Institut de Biologie Structurale, UMR 5075, University Grenoble Alpes, CNRS, CEA, 38000, Grenoble, France.
Université Paris-Saclay, CNRS, Institut de chimie moléculaire et des matériaux d'Orsay, 91405, Orsay, France.
Nat Commun. 2022 Nov 19;13(1):7110. doi: 10.1038/s41467-022-34882-6.
Heparan sulfates are complex polysaccharides that mediate the interaction with a broad range of protein ligands at the cell surface. A key step in heparan sulfate biosynthesis is catalyzed by the bi-functional glycosyltransferases EXT1 and EXT2, which generate the glycan backbone consisting of repeating N-acetylglucosamine and glucuronic acid units. The molecular mechanism of heparan sulfate chain polymerization remains, however, unknown. Here, we present the cryo-electron microscopy structure of human EXT1-EXT2, which reveals the formation of a tightly packed hetero-dimeric complex harboring four glycosyltransferase domains. A combination of in vitro and in cellulo mutational studies is used to dissect the functional role of the four catalytic sites. While EXT1 can catalyze both glycosyltransferase reactions, our results indicate that EXT2 might only have N-acetylglucosamine transferase activity. Our findings provide mechanistic insight into heparan sulfate chain elongation as a nonprocessive process and lay the foundation for future studies on EXT1-EXT2 function in health and disease.
硫酸乙酰肝素是一种复杂的多糖，在细胞表面与广泛的蛋白配体相互作用。硫酸乙酰肝素生物合成的关键步骤由多功能糖基转移酶 EXT1 和 EXT2 催化，该酶生成由重复的 N-乙酰葡萄糖胺和葡萄糖醛酸单元组成的聚糖主链。然而，硫酸乙酰肝素链聚合的分子机制仍然未知。在这里，我们展示了人 EXT1-EXT2 的冷冻电子显微镜结构，揭示了形成一个紧密堆积的异二聚体复合物，其中包含四个糖基转移酶结构域。体外和细胞内突变研究的结合用于剖析四个催化位点的功能作用。虽然 EXT1 可以催化两种糖基转移酶反应，但我们的结果表明 EXT2 可能只具有 N-乙酰葡萄糖胺转移酶活性。我们的发现为硫酸乙酰肝素链延伸作为非连续过程提供了机制上的见解，并为 EXT1-EXT2 在健康和疾病中的功能的未来研究奠定了基础。
