Analysis of major histocompatibility complex class I folding: Novel insights into intermediate forms

Folding around a peptide ligand is integral to the antigen presentation function of major histocompatibility complex (MHC) class I molecules. Several lines of evidence indicate that the broadly cross-reactive 34-1-2 antibody is sensitive to folding of the MHC class I peptide-binding groove. Here, we show that peptide-loading complex proteins associated with the murine MHC class I molecule K ^d are found primarily in association with the 34-1-2 ⁺ form. This led us to hypothesize that the 34-1-2 antibody may recognize intermediately, as well as fully, folded MHC class I molecules. To further characterize the form(s) of MHC class I molecules recognized by 34-1-2, we took advantage of its cross-reactivity with L ^d. Recognition of the open and folded forms of L ^d by the 64-3-7 and 30-5-7 antibodies, respectively, has been extensively characterized, providing us with parameters against which to compare 34-1-2 reactivity. We found that the 34-1-2 ⁺L ^d molecules displayed characteristics indicative of incomplete folding, including increased tapasin association, endoplasmic reticulum retention, and instability at the cell surface. Moreover, we show that an L ^d-specific peptide induced folding of the 34-1-2 ⁺L ^d intermediate. Altogether, these results yield novel insights into the nature of MHC class I molecules recognized by the 34-1-2 antibody.