Siegbahn Anna, Thorsheim Karin, Ståhle Jonas, Manner Sophie, Hamark Christoffer, Persson Andrea, Tykesson Emil, Mani Katrin, Westergren-Thorsson Gunilla, Widmalm Göran, Ellervik Ulf
Center for Analysis and Synthesis, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden.
Org Biomol Chem. 2015 Mar 21;13(11):3351-62. doi: 10.1039/c4ob02632b.
Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase β4GalT7. Some β-d-xylosides, such as 2-naphthyl β-d-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for β4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for β4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with β-glycosidic linkages function as good substrates for β4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by β4GalT7 is dependent on subtle differences in orientation of the xylose moiety.
蛋白聚糖（PGs）是由长链线性多糖即糖胺聚糖（GAG）链通过碳水化合物木糖共价连接到核心蛋白上组成的大分子。GAG链的生物合成始于核心蛋白的木糖基化，随后由半乳糖基转移酶β4GalT7进行半乳糖基化。一些β - D - 木糖苷，如2 - 萘基β - D - 木吡喃糖苷，可以作为β4GalT7的受体底物诱导GAG合成，从而也与核心蛋白上的GAG合成竞争。在此，我们利用酶促测定、细胞研究和分子对接模拟，展示了β4GalT7和具有芳香苷元修饰的木糖苷的构效关系。结果表明，苷元位于酶活性位点的外侧，并且相当大的苷元是可以接受的。通过用连接子将芳香苷元与木糖部分分开，观察到随着连接子长度增加半乳糖基化增加的趋势。半乳糖基化受芳香骨架中取代基的种类和位置影响，一般来说，只有具有β - 糖苷键的木糖苷才是β4GalT7的良好底物。我们还表明，将异头氧替换为硫会增加木糖苷的半乳糖基化能力，但替换为碳则会降低。最后，我们提出β4GalT7半乳糖基化的反应动力学取决于木糖部分取向的细微差异。
