Izumikawa Tomomi, Koike Toshiyasu, Shiozawa Shoko, Sugahara Kazuyuki, Tamura Jun-ichi, Kitagawa Hiroshi
Department of Biochemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan.
J Biol Chem. 2008 Apr 25;283(17):11396-406. doi: 10.1074/jbc.M707549200. Epub 2008 Mar 3.
Recently, we demonstrated that chondroitin polymerization is achieved by any two combinations of human chondroitin synthase-1 (ChSy-1), ChSy-2 (chondroitin sulfate synthase 3, CSS3), and chondroitin-polymerizing factor (ChPF). Although an additional ChSy family member, called chondroitin sulfate glucuronyltransferase (CSGlcA-T), has been identified, its involvement in chondroitin polymerization remains unclear because it possesses only glucuronyltransferase II activity responsible for the elongation of chondroitin sulfate (CS) chains. Herein, we report that CSGlcA-T exhibits polymerization activity on alpha-thrombomodulin bearing the truncated linkage region tetrasaccharide through its interaction with ChSy-1, ChSy-2 (CSS3), or ChPF, and the chain length of chondroitin formed by the co-expressed proteins in various combinations is different. In addition, ChSy family members co-expressed in various combinations exhibited distinct but overlapping acceptor substrate specificities toward the two synthetic acceptor substrates, GlcUAbeta1-3Galbeta1-O-naphthalenemethanol and GlcUAbeta1-3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, both of which share the disaccharide sequence with the glycosaminoglycan-protein linkage region tetrasaccharide. Moreover, overexpression of CSGlcA-T increased the amount of CS in HeLa cells, whereas the RNA interference of CSGlcA-T resulted in a reduction of the amount of CS in the cells. Furthermore, the analysis using the CSGlcA-T mutant that lacks any glycosyltransferase activity but interacts with other ChSy family members showed that the glycosyltransferase activity of CSGlcA-T plays an important role in chondroitin polymerization. Overall, these results suggest that chondroitin polymerization is achieved by multiple combinations of ChSy-1, ChSy-2, CSGlcA-T, and ChPF and that each combination may play a unique role in the biosynthesis of CS. Based on these results, we renamed CSGlcA-T chondroitin synthase-3 (ChSy-3).
最近，我们证明了软骨素聚合可通过人软骨素合酶-1（ChSy-1）、ChSy-2（硫酸软骨素合酶3，CSS3）和软骨素聚合因子（ChPF）的任意两种组合来实现。尽管已鉴定出另一个ChSy家族成员，称为硫酸软骨素葡萄糖醛酸基转移酶（CSGlcA-T），但其在软骨素聚合中的作用仍不清楚，因为它仅具有负责硫酸软骨素（CS）链延长的葡萄糖醛酸基转移酶II活性。在此，我们报告CSGlcA-T通过与ChSy-1、ChSy-2（CSS3）或ChPF相互作用，对带有截短连接区四糖的α-血栓调节蛋白表现出聚合活性，并且由共表达蛋白以各种组合形成的软骨素链长度不同。此外，以各种组合共表达的ChSy家族成员对两种合成受体底物GlcUAbeta1-3Galbeta1-O-萘甲醇和GlcUAbeta1-3Galbeta1-O-C(2)H(4)NH-苄氧羰基表现出不同但重叠的受体底物特异性，这两种底物均与糖胺聚糖-蛋白质连接区四糖共享二糖序列。此外，CSGlcA-T的过表达增加了HeLa细胞中CS的量，而CSGlcA-T的RNA干扰导致细胞中CS的量减少。此外，使用缺乏任何糖基转移酶活性但与其他ChSy家族成员相互作用的CSGlcA-T突变体进行的分析表明，CSGlcA-T的糖基转移酶活性在软骨素聚合中起重要作用。总体而言，这些结果表明软骨素聚合是通过ChSy-1、ChSy-2、CSGlcA-T和ChPF的多种组合实现的，并且每种组合可能在CS的生物合成中发挥独特作用。基于这些结果，我们将CSGlcA-T重新命名为软骨素合酶-3（ChSy-3）。
