Abstract
Myeloperoxidase (MPO) plays essential roles in neutrophil-mediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic under-processed and hyper-truncated glycans. Occlusion of the Asn355/Asn391-glycosylation sites and the Asn323-/Asn483-glycans, located in the MPO dimerisation zone, was found to affect the local glycan processing, thereby providing a molecular basis of the site-specific nMPO glycosylation. Native mass spectrometry, mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Longitudinal profiling of maturing, mature, granule-separated and pathogen-stimulated neutrophils demonstrated that nMPO is dynamically expressed during granulopoiesis, unevenly distributed across granules and degranulated upon activation. We also show that proMPO-to-MPO maturation occurs during early/mid-stage granulopoiesis. While similar global MPO glycosylation was observed across conditions, the conserved Asn355-/Asn391-sites displayed elevated glycan hyper-truncation, which correlated with higher enzyme activities of MPO in distinct granule populations. Enzymatic trimming of the Asn355-/Asn391-glycans recapitulated the activity gain and showed that nMPO carrying hyper-truncated glycans at these positions exhibits increased thermal stability, polypeptide accessibility and ceruloplasmin-mediated inhibition potential relative to native nMPO. Finally, molecular modelling revealed that hyper-truncated Asn355-glycans positioned in the MPO-ceruloplasmin interface are critical for uninterrupted inhibition. Here, through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity.
Highlights
Myeloperoxidase (MPO) plays essential roles in neutrophilmediated immunity via the generation of reactive oxidation products
Facilitated by its peroxidase activity, MPO is known to catalyse the formation of reactive oxidation products including hypochlorous acid (HOCl) from chloride ions (Cl−) and hydrogen peroxide (H2O2), substrates found in the maturing phagosomes [15, 16]
Uncommon monoantennary complex-type (FA1G1S1a), under-processed oligomannosidic (M5–M6) and hyper-truncated paucimannosidic (M2Fa-M3F) and chitobiose core-type (GlcNAc1–GlcNAc1F) structures were found to be characteristic N-glycans of neutrophil MPO (nMPO). These N-glycans are congruent with structures residing in the neutrophil granules [38] and those carried by other granuleresident glycoproteins including neutrophil cathepsin G and elastase [32, 39, 40]
Summary
Myeloperoxidase (MPO) plays essential roles in neutrophilmediated immunity via the generation of reactive oxidation products. Complex carbohydrates decorate MPO at discrete sites, but their functional relevance remains elusive. To this end, we have characterised the structure–biosynthesis–activity relationship of neutrophil MPO (nMPO). Mass spectrometry demonstrated that nMPO carries both characteristic underprocessed and hyper-truncated glycans. Mass photometry and glycopeptide profiling revealed significant molecular complexity of diprotomeric nMPO arising from heterogeneous glycosylation, oxidation, chlorination and polypeptide truncation variants and a previously unreported low-abundance monoprotomer. Through an innovative and comprehensive approach, we report novel functional roles of MPO glycans, providing new insight into neutrophil-mediated immunity. MPO generates highly reactive hypohalous acids and nitrogen dioxide, which readily react to form diverse reactive oxygen species, key microbicidal and immuneregulatory products of the neutrophil MPO-halide system
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