Glycosite mapping and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

Mucin-domain glycoproteins are densely O-glycosylated and play key roles in a host of biological functions. In particular, the T-cell immunoglobulin and mucin-domain containing family of proteins (TIM-1, 3, 4) decorate immune cells and act as key checkpoint inhibitors in cancer. However, their dense O-glycosylation remains enigmatic both in glycoproteomic landscape and structural dynamics, primarily due to the challenges associated with studying mucin domains. Here, we present a mucinase (SmE) and demonstrate its ability to selectively cleave along the mucin glycoprotein backbone, similar to others of its kind. Unlike other mucinases, though, SmE harbors the unique ability to cleave at residues bearing extremely complex glycans, which enabled improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we performed molecular dynamic (MD) simulations of TIM-3 and 4 to demonstrate how glycosylation affects structural features of these proteins. Overall, we present a powerful workflow to better understand the detailed molecular structures of the mucinome.

粘蛋白结构域糖蛋白（Mucin-domain glycoproteins）普遍带有高度密集的O-糖基化修饰，在众多生物学过程中发挥关键作用。其中，T细胞免疫球蛋白粘蛋白结构域蛋白家族（TIM-1、3、4）分布于免疫细胞表面，作为癌症免疫中的关键免疫检查点抑制剂（checkpoint inhibitor）发挥功能。然而，由于粘蛋白结构域的研究存在诸多技术挑战，这类蛋白的高度密集O-糖基化修饰在糖蛋白质组学图谱与结构动力学层面仍存在诸多未解之谜。本研究报道了粘蛋白酶（mucinase）SmE，并证实其可沿粘蛋白糖蛋白骨架实现特异性切割，这一特性与同类粘蛋白酶一致。但与其他粘蛋白酶不同的是，SmE具备独特的切割活性，可在携带极复杂糖基化修饰的氨基酸残基处完成切割，这一特性显著提升了包括完整TIM家族在内的多种粘蛋白的质谱分析效能。依托上述研究成果，我们针对TIM-3与TIM-4开展了分子动力学（MD）模拟实验，阐明了糖基化修饰对这两类蛋白结构特征的调控机制。综上，本研究构建了一套高效可靠的研究流程，可为深入解析粘蛋白组（mucinome）的精细分子结构提供有力支撑。