Hyper-truncated Asn355- And Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
Journal article, 2021

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.


Harry C. Tjondro

Macquarie University

Julian Ugonotti

Macquarie University

Rebeca Kawahara

Macquarie University

Sayantani Chatterjee

Macquarie University

Siyun Chen

University of Oxford

Fabian Soltermann

University of Oxford

Hannes Hinneburg

Macquarie University

Benjamin L. Parker

University of Melbourne

V. Venkatakrishnan

University of Gothenburg

Ian Loke

Cordlife Group

Regis Dieckmann

University of Gothenburg

Oliver C. Grant

University of Georgia

Johan Bylund

University of Gothenburg

Alison Rodger

Macquarie University

Robert J. Woods

University of Georgia

Anna Karlsson-Bengtsson

Chalmers, Biology and Biological Engineering, Chemical Biology

University of Gothenburg

Weston B. Struwe

University of Oxford

Morten Thaysen-Andersen

Macquarie University

Journal of Biological Chemistry

0021-9258 (ISSN) 1083-351X (eISSN)

Vol. 296 016342

The glycome of emergency neutrophils and implications in sepsis

Swedish Research Council (VR) (2018-03077), 2019-01-01 -- 2024-12-31.

Subject Categories

Biochemistry and Molecular Biology

Other Basic Medicine

Medicinal Chemistry





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