Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
| Autor: | 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, Sayantani Chatterjee |
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| Rok vydání: | 2020 |
| Předmět: |
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 |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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