Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans
Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans
复制标题
DOI：
10.1074/jbc.273.12.6615
复制
发表时间：
1998-03-20
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;Tone, Y;Sugahara, K
本研究采用简并逆转录-聚合酶链式反应方法，从人胎盘组织中分离到一个新的葡萄糖醛酸基转移酶基因，并根据参与糖类表位合成的大鼠葡萄糖醛酸基转移酶(GlcAT-P)的氨基酸序列与秀丽线虫和曼氏血吸虫可能的蛋白序列进行比对，发现该基因编码一个开放阅读框，编码335个氨基酸，具有II型跨膜蛋白拓扑结构。氨基酸序列与大鼠GlcAT-P有43%的同源性，在COOH端催化结构域的同源性最高。该蛋白的可溶性重组形式在COS-L细胞中的表达产生了具有明显特异性的Galβ1-3Galβ1-4Xy1β1-O-Ser。相反，含有Galβ1-4GlcNAc序列的asialoorosomucid是GlcAT-P的良好受体底物，而不是受体。该反应产物对β-葡萄糖醛酸苷酶敏感，与正品Glcaβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser共层析，表明该酶为β-1，3-葡萄糖醛酸基转移酶。这些结果表明，这个新的葡萄糖醛酸基转移酶基因家族的成员是以前被描述为葡萄糖醛酸基转移酶I的酶，它形成了蛋白多糖的糖胺多聚糖-蛋白质链接区Glcaβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser。
We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method, Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni, The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain, The expression of a soluble recombinant form of the protein in COS-l cells produced an active glucuronyltransferase with marked specificity for a glycoserine Gal beta 1-3Gal beta 1-4Xy1 beta 1-O-Ser. In contrast, asialoorosomucoid, which contains the Gal beta 1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to beta-glucuronidase digestion and co-chromatographed with authentic GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a beta 1,3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, of proteoglycans.