Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan chondroitin sulfate
Molecular cloning and characterization of a human uronyl 2-sulfotransferase that sulfates iduronyl and glucuronyl residues in dermatan chondroitin sulfate
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DOI：
10.1074/jbc.274.15.10474
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发表时间：
1999-04-09
影响因子：
4.8
通讯作者：
Rosenberg, RD
中科院分区：
文献类型：
--
作者：
Kobayashi, M;Sugumaran, G;Rosenberg, RD
具有与先前描述的硫酸乙酰肝素艾杜糖醛酸2-磺基转移酶同源的预测氨基酸序列的部分长度人cDNA（小林，M.，Habuchi，H.，Yoneda，M.，Habuchi，O.，和Kimata，K.，（1997）J，Biol，Chem.272，13980-13985）。使用该同源cDNA作为探针进行北方印迹分析，其证明在许多人组织和几种人癌细胞系中普遍表达5.1和2.0内切酶的信息。由于人淋巴瘤Raji细胞系具有最高水平的表达，因此使用其分离全长cDNA克隆，发现全长cDNA含有预测由406个氨基酸残基组成的IT型跨膜蛋白的开放阅读框架。将杆状病毒表达载体中的cDNA在Sf 9昆虫细胞中表达，然后将细胞提取物与3 '-磷酸腺苷5'-磷酸[S-35]硫酸盐和潜在的糖胺聚糖受体一起孵育。这证明了硫酸皮肤素具有显著的磺基转移酶活性，硫酸软骨素具有小程度的活性，但用硫酸化的N-再硫酸化肝素没有磺基转移酶活性。软骨素ABC、软骨素AC和软骨素B裂解酶处理的[S-35]硫酸盐标记的二糖产物的分析证明，该酶仅将硫酸盐转移到糖醛酸残基的α-位，硫酸皮肤素中主要是艾杜糖醛酸残基，但硫酸软骨素中有少量转移到葡萄糖醛酸残基。
A partial-length human cDNA with a predicted amino acid sequence homologous to a previously described heparan sulfate iduronyl 2-sulfotransferase (Kobayashi, M., Habuchi, H,, Yoneda, M,, Habuchi, O,, and Kimata, K, (1997) J, Biol, Chem. 272, 13980-13985) was obtained by searching the expressed sequence-tagged data bank. Northern blot analysis was performed using this homologous cDNA as a probe, which demonstrated ubiquitous expression of messages of 5.1 and 2.0 kilobases in a number of human tissues and in several human cancer cell lines. Since the human lymphoma Raji cell line had the highest level of expression, it was used to isolate a full-length cDNA clone, The full-length cDNA was found to contain an open reading frame that predicted a type IT transmembrane protein composed of 406 amino acid residues. The cDNA in a baculovirus expression vector was expressed in Sf9 insect cells, and cell extracts were then incubated together with 3'-phosphoadenosine 5'phospho[S-35]sulfate and potential glycosaminoglycan accepters. This demonstrated substantial sulfotransferase activity with dermatan sulfate, a small degree of activity with chondroitin sulfate, but no sulfotransferase activity with desulfated N-resulfated heparin, Analysis of [S-35]sulfate-labeled disaccharide products of chondroitin ABC, chondroitin AC, and chondroitin B lyase treatment demonstrated that the enzyme only transferred sulfate to the a-position of uronyl residues, which were preponderantly iduronyl residues in dermatan sulfate, but some lesser transfer to glucuronyl residues of chondroitin sulfate.