Biochemical analysis of class II antigens. Identification of a two- and a three-polypeptide chain complex of I-A locus equivalent molecules in the rat

The polypeptide chain composition of class II antigens from LEW rat spleen cells was studied utilizing cross-reactive mouse alloantiserum A. TH anti-A.TL (specificity anti-Ia^k) and the monoclonal antibodies MRC-OX6 and MRC-OX3 for immunoprecipitation. Two-dimensional gel mapping of A. TH anti-A. TL immunoprecipitates revealed that, as in the mouse, two groups of class II antigens exist corresponding to I-A and I-E locus equivalent structures. In the absence of reducing agents three monomeric chains α, 36 kDa (p36); γ, 33 kDa (p33); and β, 23 kDa (p23), were detected for I-A equivalent antigens, whereas I-E equivalent molecules separated into five monomeric chains: α, 37 kDa (p37); γ, 33 kDa (p33); and three β chains 28 (p28), 26 (p26) and 24 kDa (p24). One strong dimer component of disulfide-linked γ chains was found to be associated with products of both loci. Although slightly different in molecular weight, γ chain corresponds to the nonpolymorphic murine invariant chain Ii. Both monoclonal antibodies recognized rat homologues of the murine I-A products. Extensive sequential criss-cross-immunoprecipitation with subsequent 2-dimensional O'Farrell analysis indicated that (a) MRC-OX6 precipitated molecules which were not recognized by MRC-OX3 and vice versa; (b) MRC-OX6 precipitated a three-polypeptide chain complex composed of the polypeptides p36, p33 and p23; (c) MRC-OX3 precipitated a two-chain complex composed of p36 and p23; and (d) the respective heavy (α) and light (β) chains of both complexes possess very similar physicochemical parameters, suggesting that they are structurally related, if not identical.