Biosynthesis of heparan sulfate in EXT1-deficient cells
Biosynthesis of heparan sulfate in EXT1-deficient cells
复制标题
DOI：
10.1042/bj20100101
复制
发表时间：
2010-06-15
影响因子：
4.1
通讯作者：
Kitagawa, Hiroshi
中科院分区：
文献类型：
--
作者：
Okada, Megumi;Nadanaka, Satomi;Kitagawa, Hiroshi
HS（硫酸乙酰肝素）由EXT 1和EXT 2基因（外生骨疣素1和2）编码的HS共聚物酶合成，所述EXT 1和EXT 2基因被称为遗传性多发性外生骨疣的致病基因，所述遗传性多发性外生骨疣是一种以多发性软骨肿瘤为特征的显性遗传性遗传性疾病。已经认为异源寡聚EXT 1-EXT 2复合物是聚合酶的生物学相关形式，并且EXT 1或EXT 2的靶向缺失导致HS合成的完全缺乏。在本文中，我们意外地发现，两个不同的细胞系缺陷EXT 1表达确实产生小，但显着量的HS链。在没有EXT 1的帮助下产生的HS链比与EXT 1和EXT 2一起形成的HS链短。此外，HS在EXT 1缺陷细胞中的生物合成被EXT 2或EXTL 2（EXT-like）的敲低显著阻断，但EXTL 3的敲低不显著阻断。然后，为了研究EXTL 2在EXT 1缺陷细胞中HS生物合成中的作用，我们专注于EXTL 2的GlcNAc（N-乙酰葡糖胺）转移酶活性，该活性通过将第一个GlcNAc转移到连接区来参与HS链的起始。虽然单独的EXT 2在合成的连接区类似物GlcUA β 1-3Gal β 1-O-C2 H4 NH-苄氧基羰基上不合成乙酰肝素聚合物，但单独的EXT 2在GlcNAc α 1-4GlcUA β 1-3Gal β 1-O-C2 H4 N-苄氧基羰基上显示出显著的聚合（其中GlcHA是葡糖醛酸并且Gal是半乳糖），其通过使用重组EXTL 2将GlcNAc残基转移到GlcU β 1-3Gal β 1-O-C2 H4 NH-苄氧基羰基上而产生。这些研究结果表明，转移的第一GlcNAc残基的连接区域的EXTL 2是至关重要的HS在细胞中缺乏EXT 1的生物合成所需的。
HS (heparan sulfate) is synthesized by HS co-polymerases encoded by the EXT1 and EXT2 genes (exostosin 1 and 2), which are known as causative genes for hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumours. It has been thought that the hetero-oligomeric EXT1-EXT2 complex is the biologically relevant form of the polymerase and that targeted deletion of either EXT1 or EXT2 leads to a complete lack of HS synthesis. In the present paper we show, unexpectedly, that two distinct cell lines defective in EXT1 expression indeed produce small but significant amounts of HS chains. The HS chains produced without the aid of EXT1 were shorter than HS chains formed in concert with EXT1 and EXT2. In addition, biosynthesis of HS in EXT1-defective cells was notably blocked by knockdown of either EXT2 or EXTL2 (EXT-like), but not of EXTL3. Then, to examine the roles of EXTL2 in the biosynthesis of HS in EXT1-deficient cells, we focused on the GlcNAc (N-acetylglucosamine) transferase activity of EXTL2, which is involved in the initiation of HS chains by transferring the first GlcNAc to the linkage region. Although EXT2 alone synthesized no heparan polymers on the synthetic linkage region analogue GlcUA beta 1-3Gal beta 1-O-C2H4NH-benzyloxycarbonyl, marked polymerization by EXT2 alone was demonstrated on GlcNAc alpha 1-4GlcUA beta 1-3Gal beta 1-O-C2H4N-benzyloxycarbonyl (where GlcHA is glucuronic acid and Gal is galactose), which was generated by transferring a GlcNAc residue using recombinant EXTL2 on to GlcU beta 1-3Gal beta 1-O-C2H4NH-benzyloxycarbonyl. These findings indicate that the transfer of the first GlcNAc residue to the linkage region by EXTL2 is critically required for the biosynthesis of HS in cells deficient in EXT1.