Abstract
In this report we demonstrate that the ion pair Arg-80 alpha and Asp-57 beta, located in the peptide-binding site of nearly all class II major histocompatibility complex (MHC) proteins, is important for surface expression and function of the murine class II heterodimer I-Ad. Charge reversal at either of these two residues by site-directed mutagenesis generated mutant class II molecules that failed to appear at the cell surface. This defect in surface expression was partially reversed when the invariant chain was present or when the mutants were paired with the corresponding charge-reversed variant of the opposite chain. Surprisingly, surface expression was restored when cells expressing the single-site mutants were cultured at reduced temperature. In addition, the substitution of Asp-57 beta with residues found in alleles of class II molecules associated with diabetes resulted in heterodimers that were inefficiently expressed at the cell surface and presented foreign peptide poorly. Together, these results demonstrate that the formation of a salt-bridge between Arg-80 alpha and Asp-57 beta is required for efficient surface expression of class II MHC molecules, therefore representing an important step in the assembly and transport of functional class II heterodimers to the cell surface.
Highlights
T lymphocytes respond to foreign antigens by detecting peptide fragments of those antigens bound to products of the major histocompatibility complex (MHC)1 and displayed on the surface of antigen presenting cells
Role of Negatively Charged Residues in Surface Expression of I-Ad and Presentation of Foreign Peptide—Between 10 and 50% of COS cells transiently transfected with wild-type (WT) ␣- and -chains of I-Ad displayed significant surface staining with I-Ad-reactive mAb MK-D6 above staining with control mAb (Fig. 1)
We have demonstrated the importance of the ion pair Arg-80␣ and Asp-57 in both surface expression of and peptide presentation by class II heterodimers
Summary
T lymphocytes respond to foreign antigens by detecting peptide fragments of those antigens bound to products of the major histocompatibility complex (MHC) and displayed on the surface of antigen presenting cells (reviewed in Ref. 1). The threedimensional structures of both class I and II MHC molecules complexed with self- or foreign peptide antigens have recently been solved, revealing much about the molecular basis of peptide binding to MHC molecules [2,3,4,5,6,7,8] Both classes of MHC molecules are similar, as predicted by sequence comparison [9]; they differ in fine detail, especially in the peptide-binding site [7, 8]. Detailed functional analysis of the peptide-binding sites of both classes of molecules will be necessary to understand the role of peptide binding in determining the tertiary structure, and surface expression of MHC molecules and subsequent display of foreign antigens to the immune system. In the course of these studies, we identified an ion pair, Asp-57 of the -chain and Arg-80 of the ␣-chain of I-Ad, that plays an essential role in surface expression of I-Ad as well as peptide presentation
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