The Armenise-Harvard Laboratory of Structural Biology, Department of Biology and Biotechnology, University of Pavia, Via Ferrata 9A, 27100 Pavia, Italy.
NSW Health Pathology Randwick Genomics Laboratory, Prince of Wales Hospital, Sydney, NSW 2031, Australia.
Int J Mol Sci. 2023 Jul 7;24(13):11213. doi: 10.3390/ijms241311213.
Hydroxylysine glycosylations are post-translational modifications (PTMs) essential for the maturation and homeostasis of fibrillar and non-fibrillar collagen molecules. The multifunctional collagen lysyl hydroxylase 3 (LH3/PLOD3) and the collagen galactosyltransferase GLT25D1 are the human enzymes that have been identified as being responsible for the glycosylation of collagen lysines, although a precise description of the contribution of each enzyme to these essential PTMs has not yet been provided in the literature. LH3/PLOD3 is thought to be capable of performing two chemically distinct collagen glycosyltransferase reactions using the same catalytic site: an inverting beta-1,O-galactosylation of hydroxylysines (Gal-T) and a retaining alpha-1,2-glucosylation of galactosyl hydroxylysines (Glc-T). In this work, we have combined indirect luminescence-based assays with direct mass spectrometry-based assays and molecular structure studies to demonstrate that LH3/PLOD3 only has Glc-T activity and that GLT25D1 only has Gal-T activity. Structure-guided mutagenesis confirmed that the Glc-T activity is defined by key residues in the first-shell environment of the glycosyltransferase catalytic site as well as by long-range contributions from residues within the same glycosyltransferase (GT) domain. By solving the molecular structures and characterizing the interactions and solving the molecular structures of human LH3/PLOD3 in complex with different UDP-sugar analogs, we show how these studies could provide insights for LH3/PLOD3 glycosyltransferase inhibitor development. Collectively, our data provide new tools for the direct investigation of collagen hydroxylysine PTMs and a comprehensive overview of the complex network of shapes, charges, and interactions that enable LH3/PLOD3 glycosyltransferase activities, expanding the molecular framework and facilitating an improved understanding and manipulation of glycosyltransferase functions in biomedical applications.
羟赖氨酸糖基化是纤维状和非纤维状胶原蛋白分子成熟和动态平衡所必需的翻译后修饰（PTMs）。多功能胶原蛋白赖氨酰羟化酶 3（LH3/PLOD3）和胶原蛋白半乳糖基转移酶 GLT25D1 是已被确定负责胶原蛋白赖氨酸糖基化的人类酶，尽管文献中尚未提供每种酶对这些必需 PTM 的贡献的精确描述。LH3/PLOD3 被认为能够使用相同的催化位点进行两种化学上不同的胶原蛋白糖基转移酶反应：羟赖氨酸的反式β-1，O-半乳糖基化（Gal-T）和半乳糖基羟赖氨酸的保留α-1，2-葡萄糖基化（Glc-T）。在这项工作中，我们将间接基于发光的测定法与直接基于质谱的测定法和分子结构研究相结合，证明 LH3/PLOD3 仅具有 Glc-T 活性，而 GLT25D1 仅具有 Gal-T 活性。结构引导的突变证实，Glc-T 活性由糖基转移酶催化位点的第一壳层环境中的关键残基以及同一糖基转移酶（GT）域内的长程残基贡献定义。通过解决人 LH3/PLOD3 与不同 UDP-糖类似物复合物的分子结构并表征相互作用，我们展示了这些研究如何为 LH3/PLOD3 糖基转移酶抑制剂的开发提供见解。总之，我们的数据为直接研究胶原蛋白羟赖氨酸 PTM 提供了新工具，并全面概述了使 LH3/PLOD3 糖基转移酶活性成为可能的形状、电荷和相互作用的复杂网络，扩展了分子框架，并促进了对糖基转移酶功能在生物医学应用中的更好理解和操纵。
