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To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

Three miniature swine lines, inbred for swine leukocyte antigen (SLA) haplotypes, a, c, and d, and a recombinant line, haplotype g, were analyzed for possible restriction fragment length polymorphisms (RFLP) by Southern blot hybridization with human C2, factor B (Bf), and C4 specific probes. The search for RFLP by using a human C2 probe failed to reveal any variants. However, a Taq I polymorphism was identified with the human Bf probe and Bam HI and Pvu II polymorphisms were identified with the human C4 probe. Overlapping restriction fragments were found with the C2 and Bf probes, which strongly suggests close linkage of C2 and Bf genes in swine. Segregation analyses of the Bf and C4 polymorphisms indicated that the polymorphic fragments followed a Mendelian pattern of inheritance. The recombinant haplotype g, which expresses class I genes of haplotype c and class II genes of haplotype d, was shown to produce an identical RFLP pattern, by using the Bf and C4 probes, as haplotype d, but different from that of haplotype c. This indicates that there is a close association of [C4-Bf-C2] and class II genes in miniature swine. Although these data do not show conclusively the location of the [C4-Bf-C2] genes, it is hypothesized that swine [C4-Bf-C2] genes are located between the class II and class I genes, as has been demonstrated in mouse and man.