Abstract
In vitro translation studies indicate that the beta 2-microglobulin (beta 2-m) light chain influences the formation of intrachain disulfide bonds in class I major histocompatibility complex (MHC) molecules during their biosynthesis. We now have examined the influence of beta 2-m on class I MHC intrachain disulfide bond formation in vivo. Using beta 2-m+ and beta 2-m- derivatives of a cell line transfected with the mouse H-2Ld gene, we show that all of the H-2Ld molecules from beta 2-m+ cells have both the alpha 2 and alpha 3 intrachain disulfide bonds, whereas about 50% of the H-2Ld molecules from beta 2-m- cells have only one of these bonds. All of the free H-2Ld heavy chains from beta 2-m+ cells can undergo a peptide-induced conformational change and can bind exogenous peptide and beta 2-m stably in vitro. Only those H-2Ld molecules from beta 2-m- cells, which have both intrachain disulfide bonds, undergo a peptide- and beta 2-m-induced conformational change in vitro. These H-2Ld molecules do not bind beta 2-m and peptide stably in vitro. From these results emerges a greater understanding of the role of beta 2-m at the time of class I MHC molecule synthesis: beta 2-m promotes intrachain disulfide bond formation in the class I MHC molecule and additionally affects class I MHC structure to render it competent to form stable trimolecular complexes with peptide and beta 2-m.
Highlights
Class I MHC molecules synthesized in thaebsence of &-m &m+and P2-m-derivativesof a cell line transfected with in a cell-free translation system fail ftorm one of two internal the mouseH-2Ldgene, we show that all of €thI-e2Ldmol- disulfide bridges [9]
These disulfide bridges occur in the az eculesfrom &-m+cellshave both thae2and a3intrachain domain, which contributes to the peptide binding site, and in disulfide bonds, whereas about50% of the H-2Ld mol- the agdomain, which associates with the&-m light chain[10]
Ecules from P2-m-cells have only oneof these bonds.All Based on the reactivityof the i n vitro synthesized class I MHC of the free H-2Ldheavy chainsfrom &-m+cells can un- molecules with domain-specific monoclonal antibodies, dergo a peptide-induced conformational change andit cwanasconcluded that in the absencofe &-m the disulfide bond bind exogenous peptide and P2-mstably in vitro
Summary
Clonal antibody; PAGE,polyacrylamide gel electrophoresis; DTT, dithio- Cell Lines-The cell lines used in these studies include three transthreitol. MAb 30.5.7-reactive H-2Ldmolecules from both cell lines have a n identical electrophoretic mobility under nonreducing conditions (Fig. 1, lanes 1, 2, and 61, and reduction leads toa comparable shift imnobility on SDS-PAGE (data not shown). Under reducing conditions,the H-2Ldmolecules precipitated from HCT-Ld3and HCT-Ld/p,-m cells by mAbs 30.5.7and 64.3.7 have identicalelectrophoretic mobilities (Fig. 2, lanes 4, 6, and 8;data not shown). These results, together withthe datashowing the differential effects of pp peptide and &-mon the nonreduced forms of H-2Ldsynthesized in &-m- cells, suggest that &-m influences disulfide bond formation in vivo. 4A).Addition of 0.5 p~ human Pn-mincreases the amount of pp peptide bound but does not increase the stability of pep-
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