Busse Marta, Feta Almir, Presto Jenny, Wilén Maria, Grønning Mona, Kjellén Lena, Kusche-Gullberg Marion
Department of Biomedicine, University of Bergen, Jonas Lies vei 91, Bergen, Norway.
J Biol Chem. 2007 Nov 9;282(45):32802-10. doi: 10.1074/jbc.M703560200. Epub 2007 Aug 29.
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 glycosyl-transferase 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 EXT2-Y419X mutant.
外生骨疣素（EXT）基因家族编码参与硫酸乙酰肝素生物合成的糖基转移酶。该家族目前已克隆出五个人类成员：EXT1、EXT2、EXTL1、EXTL2和EXTL3。据信，EXT1和EXT2形成一种位于高尔基体的异源寡聚复合物，催化硫酸乙酰肝素生物合成中的链延长步骤，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何种反应尚不清楚。我们使用基因沉默策略来研究EXT1、EXT2和EXTL3在硫酸乙酰肝素链延长中的作用。将针对人类EXT1、EXT2或EXTL3 mRNA的小干扰RNA（siRNA）导入人胚肾293细胞。与转染对照siRNA的细胞相比，转染EXT1或EXT2 siRNA的细胞合成的硫酸乙酰肝素链较短，而转染EXTL3 siRNA的细胞合成的链较长。我们还构建了过表达EXT蛋白的人类细胞系。EXT1的过表达导致硫酸乙酰肝素链长度增加，在共表达EXT2的细胞中更为明显，而单独过表达EXT2对硫酸乙酰肝素链延长没有可检测到的影响。EXT1或EXT2中的突变与遗传性多发性外生骨疣有关，这是一种人类疾病，其特征是在骨骺生长板处形成软骨帽状骨赘。为了进一步研究EXT2的作用，我们构建了过表达突变型EXT2的人类细胞系。其中一个突变，EXT2-Y419X，导致一种截短蛋白。有趣的是，EXT2-Y419X突变体不具备野生型EXT2与EXT1共同增强硫酸乙酰肝素链长度的能力。
