Molecular cloning of a chondroitin polymerizing factor that cooperates with chondroitin synthase for chondroitin polymerization
Molecular cloning of a chondroitin polymerizing factor that cooperates with chondroitin synthase for chondroitin polymerization
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DOI：
10.1074/jbc.m302493200
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发表时间：
2003-06-27
影响因子：
4.8
通讯作者：
Sugahara, K
中科院分区：
文献类型：
--
作者：
Kitagawa, H;Izumikawa, T;Sugahara, K
我们最近克隆了人软骨素合酶（ChSy），其表现出葡萄糖醛酸转移酶-II（GlcAT-II）和N-乙酰半乳糖胺转移酶-II（GalNAcT-II）活性，负责硫酸软骨素重复二糖单元的生物合成，但软骨素聚合并未在体外使用重组ChSy证明。我们在这里报告，软骨素聚合活性需要伴随表达的一种新的蛋白质命名为软骨素聚合因子（ChPF）与ChSy。人ChPF由775个氨基酸组成，具有II型跨膜蛋白拓扑结构。氨基酸序列与人ChSy的氨基酸序列有23%的同源性。在COS-1细胞中可溶性重组形式的蛋白质的表达产生了几乎没有GlcAT-II或GalNAcT-II活性的蛋白质。与此相反，共表达的ChPF和ChSy产生显着增强的糖基转移酶的活动，而简单的混合的两个单独表达的蛋白质没有。此外，使用UDP-葡萄糖醛酸（GlcUA）和UDP-N-乙酰半乳糖胺（GalNAc）作为糖供体，在α-血栓调节蛋白的所谓糖胺聚糖-蛋白质连接区四糖序列上证明了软骨素聚合。这些结果表明，ChPF作为一个特定的激活因子的ChSy在软骨素聚合。ChPF的编码区被分为四个离散的外显子，并定位于染色体2 q35-q36。北方印迹分析显示，ChPF基因在不同的人体组织中表现出明显不同的表达模式，这与ChSy的表达模式相似。因此，ChPF是哺乳动物ChSy的软骨素聚合活性所必需的。
We recently cloned human chondroitin synthase (ChSy) exhibiting the glucuronyltransferase-II (GlcAT-II) and N-acetylgalactosaminyltransferase-II (GalNAcT-II) activities responsible for the biosynthesis of repeating disaccharide units of chondroitin sulfate, but chondroitin polymerization was not demonstrated in vitro using the recombinant ChSy. We report here that the chondroitin polymerizing activity requires concomitant expression of a novel protein designated chondroitin polymerizing factor (ChPF) with ChSy. The human ChPF consists of 775 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 23% identity to that of human ChSy. The expression of a soluble recombinant form of the protein in COS-1 cells produced a protein with little GlcAT-II or GalNAcT-II activity. In contrast, coexpression of the ChPF and ChSy yielded markedly augmented glycosyltransferase activities, whereas simple mixing of the two separately expressed proteins did not. Moreover, using both UDP-glucuronic acid (GlcUA) and UDP-N-acetylgalactosamine (GalNAc) as sugar donors, chondroitin polymerization was demonstrated on the so-called glycosaminoglycan-protein linkage region tetrasaccharide sequence of alpha-thrombomodulin. These results suggested that the ChPF acts as a specific activating factor for ChSy in chondroitin polymerization. The coding region of the ChPF was divided into four discrete exons and localized to chromosome 2q35-q36. Northern blot analysis revealed that the ChPF gene exhibited a markedly different expression pattern among various human tissues, which was similar to that of ChSy. Thus, the ChPF is required for chondroitin polymerizing activity of mammalian ChSy.