Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants.
Location of the glucuronosyltransferase domain in the heparan sulfate copolymerase EXT1 by analysis of Chinese hamster ovary cell mutants.
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DOI：
10.1074/jbc.m002990200
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发表时间：
2000-09
期刊：
影响因子：
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通讯作者：
G. Wei;X. Bai;M. Gabb;K. Bame;T. Koshy;P. Spear;J. Esko
中科院分区：
文献类型：
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作者：
G. Wei;X. Bai;M. Gabb;K. Bame;T. Koshy;P. Spear;J. Esko
硫酸乙酰肝素是由葡萄糖醛酸(Glca)和N-乙酰氨基葡萄糖(GlcNAc)残基共聚而成的。最近的研究表明，这些反应是由Ext1和Ext2编码的共聚酶催化的，Ext1和Ext2是与遗传性多发性骨软骨病相关的exostosin家族的成员。此前，我们鉴定了一组不能产生硫酸乙酰肝素的中国仓鼠卵巢细胞突变体(PgsD)(Lidholt，K.，Weinke，J.L.，Kiser，C.S.，Lugemwa，F.N.，Bame，K.J.，Cheifetz，S.，Massagué，J.，Lindahl，U，和Esko，J.D.(1992)Proc。娜塔莉。阿卡德。SCI。美国，89,2267-2271)。在这里，我们证明了pgsD突变体包含选择性地改变Glca转移酶活性的突变，或者同时影响GlcNAc和Glca转移酶活性的突变。EXT1的表达纠正了突变体中的缺陷，而Ext2和相关的EXT样cDNA则不能。对EXT1突变等位基因的分析显示，在一个包含(D/E)X(D/E)基序的区域中出现了聚集的错义突变，该基序被认为与其他转移酶研究中的核苷酸糖结合在一起。这些发现提供了对该酶的GLCA转移酶亚域的位置的洞察，并表明GLCA转移酶亚域的丢失可能足以导致遗传性多发性外骨质瘤。
Heparan sulfate formation occurs by the copolymerization of glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) residues. Recent studies have shown that these reactions are catalyzed by a copolymerase encoded by EXT1 and EXT2, members of the exostosin family of putative tumor suppressors linked to hereditary multiple exostoses. Previously, we identified a collection of Chinese hamster ovary cell mutants (pgsD) that failed to make heparan sulfate (Lidholt, K., Weinke, J. L., Kiser, C. S., Lugemwa, F. N., Bame, K. J., Cheifetz, S., Massagué, J., Lindahl, U., and Esko, J. D. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2267-2271). Here, we show that pgsD mutants contain mutations that either alter GlcA transferase activity selectively or that affect both GlcNAc and GlcA transferase activities. Expression of EXT1 corrects the deficiencies in the mutants, whereas EXT2 and the related EXT-like cDNAs do not. Analysis of the EXT1 mutant alleles revealed clustered missense mutations in a domain that included a (D/E)X(D/E) motif thought to bind the nucleotide sugar from studies of other transferases. These findings provide insight into the location of the GlcA transferase subdomain of the enzyme and indicate that loss of the GlcA transferase domain may be sufficient to cause hereditary multiple exostoses.