Allele-independent protein turnover enables codominant expression of HLA class Ia alleles in human APCs

Background and aims: MHC class I (MHCI) heavy chains (HC) are expressed from three loci in the MHC (HLA-A, -B, and -C in humans), whose extensive polymorphism maps to the antigen-binding groove, diversifying the bound peptide repertoire. Maternally and paternally derived MHCI alleles are codominantly expressed, but how this is achieved at the protein level is poorly understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules. Methods: Human antigen-presenting cell (APC) lines with functional MHCI peptide loading pathways (EBV-B cells and the myeloid cell line, KG-1), as well as monocyte-derived DCs (MoDCs), were labeled biosynthetically with heavy water (2H2O). Folded MHCI molecules were immunoprecipitated with the W6/32 mAb, and tryptic digests analysed by mass spectrometry. MHCI-derived peptides were assigned to specific alleles and isotypes, and turnover rates quantified by 2H incorporation, after correcting for cell growth. Results: MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type (MoDCs > KG-1 cells > EBV-B cells), activation state (slow-down post LPS in MoDCs), donor variation, and MHCI isotype (somewhat faster turnover for HLA-C). However, in all settings, the turnover half-lives of alleles of the same isotype were similar. This included the HLA-B27 alleles associated with spondyloarthritides. Conclusions: MHCI protein turnover rates in APCs with functional peptide loading pathways are largely allele-independent. This may be an important feature enabling the normal function and codominant expression of MHCI alleles. B27 misfolding, previously implicated in the pathogenesis of spondyloarthropathy, arises despite normal turnover kinetics of folded B27 molecules. Lastly, this is one of very few studies to date using stable isotope/mass spectrometric techniques to assess the effect of structural polymorphisms on protein turnover. In this regard, MHCI structural diversity provi