Position-specific N- and O-glycosylation of the reactive centre loop impacts neutrophil elastase-mediated proteolysis of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) delivers anti-inflammatory cortisol to inflamed tissues through the proteolytic cleavage of an exposed reactive centre loop (RCL) by neutrophil elastase (NE). We previously demonstrated that RCL-localised Asn347-linked N-glycans impact NE proteolysis, but a comprehensive structure-function characterisation of the RCL glycosylation is still required to better understand CBG glycobiology. Herein, we firstly performed RCL-centric glycoprofiling of serum-derived CBG to elucidate the Asn347-glycans and then used molecular dynamics (MD) simulations to study their impact on NE proteolysis. Importantly, we also identified novel 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 sites was experimentally observed and supported in silico by modelling of a CBG-GalNAc-transferase (GalNAc-T) complex with various RCL glycans. GalNAc-T2 and -T3 abundantly expressed by liver and gallbladder, respectively, showed in vitro a capacity to transfer GalNAc (Tn) to multiple RCL sites suggesting their involvement in RCL O-glycosylation. Recombinant CBG (rCBG) was then used to determine roles of RCL O-glycosylation through longitudinal NE-centric proteolysis experiments, which demonstrated that both sialo- (disialyl T) and asialo-glycans (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. MD substantiated that short Thr345-linked O-glycans abrogate NE interactions. In conclusion, we report on strategically-positioned and biologically-relevant CBG RCL glycans, which improve our understanding of mechanisms governing cortisol delivery to inflamed tissues.

皮质类固醇结合球蛋白（Corticosteroid-binding globulin, CBG）可通过中性粒细胞弹性蛋白酶（neutrophil elastase, NE）对暴露的反应中心环（reactive centre loop, RCL）进行蛋白水解切割，将抗炎皮质醇递送至炎症组织。我们此前的研究证实，定位于RCL的天冬酰胺347连接的N-聚糖会影响NE的蛋白水解过程，但仍需对RCL糖基化开展全面的结构-功能表征，以进一步阐明CBG的糖生物学机制。本研究首先对血清来源的CBG进行以RCL为核心的糖谱分析，以明确天冬酰胺347连接的聚糖组成；随后借助分子动力学（molecular dynamics, MD）模拟，探究此类聚糖对NE蛋白水解过程的影响。值得注意的是，我们还在血清CBG的四个RCL位点（苏氨酸338、苏氨酸342、苏氨酸345、丝氨酸350）上发现了新型O-糖基化修饰（双唾液酸T抗原），该修饰紧邻NE的靶向切割位点缬氨酸344-苏氨酸345。实验中观察到RCL上存在受限的N-与O-聚糖共现模式，仅涉及天冬酰胺347与苏氨酸338两个位点；该结果通过计算机模拟构建结合不同RCL聚糖的CBG-N-乙酰半乳糖胺转移酶（GalNAc-transferase, GalNAc-T）复合物得到了验证。分别在肝脏与胆囊中高表达的GalNAc-T2与GalNAc-T3，在体外实验中可将GalNAc（Tn抗原）转移至多个RCL位点，提示二者参与了RCL的O-糖基化过程。随后我们利用重组CBG（recombinant CBG, rCBG）开展以NE为核心的纵向蛋白水解实验，以明确RCL O-糖基化的功能；结果显示，修饰于苏氨酸345的唾液酸化聚糖（双唾液酸T抗原）与去唾液酸化聚糖（T抗原）均可抑制NE的蛋白水解活性。合成的RCL O-糖肽进一步拓展了上述研究结论：苏氨酸345连接的Tn抗原与苏氨酸342连接的Tn抗原可分别对NE切割产生强效与中度的保护作用。分子动力学模拟证实，苏氨酸345连接的短链O-聚糖会阻断NE与CBG的相互作用。综上，本研究报道了具有策略性定位且具备生物学功能相关性的CBG RCL糖基化修饰，加深了我们对皮质醇向炎症组织递送机制的理解。