Peptide map analyses of murine Ia antigens of the I–E subregion using HPLC

Abstract

THE I REGION of the major histocompatibility complex (H–2) of the mouse¹ controls various immunologically related functions including immune responsiveness and T-cell, B-cell and macrophage interactions^2,3. The only gene products of the I region which have been identified by immunochemical techniques are the Ia antigens which are encoded by loci mapping to the I–A and I–E subregions of the I region⁴. Previously there has been ambiguity over whether the I–E and/or I–C subregion(s) control molecules detected by immunoprecipitation. Recent evidence suggests that the former, not the latter, controls these molecules^5,6. The Ia antigens are highly polymorphic by serological analysis and are expressed predominantly on B lymphocytes and epidermal cells⁷. Ia molecules are integral cell-surface glycoproteins and consist of two subunits of approximate molecular weights 35,000 (α) and 28,000 (β) which are noncovalently associated⁷. A general nomenclature has been agreed upon for the Ia polypeptides⁸. For example, the α polypeptide from the I–E subregion of a mouse of the H–2^d haplotype is denoted E_α^d. Partial N-terminal amino acid sequence analyses of Ia polypeptides from the I–E and I–A subregions of mice^8–14, humans^14,15 and guinea pigs¹⁶ have led to several important observations. (1) The E_α polypeptides of mice show striking homology with human α chains (p34) at least at their N-termini. The Eβ polypeptides of mice also demonstrate some homology with their guinea pig and human counterparts. (2) The Ia polypeptides from the I–A subregion of mouse show little apparent homology with their human, guinea pig and I–E-subregion counterparts. (3) The A_α, A_β and E_β polypeptides of various haplotypes differ by one or more residues at their N-termini. However, for the E_α^d and E_α^k polypeptides no amino acid differences have been unequivocally demonstrated. To determine whether the N-terminal differences in the E_β polypeptides extend throughout other regions of these polypeptides and whether the E_α polypeptides are identical, we have undertaken tryptic peptide analyses of two Ia molecules from the I–E subregion. We report here the application of high-pressure liquid chromatography (HPLC) to the separation of tryptic peptides and present comparative tryptic peptide maps for I–E subregion products from the H–2^d and H–2^k haplotypes.