Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation
Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain elongation
复制标题
DOI：
10.1074/jbc.m703560200
复制
发表时间：
2007-11-09
影响因子：
4.8
通讯作者：
Kusche-Gullberg, Marion
中科院分区：
文献类型：
--
作者：
Busse, Marta;Feta, Almir;Kusche-Gullberg, Marion
外泌素（EXT）家族基因编码参与硫酸肝素生物合成的糖基转移酶。迄今为止，已克隆出该家族的5个人类成员：EXT1、EXT2、EXTL1、EXTL2和EXTL3。EXT1和EXT2被认为形成高尔基异聚物复合物，催化硫酸肝素生物合成中的链延伸步骤，而EXTL蛋白在体外表现出重叠的糖基转移酶活性，因此它们在体内催化的反应并不明显。我们使用基因沉默策略来研究EXT1、EXT2和EXTL3在硫酸肝素链延伸中的作用。将针对人EXT1、EXT2或EXTL3 mrna的小干扰rna （sirna）引入人胚胎肾293细胞。与转染对照siRNA的细胞相比，转染EXT1或EXT2 siRNA的细胞合成的硫酸肝素链较短，而转染EXTL3 siRNA的细胞合成的硫酸肝素链较长。我们还生成了过表达EXT蛋白的人细胞系。过表达EXT1导致HS链长度增加，在共表达EXT2的细胞中更为明显，而单独过表达EXT2对硫酸肝素链伸长无明显影响。EXT1或EXT2的突变与遗传性多发性外生骨病有关，多发性外生骨病是一种人类疾病，其特征是在骨骺生长板处形成软骨覆盖的骨外生物。为了进一步研究EXT2的作用，我们构建了过表达EXT2突变体的人细胞系。其中一个突变，EXT2-Y419X，导致了一个截断的蛋白质。有趣的是，野生型EXT2与EXT1一起增加HS链长度的能力并没有被EXT2Y419X突变体所共享。
The exostosin (EXT) family of genes encodes glycosyltransferases involved in heparan sulfate biosynthesis. Five human members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are believed to form a Golgi-located hetero-oligomeric complex that catalyzes the chain elongation step in heparan sulfate biosynthesis, whereas the EXTL proteins exhibit overlapping glycosyltransferase activities in vitro, so that it is not apparent what reactions they catalyze in vivo. We used gene-silencing strategies to investigate the roles of EXT1, EXT2, and EXTL3 in heparan sulfate chain elongation. Small interfering RNAs (siRNAs) directed against the human EXT1, EXT2, or EXTL3 mRNAs were introduced into human embryonic kidney 293 cells. Compared with cells transfected with control siRNA, those transfected with EXT1 or EXT2 siRNA synthesized shorter heparan sulfate chains, and those transfected with EXTL3 siRNA synthesized longer chains. We also generated human cell lines overexpressing the EXT proteins. Overexpression of EXT1 resulted in increased HS chain length, which was even more pronounced in cells coexpressing EXT2, whereas overexpression of EXT2 alone had no detectable effect on heparan sulfate chain elongation. Mutations in either EXT1 or EXT2 are associated with hereditary multiple exostoses, a human disorder characterized by the formation of cartilage-capped bony outgrowths at the epiphyseal growth plates. To further investigate the role of EXT2, we generated human cell lines overexpressing mutant EXT2. One of the mutations, EXT2-Y419X, resulted in a truncated protein. Interestingly, the capacity of wild type EXT2 to enhance HS chain length together with EXT1 was not shared by the EXT2Y419X mutant.