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 remain 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.

中文翻译：

---

超截短的 Asn355- 和 Asn391- 聚糖调节中性粒细胞颗粒髓过氧化物酶的活性

髓过氧化物酶 (MPO) 通过生成反应性氧化产物，在中性粒细胞介导的免疫中发挥重要作用。复杂碳水化合物在离散位点装饰 MPO，但它们的功能相关性仍然难以捉摸。为此，我们表征了中性粒细胞 MPO (nMPO) 的结构-生物合成-活性关系。质谱分析表明，nMPO 携带特征性的未加工聚糖和超截短聚糖。发现位于 MPO 二聚化区域的 Asn355/Asn391 糖基化位点和 Asn323-/Asn483-聚糖的封闭会影响局部聚糖加工，从而提供位点特异性 nMPO 糖基化的分子基础。天然质谱、质量光度测定和糖肽分析揭示了二原聚体 nMPO 的显着分子复杂性，该二原聚体由异质糖基化、氧化、氯化和多肽截短变体以及先前未报道的低丰度单原聚体引起。成熟、成熟、颗粒分离和病原体刺激的中性粒细胞的纵向分析表明，nMPO 在粒细胞生成过程中动态表达，在颗粒中分布不均匀，并在激活时脱颗粒。我们还表明，proMPO 到 MPO 的成熟发生在早期/中期粒细胞生成过程中。虽然在不同条件下观察到类似的整体 MPO 糖基化，但保守的 Asn355-/Asn391-位点显示出升高的聚糖超截短，这与不同颗粒群体中 MPO 较高的酶活性相关。Asn355-/Asn391-聚糖的酶促修剪概括了活性增益，并表明在这些位置携带超截短聚糖的 nMPO 相对于天然 nMPO 表现出更高的热稳定性、多肽可及性和铜蓝蛋白介导的抑制潜力。最后，分子模型表明，位于 MPO-铜蓝蛋白界面的超截短 Asn355-聚糖对于不间断的抑制至关重要。在这里，通过创新和全面的方法，我们报告了 MPO 聚糖的新功能作用，为中性粒细胞介导的免疫提供了新的见解。