Immunopeptidomic Analysis Reveals That Deamidated HLA-bound Peptides Arise Predominantly from Deglycosylated Precursors

Abstract

<jats:p>The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Although immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the <jats:italic>N</jats:italic>-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.</jats:p>

CD8+ T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph

“…To identify new A24/influenza-derived epitopes, we utilized an immunopeptidomics approach to sequence HLA-bound peptides on influenza-infected cells by liquid chromatography-tandem mass spectrometry (LC–MS/MS) 24 . Experiments were performed with the class I-reduced C1R B-lymphoblastoid cell line (minimal HLA-B*35:03 surface expression but normal levels of HLA-C*04:01 expression 33 ) and an HLA-A*24:02-transfected C1R cell line (C1R.A24) 34 . Initial experiments were performed in both cell lines at 16 h post-infection with and without the HKx31 IAV virus, isolating peptide-bound HLA-I molecules post-lysis utilizing the pan HLA-I antibody W6/32 (Supplementary Data 1 ).…”

CD8+T-cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph

“…To identify new A24/influenza-derived epitopes, we utilized an immunopeptidomics approach to sequence HLA-bound peptides on influenza-infected cells by liquid chromatography with tandem mass spectrometry (LC-MS/MS) 27 . Experiments were performed with class I-reduced (CIR) B-lymphoblastoid cell line (minimal HLA-B*35:03 surface expression but normal levels of HLA-C*04:01 expression 36 ) and an HLA-A*24:02-transfected CIR cell line (CIR.A24) 37 . Initial experiments were performed in both cell lines at 16 hours post-infection with and without the HKx31 IAV virus, isolating peptide-bound HLA-I molecules post-lysis utilizing the pan HLA-I antibody W6/32 ( Supplementary Data 1 ).…”

Does Antigen Glycosylation Impact the HIV-Specific T Cell Immunity?

“…For this reason, we attempted to evaluate the effect of glycosylation on T cell responses using b-O-glycosylated synthetic peptides. Additionally, since N-glycosylation followed by complete N-deglycosylation causes a change of an Asn (N) residue to Asp (D), we also tested OLP sequence variants that contained N to D substitutions to account for potential responses to N-deglycosylated neo-epitopes (64).…”