Allele-Independent Turnover of Human Leukocyte Antigen (HLA) Class Ia Molecules

Abstract

Major histocompatibility complex class I (MHCI) glycoproteins present cytosolic peptides to CD8+ T cells and regulate NK cell activity. Their heavy chains (HC) are expressed from up to three MHC gene loci (human leukocyte antigen [HLA]-A, -B, and -C in humans), whose extensive polymorphism maps predominantly to the antigen-binding groove, diversifying the bound peptide repertoire. Codominant expression of MHCI alleles is thus functionally critical, but how it is regulated is not fully understood. Here, we have examined the effect of polymorphism on the turnover rates of MHCI molecules in cell lines with functional MHCI peptide loading pathways and in monocyte-derived dendritic cells (MoDCs). Proteins were labeled biosynthetically with heavy water (2H2O), folded MHCI molecules immunoprecipitated, 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. MHCI turnover half-lives ranged from undetectable to a few hours, depending on cell type, activation state, donor, and MHCI isotype. However, in all settings, the turnover half-lives of alleles of the same isotype were similar. Thus, MHCI protein turnover rates appear to be allele-independent in normal human cells. We propose that this is an important feature enabling the normal function and codominant expression of MHCI alleles.

Molluscum contagiosum virus MC80 sabotages MHC-I antigen presentation by targeting tapasin for ER-associated degradation

“…The cell surface half-life of MHC-I can be greater than 24 hours depending on the specific cell line and peptide(s) displayed [42,43]. We investigated the kinetics of MHC-I surface downregulation using a transient transfection system, finding that maximal expression of MC80 at 24 hours post transfection (hpt) did not coincide with maximal downregulation of MHC-I surface expression (Fig 1B).…”

Most non-canonical proteins uniquely populate the proteome or immunopeptidome

“…MHC-I-peptide complexes are transported to the cell surface to enable T cell immunosurveillance of infected and neoplastic cells. Cell surface MHC-I-associated peptides (MAPs) exhibit half-lives on the order of 12 h (Blaha et al, 2019;Prevosto et al, 2016), far longer than their source polypeptides in the case of SLiPs and DRiPs (Dersh et al, 2021). Thus, MHC-I serves as a sink for peptides whose source protein translation would otherwise be invisible to MS due to their rapid degradation.…”

Quality control of HLA-DR molecules by the lysosomal aspartyl protease, cathepsin D

“…As a specificity control, we previously reported that HLA class I molecules are not detectably rescued by PepA (or by any other protease inhibitors) in KG-1 cells (45). This suggests that CatD has no role in HLA class I degradation in this cell line, or that this enzyme acts redundantly with other lysosomal cathepsins on class I molecules.…”

“…KG-1 is an acute myleloid leukemia cell line (85) with features resembling immature DCs (86,87), expressing DRB1*11 and *14 (88). Cells were grown at 37 ˚C in a humidified incubator, in RPMI1640 medium supplemented with 10% FBS, 2 mM L-glutamine, and penicillin/streptomycin, except for KG-1 cells, which were grown in IMDM with 20% Monocyte-derived dendritic cell culture Monocyte-derived dendritic cells were derived from healthy donor blood as described (45) with modifications as follows. Briefly, volunteers were recruited with ethical approval For total (surface plus intracellular) DR staining, cells were washed in PBS, fixed and permeabilized using the Cytofix/Cytoperm kit (BD), washed in 1× Perm/Wash buffer, and subsequently processed as for live staining, except that all reagents were diluted, and cells washed, in 1× Perm/Wash buffer.…”

“…Bands of interest were subjected to in-gel tryptic digestion and analysed by UPLC-MS/MS on an Orbitrap Velos mass spectrometer as previously described (45,104). For the identification of CatD cleavage products, we exploited the orthogonal cleavage specificities of trypsin (after Lys/Arg) and CatD (after large hydrophobic residues (58,105) (www.ebi.ac.uk/MEROPS database entry A01.009 (106)).…”