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The early studies o f Forsgren and Sjoquist (1966) had shown that the immune interaction between IgG and protein A was non-specific, with the protein A binding to the Fc-region of the IgG molecule and not the Fab-region. A more precise localisation of the protein A reactivity was subsequendy obtained by Kronvall and Frommel (1970), when examination of isolated F(ab’)2» Fab, Fc and F c’ fragments, along with pepsin component II and III (see Figure 1.1), showed the binding site for SpA to be located on pepsin component II; this corresponds mainly to the C „ 2 domain of IgG.

Attempts to further identify the exact region of the Fc fragment involved in the binding reaction between IgG and SpA, and the amino acid residues involved have been carried out using chemical modification techniques, but with only a limited amount of success. Results did, however, suggest that the presence o f covalently linked CH2 and CH3 were necessary for protein A binding (Endrescn and Grov, 1976; Stewart et al., 1978; Tarkhanova et al., 1980). X-ray crystallographic investigations carried out by Deisenhofer et al. (1978) and Deisenhofer (1981), have defined both the secondary structure o f a purified fragment B o f protein A and the Fc fragment o f IgG and the nature of the interaction between these two fragments. The Cpj2 and Cpj3 domains of the Fc fragment of IgG consists of 2 layers of anti-parallel ^-pleated sheets, enclosing a mainly non-polar interior. The Cjj2 chains are more disordered than the Cjj3 chains as they do not form a lateral contact. This dissimilarity between CH2 and Cj j3 gives a flexible interface between the two regions.

Figure 1.1 Diagrammatic representation o f the structure of human IgGl

This figure was adapted from Edelmann et al., (1969) and shows the different domains o f the heavy (H) and light (L) chains; the va ria b le regions (V) and the constant r eg io n s (C). The intrachain and in terchain disulphide bonds are shown. The cleavage sites for the proteolytic enzymes papain and pepsin are indicated; the various peptide fragments produced and their nomenclature are marked.

Fragment B of protein A is a small globular protein consisting of three helices which form a regular triangular array. There are two antiparallel a helices consisting of three turns each, and a C-terminal helix consisting of two turns, with the remainder of the molecule being elongated.

The data of Deisenhofer (1981) indicated two contacts between fragment B and Fc, however, it is thought that only one is normally formed under physiological conditions and the other is a crystal contact only. The former contact, which is predominantly hydrophobic, involves residues from both the N-terminal helices and the Cpj2 and Cjj3 domains of Fc. Investigations by Mota et al. (1978) and Langone et al. (1978 a&b), have shown that a complete protein A molecule, containing IgG binding regions E, A, B, C and D, can bind two IgG molecules. More recent studies (Hanson and Schumaker, 1984; Moks et al., 1986) indicate that IgG is functionally bivalent for SpA, which itself is at least tetravalent and possibly even pentavalent. Hanson and Schumaker propose that the composition of the IgG-SpA complex varies according to the ratio of IgG and SpA molecules present, with an IgG j-SpA j, and IgG2-SpA2 complex being favoured at high SpA ratios (4:1 and above) and a IgG4-SpA2 complex being favoured at higher IgG to SpA ratio (1.7:1) to (4:1).

In addition to an Fc binding ability, SpA also exhibits a weak reactivity to the Fab fragment of many classes o f immunoglobulins (Endresen, 1979; Inganas et al., 1980). This interaction does not involve the antigen combining site and is also able to act independently of the Fc activity o f a single IgG-SpA domain (Inganas et al., 1981). It has been shown that in order to precipitate SpA, and for complement activation by SpA, the IgG involved m ust have both Fc and Fab reactivities, (Endresen, 1979; Inganas and Nilsson, 1981).

The binding specificities o f protein A for the Fc fragment o f the different types of immunoglobulin and their sub-classes are summarised in Table 1.1. W ith few exceptions SpA reacts stro n g ly with the IgG o f alm ost all mammals. Notable

TABLE 1.]

Binding Specifities of Protein A to the Fc Fragm ent of the Immunoglobulin

Species Type of Immunoglobulin Subgroup

Man IgG 1,2.4,

(3 variable)

IgA 2

IgM 2

Rabbit IgG (Soluble complex) •

Mouse IgM (weakly) -

IgG 1 (weakly)

2a, 2b, 3

Rat IgG 1.2c

Guinea pig IgG 1.2

Cow IgG 2 (weakly)

Sheep IgG 2 (weakly)

Goat IgG 2 (weakly)

Horse IgG a, b, c (T)

Dog IgG

IgA (some) IgM (some)

a, b, c, d

Protein A does not bind Avian IgG, though it has been reported to bind to small amounts of chicken IgM (3%).

The table was adapted from review articles by Langone (1982a) and Surolia et al.,

exceptions are ruminant IgG and subclass 3 of human IgG (Langone, 1982a). The failure of most human IgG 3 immunoglobulins to bind to SpA is attributed to the presence of an amino acid change at position 435 in the Fc fragment, where arginine has been substituted for histidine. When the substitution was included in the models of Deisenhofer (1981) it was shown that arginine would considerably affect the normal Fc-SpA complex as the arginine side chain could form additional contacts with other amino acids of the complex, while its positive charge would also remain unbalanced. Such major affects would prevent arginine fitting into the normal Fc-SpA complex. Other studies have shown that SpA can bind to certain human IgA’s and IgM’s, Table 1.1 (reviewed by Langone, 1982a). SpA also binds to some IgA and IgM groups in some mammalian species.

Initial reports on the binding of SpA to the immunoglobulins o f non-mammalian species (Kronvall et al., 1970; 1974) indicated that only the serum from two primative flightless birds (Rhea americana and Pterocnemia pennata) showed positive reactions. No reactions were recorded among Amphibian, Reptilian, Fish or among all other birds tested. However, Zikan et al. (1980) reported that small amounts o f non-mammalian serum IgM o f chicken, clawed toad and carp bound to SpA. Subsequent tests on purified carp IgM appeared to locate the majority of the binding activity to the Fab region. Although protein A has been reported to bind weakly to the Fab fragments of some IgG and IgE globulins. These particular IgM and Fab-SpA binding results were considered dubious by Langone (1982a) who concluded that the binding seen may have been due to Fab antibody activity against SpA.