Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.
Position-specific N- and O-glycosylation of the reactive center loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin.
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DOI：
10.1016/j.jbc.2023.105519
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发表时间：
2024-01
影响因子：
4.8
通讯作者：
Thaysen-Andersen, Morten
中科院分区：
文献类型：
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作者：
Chernykh, Anastasia;Abrahams, Jodie L;Grant, Oliver C;Kambanis, Lucas;Sumer-Bayraktar, Zeynep;Ugonotti, Julian;Kawahara, Rebeca;Corcilius, Leo;Payne, Richard J;Woods, Robert J;Thaysen-Andersen, Morten
皮质类固醇结合球蛋白（CBG）通过中性粒细胞弹性蛋白酶（NE）对暴露的反应中心环（RCL）进行蛋白水解，将抗炎皮质醇递送至炎症组织。我们先前证明了RCL定位的Asn 347连接的N-聚糖影响NE蛋白水解，但是仍然需要RCL糖基化的全面结构-功能表征以更好地理解CBG糖生物学。在此，我们首先进行了RCL中心的血清来源的CBG的糖分析，以阐明Asn 347-聚糖，然后使用分子动力学模拟研究它们对NE蛋白水解的影响。重要的是，我们还在靠近NE靶向的Val 344-Thr 345切割位点的血清CBG的四个RCL位点（Thr 338/Thr 342/Thr 345/Ser 350）上鉴定了O-糖基化（二/唾液酸T）。通过实验观察到RCL上仅涉及Asn 347和Thr 338糖基化的限制性N-和O-聚糖共存模式，并通过CBG-GalNAc-转移酶（GalNAc-T）与各种RCL聚糖复合物的建模进行计算机模拟支持。分别由肝脏和胆囊大量表达的GalNAc-T2和GalNAc-T3在体外显示出将GalNAc（Tn）转移到多个RCL位点的能力，表明它们参与RCL O-糖基化。然后，重组CBG用于通过纵向NE中心蛋白水解实验确定RCL O-糖基化的作用，这表明修饰Thr 345的唾液酸聚糖（disialyl T）和去唾液酸聚糖（T）都抑制NE蛋白水解。合成的RCL O-糖肽通过显示Thr 345-Tn和Thr 342-Tn分别赋予针对NE切割的强保护和中等保护来扩展这些发现。分子动力学证实，短Thr 345连接的O-聚糖废除NE相互作用。总之，我们报告的生物学相关的CBG RCL糖基化事件，这提高了我们对皮质醇输送到炎症组织的机制的理解。
Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through proteolysis of an exposed reactive center loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localized Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure–function characterization of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we first performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics simulations to study their impact on NE proteolysis. Importantly, we also identified O-glycosylation (di/sialyl T) across four RCL sites (Thr338/Thr342/Thr345/Ser350) of serum CBG close to the NE-targeted Val344–Thr345 cleavage site. A restricted N- and O-glycan co-occurrence pattern on the RCL involving exclusively Asn347 and Thr338 glycosylation was experimentally observed and supported in silico by modeling of a CBG–GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and GalNAc-T3 abundantly expressed by liver and gall bladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialoglycans (disialyl T) and asialoglycans (T) decorating Thr345 inhibit NE proteolysis. Synthetic RCL O-glycopeptides expanded on these findings by showing that Thr345-Tn and Thr342-Tn confer strong and moderate protection against NE cleavage, respectively. Molecular dynamics substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on biologically relevant CBG RCL glycosylation events, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.