Kb, Kd, and Ld Molecules Share Common Tapasin Dependencies as Determined Using a Novel Epitope Tag

Abstract

The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, Ld, Kd, and Kb. Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.

Biochemical Features of the MHC-Related Protein 1 Consistent with an Immunological Function

“…A very comparable staining pattern was observed with mMR1 expressed in a murine fibroblast line (B6/WT-3). The size of the intracellular pool of MR1 in both cell lines was quite similar to that previously observed with epitope-tagged forms of murine (K b , K d , and H2-M3) and human (HLA-B27) class I molecules (33,34,42). In contrast, cell surface levels of 64-3-7-reactive classical class I molecules are typically much higher than we observed with hMR1 or mMR1.…”

“…Although all class I-and class I-like molecules that we have analyzed to date undergo a transition to a 64-3-7-negative state after synthesis (31,33,34,42), it remains formally possible that MR1 is an exception and does not achieve a 64-3-7-negative conformation, even if it binds a peptide or other specific ligand. In fact, this could explain the result from the denaturation experiment described above.…”

“…However, because we used mAb 64-3-7 to detect expression (specific for open class I H chains), it remained possible that there existed a fraction of surface-expressed MR1 that is in a 64-3-7-negative conformation. Indeed, for epitope-tagged classical class I molecules, Abs specific for the folded conformation reveal that most molecules at the cell surface are folded, reflecting occupancy with a high-affinity peptide (31,33,34,42). Thus, we sought to investigate whether MR1 also exists in a 64-3-7-negative conformation.…”

“…After lysis for 30 min on ice, postnuclear lysates were incubated with protein A-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden) for 1 h. Beads were washed four times in PBS ϩ 0.1% digitonin, and bound proteins were eluted by clearing the precipitating Ab from the samples overnight at 4°C by competition with excess peptide EPQAPWM, which corresponds to the 64-3-7 epitope (42). For denaturation/64-3-7 immunoprecipitation experiments, postnuclear lysates were generated in 1% Nonidet P-40 (in TBS) and each sample was split into two equal aliquots.…”

Identification of naturally processed peptides bound to the class I MHC molecule H‐2Kd of normal and TAP‐deficient cells

“…Results were plotted as the concentration of the test peptide needed to achieve a threefold increase in the mean fluorescence intensity (MFI) over the background cell surface Kd levels. We assumed, based on the results of others, that the increase in Kd reflects the binding properties of the peptide for Kd [59][60][61][62].…”