Comparative analysis of the impact of a free cysteine in tapasin on the maturation and surface expression of murine MHC class I allotypes

Abstract

Tapasin is a key molecule in the major histocompatibility complex (MHC) class I peptide-loading complex, interacting with several other proteins in the complex. An amino acid substitution at a free cysteine position in tapasin has been shown to disrupt the covalent association of tapasin with ERp57. In this study, we mutated the free cysteine in mouse tapasin, and analysed the effects on the cell surface expression of the mouse MHC class I molecules K(d) and K(b). The C95S substitution in mouse tapasin increased the proportion of open forms relative to folded forms for both types of MHC class I molecules at the cell surface. Furthermore, the C95S substitution resulted in increased association of tapasin with folded K(d). Overall, our studies with these mouse MHC class I allotypes have revealed that the free cysteine 95 in mouse tapasin influences stable expression at the plasma membrane for both MHC class I allotypes, and have shown that tapasin's interaction with folded K(d) is elevated by the C95S substitution in tapasin.

Figure 1

(A) Wild type and mutant tapasin expression levels were matched among paired transfectants expressing each type of MHC class I heavy chain (K^d or K^b). Aliquots of whole cell lysates were electrophoresed on a 10% acrylamide Tris-glycine gel. The proteins were subsequently transferred from the gel to a blot and probed with a mAb specific for mouse tapasin (Tsn). (B) More folded K^d was associated with tapasin C95S than with wild type tapasin. Immunoprecipitations were performed with monoclonal Ab 34-1-2 on lysates of the indicated cell types. The immunoprecipitated proteins were electrophoresed on a 10% acrylamide Tris-glycine gel, transferred to a membrane, and probed with anti-tapasin Ab (indicated as Tsn). (C) Cell surface K^b molecules on cells expressing mouse tapasin C95S had a slightly faster turnover rate, relative to cell surface K^b molecules on cells expressing wild type tapasin. MF cells that expressed mouse wild type tapasin or tapasin C95S were cultured in medium containing 2 μg/ml brefeldin A for 0, 3, 6, 9, 12, or 15 hours. Following incubation in the brefeldin A-containing medium, the cells were stained with anti-K^b mAb B8-24-3 and phycoerythrin-labeled secondary antibody, and analyzed on a FACS Calibur.

Figure 2

Flow cytometric analysis was performed with the 64-3-7 mAb (for open forms) and either mAb B8-24-3 (for K^b folded forms) or mAb 34-1-2 (for K^d folded forms) and a phycoerythrin-conjugated secondary Ab on MF cells expressing wild type tapasin or tapasin C95S. Samples were assayed on a FACS Calibur flow cytometer (BD Biosciences). The ratios of the mean fluorescence intensity values for K^b and K^d were calculated and are shown on the graphs. Background staining with secondary Ab only was <10 channels for all the cell types included in the analysis. The results shown in A and B were obtained with cells cultured in medium containing fetal bovine serum, and the results shown in C and D were obtained with cells cultured in medium containing a serum substitute.