Department of Chemistry, Yale University, New Haven, CT, 06511, USA.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, 92093, USA.
Nat Commun. 2023 Oct 4;14(1):6169. doi: 10.1038/s41467-023-41756-y.
Mucin-domain glycoproteins are densely O-glycosylated and play critical 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 regulators in cellular immunity. However, their dense O-glycosylation remains enigmatic, primarily due to the challenges associated with studying mucin domains. Here, we demonstrate that the mucinase SmE has a unique ability to cleave at residues bearing very complex glycans. SmE enables improved mass spectrometric analysis of several mucins, including the entire TIM family. With this information in-hand, we perform molecular dynamics (MD) simulations of TIM-3 and -4 to understand how glycosylation affects structural features of these proteins. Finally, we use these models to investigate the functional relevance of glycosylation for TIM-3 function and ligand binding. Overall, we present a powerful workflow to better understand the detailed molecular structures and functions of the mucinome.
粘蛋白结构域糖蛋白高度 O-糖基化，在多种生物学功能中发挥关键作用。特别是 T 细胞免疫球蛋白和粘蛋白结构域包含蛋白家族（TIM-1、-3、-4）装饰免疫细胞，并作为细胞免疫的关键调节剂。然而，它们高度复杂的 O-糖基化仍然很神秘，主要是因为研究粘蛋白结构域相关的挑战。在这里，我们证明粘蛋白酶 SmE 具有独特的在含有非常复杂聚糖的残基上切割的能力。SmE 使包括整个 TIM 家族在内的几种粘蛋白的质谱分析得到改善。有了这些信息，我们对 TIM-3 和 -4 进行分子动力学 (MD) 模拟，以了解糖基化如何影响这些蛋白质的结构特征。最后，我们使用这些模型来研究糖基化对 TIM-3 功能和配体结合的功能相关性。总的来说，我们提出了一种强大的工作流程，以更好地了解粘蛋白组的详细分子结构和功能。
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