AUTHOR=Goodson Hayley , Kawahara Rebeca , Chatterjee Sayantani , Goncalves Gabriel , Fehring Joshua , Purcell Anthony W. , Croft Nathan P. , Thaysen-Andersen Morten 

TITLE=Profound N-glycan remodelling accompanies MHC-II immunopeptide presentation

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1258518

DOI=10.3389/fimmu.2023.1258518

ISSN=1664-3224

ABSTRACT=<p>Immunopeptidomics, the study of peptide antigens presented on the cell surface by the major histocompatibility complex (MHC), offers insights into how our immune system recognises self/non-self in health and disease. We recently discovered that hyper-processed (remodelled) <italic>N</italic>-glycans are dominant features decorating viral spike immunopeptides presented via MHC-class II (MHC-II) molecules by dendritic cells pulsed with SARS-CoV-2 spike protein, but it remains unknown if endogenous immunopeptides also undergo <italic>N</italic>-glycan remodelling. Taking a multi-omics approach, we here interrogate published MHC-II immunopeptidomics datasets of cultured monocyte-like (THP-1) and breast cancer-derived (MDA-MB-231) cell lines for overlooked <italic>N</italic>-glycosylated peptide antigens, which we compare to their source proteins in the cellular glycoproteome using proteomics and <italic>N</italic>-glycomics data from matching cell lines. Hyper-processed chitobiose core and paucimannosidic <italic>N-</italic>glycans alongside under-processed oligomannosidic <italic>N</italic>-glycans were found to prevalently modify MHC-II-bound immunopeptides isolated from both THP-1 and MDA-MB-231, while complex/hybrid-type <italic>N</italic>-glycans were (near-)absent in the immunopeptidome as supported further by new <italic>N</italic>-glycomics data generated from isolated MHC-II-bound peptides derived from MDA-MB-231 cells. Contrastingly, the cellular proteomics and <italic>N</italic>-glycomics data from both cell lines revealed conventional <italic>N</italic>-glycosylation rich in complex/hybrid-type <italic>N</italic>-glycans, which, together with the identification of key lysosomal glycosidases, suggest that MHC-II peptide antigen processing is accompanied by extensive <italic>N</italic>-glycan trimming. <italic>N</italic>-glycan remodelling appeared particularly dramatic for cell surface-located glycoproteins while less remodelling was observed for lysosomal-resident glycoproteins. Collectively, our findings indicate that both under- and hyper-processed <italic>N</italic>-glycans are prevalent features of endogenous MHC-II immunopeptides, an observation that demands further investigation to enable a better molecular-level understanding of immune surveillance.</p>