Disparate interaction of peptide ligand with nascent versus mature class I major histocompatibility complex molecules: comparisons of peptide binding to alternative forms of Ld in cell lysates and the cell surface.

Abstract

SummaryTo determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the L d molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular L a molecules can be detected in an alternative conformation designated Laalt, Ldalt molecules are non-ligand associated and show weak if any 32-microglobulin (32m) association. We report here that L a molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs L d surface half-life. By contrast, Laalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with L a, live cells were incubated with iodinated peptides and L d molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface L a molecules was found not to depend upon exchange with exogenous 32m, but did correlate with the level of 32m association. To study the intracellular interaction of peptide with L d, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded L d. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to L d was found to affect almost exclusively immature (Endo H s) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Laalt molecules are precursors awaiting constituents such as peptide and 32m that influence folding, whereas surface Laalt molecules appear refractory to association with peptide, 32m, and consequent folding.

TCRβ Chain That Forms Peptide-Independent Alloreactive TCR Transfers Reduced Reactivity with Irrelevant Peptide/MHC Complex

“…TAP-proficient cells in general have more peptide-free MHC class I molecules at the cell surface than the TAP-deficient cells, even though the proportion of peptide-free molecules is larger in TAPdeficient cells (27). This is also true in our model, because staining of P815 cells with an Ab specific for peptide-free H-2L d (28,29) is brighter than that of RMA-S-L d (Fig. 4C).…”

Polymorphism at Position 97 in MHC Class I Molecules Affects Peptide Specificity, Cell Surface Stability, and Affinity for β2-Microglobulin

“…Analysis of the chimeric D d -L d MHC class I molecule, D dm1 suggested that the amino-terminal half of L d (the ␣1 and ␣2 domains) was mostly responsible for its slow trafficking and assembly and weak ␤ 2 m association (2), in agreement with the structural studies cited above. Third, L d has a shorter half-life on the cell surface (3,10,11), and a higher proportion of the total L d molecules on the cell surface exists as open (mAb 64-3-7 ϩ ) forms (3,12). These latter findings suggest that peptide ligands associated with cell surface forms of L d tend to dissociate comparatively quickly to generate transient open forms of L d .…”

Tapasin−/− and TAP1−/− Macrophages Are Deficient in Vacuolar Alternate Class I MHC (MHC-I) Processing due to Decreased MHC-I Stability at Phagolysosomal pH

“…This observation implies that the processing mechanism includes the exchange of stabilizing peptide for antigenic peptide derived from exogenous Ag. Peptide exchange is possible because components of the MHC-I complex (MHC-I heavy chain, ␤ 2 -microglobulin, and peptide) bind each other in a reversible and dynamic fashion (37,(45)(46)(47). It may seem paradoxical that the binding of high affinity peptides to MHC-I promotes a processing mechanism based on peptide exchange, but enhanced MHC-I survival conferred by stabilizing peptide enables MHC-I to survive for participation in processing, which apparently outweighs considerations of peptide competition.…”