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Hyper-truncated Asn355- and Asn391-glycans modulate the activity of neutrophil granule myeloperoxidase
Harry C Tjondro1, Julian Ugonotti1, Rebeca Kawahara1
1Department of Molecular Sciences, Macquarie University, Sydney, New South Wales, Australia; Biomolecular Discovery Research Centre, Macquarie University, Sydney, New South Wales, Australia.
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.
Insights
Complex carbohydrates on neutrophil myeloperoxidase (MPO) impact its function. Hyper-truncated glycans at specific sites enhance MPO activity and stability, revealing new roles in immunity.
Area of Science:
- Immunology
- Glycobiology
- Proteomics
Background:
- Neutrophil myeloperoxidase (nMPO) is crucial for immunity, generating reactive oxidation products.
- The functional significance of complex carbohydrates (glycans) decorating nMPO remains largely unknown.
- Understanding nMPO glycosylation is key to elucidating its role in neutrophil function.
Purpose of the Study:
- To characterize the structure-biosynthesis-activity relationship of neutrophil myeloperoxidase (nMPO).
- To investigate the functional relevance of nMPO glycosylation, particularly at specific sites.
- To explore the dynamic expression and distribution of nMPO during granulopoiesis and neutrophil activation.
Main Methods:
- Mass spectrometry (native and glycopeptide profiling) to analyze nMPO structure and glycosylation.
- Mass photometry to assess molecular complexity.
- Longitudinal profiling of neutrophils at various maturation and activation states.
- Molecular modeling to understand glycan interactions.
Main Results:
- nMPO exhibits heterogeneous glycosylation, including under-processed and hyper-truncated glycans.
- Specific glycosylation sites (Asn355/Asn391) influence local glycan processing and correlate with higher MPO activity.
- Hyper-truncated glycans at Asn355/Asn391 enhance nMPO thermal stability, polypeptide accessibility, and ceruloplasmin inhibition.
- nMPO is dynamically expressed and distributed, with maturation occurring during granulopoiesis.
Conclusions:
- Novel functional roles for MPO glycans in neutrophil-mediated immunity have been identified.
- Hyper-truncated glycans at specific sites are critical for modulating nMPO activity and stability.
- These findings provide new insights into the regulation of neutrophil function and MPO's role in immunity.
