenon is called allelic exclusion.

The third type of variation is attributable to antigenic determinants that are unique to each particular antibody molecule produced by an individual. These markers are called idiotypic determinants, and they are associated with a single species of antibody. The antiidiotypic antibodies that recognize a particular idiotype will not react with any other immunoglobulins in the donor other than the purified anti body that was used to raise the antiidiotype antibody. In most cases, the immune response to an antigen results in a mixture of several antibodies, each of which has identical binding specificity but distinct idiotypic determinants. Thus there can be many idiotypes for a given antigenic specificity, which has been interpreted as being a reflection of physical heterogeneity in or near the antibody combining site, for example, in the variable region domains. In some species (notably certain strains of mice), the response to antigen results in a predominant idiotype on all antibodies of a given specificity. Because this quality is inherited, the idiotypes are called major, cross-reactive, or public. Some public idiotypes have been found in certain species (again, most notably mice) to be genetically linked to allotypes. Three kinds of antiidiotype antibodies have been described: those that function as an internal image of the original antigen by mimicking the antigen structure, those that recognize antibody combining site-associated idiotypes, and those that are specific for framework-associated determinants. The internal image antiidiotypic antibodies are of clinical interest.

Every immunoglobulin is a glycoprotein, and the critical glycan is attached to the H chain in the Fc domain at the conserved asparagine at position 297 (Asn297). This single, N-linked glycan is essential for maintaining an open conformation of the two H chains as it lines the opposing faces of the pair of C H2 subdomains of Fc (see Fig. 2B ). The core structure of the N-linked glycan is a biantennary heptapolysaccharide containing N-acetylglucosamine plus additional sugars (fucose, galactose), with bisecting N-acetylglucosamine and sialic acid variably present. Effector functions depend on the Asn297-linked glycan and are influenced by its structure. Deglycosylated IgG does not interact effectively with Fc γ receptors (Fc γ Rs) and cannot support in vivo effector responses, including antibody-dependent cell-mediated cytotoxicity or complement-dependent cytotoxicity Individual glycoforms contribute to modulating inflammatory responses and have disease association. For example, glycosylation differs in patients with rheumatoid arthritis or vasculitis compared with the nor mal population. Addition of sialic acid to the N-linked glycan reduces binding of IgG to Fc γ Rs and reduces in vivo cytotoxicity. Regulation of sialylation of IgG contributes to the anti-inflammatory homeostasis of serum IgG. Upon antigen challenge, reduced sialic acid–IgG can mediate immune clearance and protective immunity through interaction with subclass-specific Fc γ Rs. Kaneko et al. have proposed that the protective effect of intravenous immunoglobulin (IVIg) therapy is attributable to the minor fraction of sialylated IgG species in the total IVIg preparation and that the high doses required (1 to 3 g/kg body weight) for antiinflammatory activity could be significantly reduced by increasing the percentage of sialylated IgG.

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