An Ion Pair in Class II Major Histocompatibility Complex Heterodimers Critical for Surface Expression and Peptide Presentation

Abstract

In this report we demonstrate that the ion pair Arg-80␣ and Asp-57␤, 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-A d . 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␤ 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␣ and Asp-57␤ 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.

The MHC Class II Molecule I-Ag7 Exists in Alternate Conformations That Are Peptide Dependent

“…Class II molecules that have unstable interactions with peptide are thought to have more rapid turnover rates within APCs. I-A g7 has been shown to both bind peptide poorly and to have an atypically short half-life (19,52,53). Thus, the minor population of I-A g7 that interacts only with 40M may persist at the cell surface, possibly due to its more stable interaction with peptide.…”

On the perils of poor editing: regulation of peptide loading by HLA-DQ and H2-A molecules associated with celiac disease and type 1 diabetes

“…Pulse/chase labelling studies show no major defects in assembly, early maturation, and ER to Golgi export of A g7 (130), but defects are detectable in other assays. In Ii-deficient cells, substitution of Asp57β to Ser in A d (as in A g7 ) impairs surface expression (131). Thus, lack of inter-chain salt bridging may compromise αβ heterodimer assembly in non-Asp57β alleles.…”

“…Thus, lack of inter-chain salt bridging may compromise αβ heterodimer assembly in non-Asp57β alleles. However, as murine APC populations in vivo typically express Ii, and Ii transfection overcomes the expression defect of A d (Asp β 57→Ser) (131), assembly defects are unlikely to contribute much to immune defects associated with lack of Aspβ57 in vivo . Moreover, A g7 αβ heterodimers expressed as recombinant soluble molecules in insect cells have been used in peptide binding and crystallization studies without evidence of instability (132).…”

Recombinant NFAT1 (NFATp) Is Regulated by Calcineurin in T Cells and Mediates Transcription of Several Cytokine Genes

“…Jurkat cells were transfected by electroporation in serum-free medium in 0.4-cm cuvettes with settings of 250 V and 960 F, using a Bio-Rad Gene Pulser. COS cells were transfected by the DEAE-dextran method (38). One day after transfection, cells were treated overnight by adding ionomycin or PMA to cell culture media; solvent was added as a control.…”