1. Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
- Author
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Rebeca Kawahara, Fabian Soltermann, Hannes Hinneburg, Siyun Chen, Robert J. Woods, Regis Dieckmann, Morten Thaysen-Andersen, Ian Loke, Vignesh Venkatakrishnan, Weston B. Struwe, Oliver C. Grant, Alison Rodger, Julian Ugonotti, Harry C. Tjondro, Johan Bylund, Benjamin L. Parker, Anna Karlsson-Bengtsson, and Sayantani Chatterjee
- Subjects
0301 basic medicine ,Glycosylation ,Az-MPO, azurophilic granule-resident MPO ,Neutrophils ,GlcNAc, N-acetyl-β-D-glucosamine ,HOCl, hypochlorous acid ,MPO, myeloperoxidase ,N-glycosylation ,Biochemistry ,SN, maturing neutrophil with segmented nuclei ,chemistry.chemical_compound ,N-linked glycosylation ,Sp granule, specific granule ,PMN-nMPO, myeloperoxidase from derived from resting (circulating) neutrophils ,Sp-MPO, specific granule-resident MPO ,Se/Pl-MPO, secretory vesicle/plasma membrane-resident MPO ,CD, circular dichroism ,Se/Pl, secretory vesicle and plasma membrane fraction ,PMN, polymorphonuclear cell (neutrophil) ,biology ,granulopoiesis ,LDH, lactate dehydrogenase ,LFQ, label-free quantitation ,Glycopeptides ,neutrophil ,RMSF, root mean squared fluctuation ,Editors' Pick ,MD, molecular dynamics ,inhibition ,Cell biology ,ceruloplasmin ,myeloperoxidase ,BN, band neutrophil ,Man, α/β-D-mannose ,Myeloperoxidase ,H2O2, hydrogen peroxide ,PGC, porous graphitised carbon ,nMPO, neutrophil-derived myeloperoxidase (unfractionated) ,Research Article ,Glycan ,Endo H, endoglycosidase H ,TMB, 3,3ʹ,5,5ʹ-tetramethylbenzidine ,Ge-MPO, gelatinase granule-resident MPO ,Cytoplasmic Granules ,Granulopoiesis ,ER, endoplasmic reticulum ,MOI, multiplicity-of-infection ,03 medical and health sciences ,Azurophilic granule ,Endoglycosidase H ,Polysaccharides ,PDB, Protein Data Bank ,Humans ,RMSD, root mean squared deviation ,KRG buffer, Krebs-Ringer buffer with glucose ,Molecular Biology ,Az granule, azurophilic granule ,Dg-MPO, degranulated MPO ,Peroxidase ,PM, promyelocyte ,degranulation ,030102 biochemistry & molecular biology ,activity ,LC-MS/MS, liquid chromatography tandem mass spectrometry ,MM, metamyelocyte ,Cell Biology ,granule ,Fuc (F), α-L-fucose ,EIC, extracted ion chromatogram ,CytB/I, cytochalasin B and ionomycin ,030104 developmental biology ,Specific granule ,chemistry ,biology.protein ,AUC, area-under-the-curve ,biosynthesis ,SD, standard deviation ,Ge granule, gelatinase granule - 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.
- Published
- 2020