Gulberti Sandrine, Lattard Virginie, Fondeur Magali, Jacquinet Jean-Claude, Mulliert Guillermo, Netter Patrick, Magdalou Jacques, Ouzzine Mohamed, Fournel-Gigleux Sylvie
UMR 7561 CNRS-Université Henri Poincaré Nancy 1, Faculté de Médecine, 54505 Vandoeuvre-lès-Nancy, France.
J Biol Chem. 2005 Jan 14;280(2):1417-25. doi: 10.1074/jbc.M411552200. Epub 2004 Nov 1.
We determined whether the two major structural modifications, i.e. phosphorylation and sulfation of the glycosaminoglycan-protein linkage region (GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1), govern the specificity of the glycosyltransferases responsible for the biosynthesis of the tetrasaccharide primer. We analyzed the influence of C-2 phosphorylation of Xyl residue on human beta1,4-galactosyltransferase 7 (GalT-I), which catalyzes the transfer of Gal onto Xyl, and we evaluated the consequences of C-4/C-6 sulfation of Galbeta1-3Gal (Gal2-Gal1) on the activity and specificity of beta1,3-glucuronosyltransferase I (GlcAT-I) responsible for the completion of the glycosaminoglycan primer sequence. For this purpose, a series of phosphorylated xylosides and sulfated C-4 and C-6 analogs of Galbeta1-3Gal was synthesized and tested as potential substrates for the recombinant enzymes. Our results revealed that the phosphorylation of Xyl on the C-2 position prevents GalT-I activity, suggesting that this modification may occur once Gal is attached to the Xyl residue of the nascent oligosaccharide linkage. On the other hand, we showed that sulfation on C-6 position of Gal1 of the Galbeta1-3Gal analog markedly enhanced GlcAT-I catalytic efficiency and we demonstrated the importance of Trp243 and Lys317 residues of Gal1 binding site for enzyme activity. In contrast, we found that GlcAT-I was unable to use digalactosides as acceptor substrates when Gal1 was sulfated on C-4 position or when Gal2 was sulfated on both C-4 and C-6 positions. Altogether, we demonstrated that oligosaccharide modifications of the linkage region control the specificity of the glycosyltransferases, a process that may regulate maturation and processing of glycosaminoglycan chains.
我们确定了两种主要的结构修饰，即糖胺聚糖 - 蛋白质连接区域（GlcAbeta1 - 3Galbeta1 - 3Galbeta1 - 4Xylbeta1）的磷酸化和硫酸化，是否控制负责四糖引物生物合成的糖基转移酶的特异性。我们分析了木糖残基C - 2位磷酸化对人β1,4 - 半乳糖基转移酶7（GalT - I）的影响，该酶催化半乳糖转移到木糖上，并且我们评估了Galbeta1 - 3Gal（Gal2 - Gal1）的C - 4/C - 6硫酸化对负责完成糖胺聚糖引物序列的β1,3 - 葡糖醛酸基转移酶I（GlcAT - I）的活性和特异性的影响。为此，合成了一系列磷酸化木糖苷以及Galbeta（1 - 3）Gal的硫酸化C - 4和C - 6类似物，并作为重组酶的潜在底物进行测试。我们的结果表明，木糖C - 2位的磷酸化会阻止GalT - I的活性，这表明这种修饰可能在半乳糖连接到新生寡糖连接区域的木糖残基后发生。另一方面，我们表明Galbeta1 - 3Gal类似物中Gal1的C - 6位硫酸化显著提高了GlcAT - I的催化效率，并且我们证明了Gal1结合位点的Trp243和Lys317残基对酶活性的重要性。相反，我们发现当Gal1在C - 4位硫酸化或Gal2在C - 4和C - 6位都硫酸化时，GlcAT - I无法将二半乳糖苷用作受体底物。总之，我们证明了连接区域的寡糖修饰控制糖基转移酶的特异性，这一过程可能调节糖胺聚糖链的成熟和加工。
