Shworak N W, Liu J, Fritze L M, Schwartz J J, Zhang L, Logeart D, Rosenberg R D
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 1997 Oct 31;272(44):28008-19. doi: 10.1074/jbc.272.44.28008.
The cellular rate of anticoagulant heparan sulfate proteoglycan (HSPGact) generation is determined by the level of a kinetically limiting microsomal activity, HSact conversion activity, which is predominantly composed of the long sought heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST) (Shworak, N. W., Fritze, L. M. S., Liu, J., Butler, L. D., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27063-27071; Liu, J., Shworak, N. W., Fritze, L. M. S., Edelberg, J. M., and Rosenberg, R. D. (1996) J. Biol. Chem. 271, 27072-27082). Mouse 3-OST cDNAs were isolated by proteolyzing the purified enzyme with Lys-C, sequencing the resultant peptides as well as the existing amino terminus, employing degenerate polymerase chain reaction primers corresponding to the sequences of the peptides as well as the amino terminus to amplify a fragment from LTA cDNA, and utilizing the resultant probe to obtain full-length enzyme cDNAs from a lambda Zap Express LTA cDNA library. Human 3-OST cDNAs were isolated by searching the expressed sequence tag data bank with the mouse sequence, identifying a partial-length human cDNA and utilizing the clone as a probe to isolate a full-length enzyme cDNA from a lambda TriplEx human brain cDNA library. The expression of wild-type mouse 3-OST as well as protein A-tagged mouse enzyme by transient transfection of COS-7 cells and the expression of both wild-type mouse and human 3-OST by in vitro transcription/translation demonstrate that the two cDNAs directly encode both HSact conversion and 3-OST activities. The mouse 3-OST cDNAs exhibit three different size classes because of a 5'-untranslated region of variable length, which results from the insertion of 0-1629 base pairs (bp) between residues 216 and 217; however, all cDNAs contain the same open reading frame of 933 bp. The length of the 3'-untranslated region ranges from 301 to 430 bp. The nucleic acid sequence of mouse and human 3-OST cDNAs are approximately 85% similar, encoding novel 311- and 307-amino acid proteins of 35,876 and 35,750 daltons, respectively, that are 93% similar. The encoded enzymes are predicted to be intraluminal Golgi residents, presumably interacting via their C-terminal regions with an integral membrane protein. Both 3-OST species exhibit five potential N-glycosylation sites, which account for the apparent discrepancy between the molecular masses of the encoded enzyme (approximately 34 kDa) and the previously purified enzyme (approximately 46 kDa). The two 3-OST species also exhibit approximately 50% similarity with all previously identified forms of the heparan biosynthetic enzyme N-deacetylase/N-sulfotransferase, which suggests that heparan biosynthetic enzymes share a common sulfotransferase domain.
抗凝血硫酸乙酰肝素蛋白聚糖（HSPGact）的细胞生成速率由一种动力学上起限制作用的微粒体活性水平决定，即HSact转化活性，其主要由长期以来寻找的硫酸乙酰肝素D - 葡糖胺基3 - O - 磺基转移酶（3 - OST）组成（Shworak，N. W.，Fritze，L. M. S.，Liu，J.，Butler，L. D.，和Rosenberg，R. D.（1996）J. Biol. Chem. 271，27063 - 27071；Liu，J.，Shworak，N. W.，Fritze，L. M. S.，Edelberg，J. M.，和Rosenberg，R. D.（1996）J. Biol. Chem. 271，27072 - 27082）。通过用Lys - C对纯化的酶进行蛋白酶解、对所得肽段以及现有的氨基末端进行测序、使用与肽段和氨基末端序列对应的简并聚合酶链反应引物从LTA cDNA扩增片段，并利用所得探针从λZap Express LTA cDNA文库中获得全长酶cDNA来分离小鼠3 - OST cDNA。通过用小鼠序列搜索表达序列标签数据库、鉴定部分长度的人cDNA并利用该克隆作为探针从λTriplEx人脑cDNA文库中分离全长酶cDNA来分离人3 - OST cDNA。通过瞬时转染COS - 7细胞表达野生型小鼠3 - OST以及带有蛋白A标签的小鼠酶，以及通过体外转录/翻译表达野生型小鼠和人3 - OST，证明这两种cDNA直接编码HSact转化活性和3 - OST活性。由于5' - 非翻译区长度可变，小鼠3 - OST cDNA表现出三种不同的大小类别，这是由于在残基216和217之间插入了0 - 1629个碱基对（bp）；然而，所有cDNA都包含相同的933 bp开放阅读框。3' - 非翻译区的长度范围为301至430 bp。小鼠和人3 - OST cDNA的核酸序列约85%相似，分别编码35,876和35,750道尔顿的新型311和307个氨基酸的蛋白质，它们93%相似。预测编码的酶是高尔基体腔内驻留蛋白，可能通过其C末端区域与一种整合膜蛋白相互作用。两种3 - OST都有五个潜在的N - 糖基化位点，这解释了编码酶的分子量（约34 kDa）与先前纯化的酶（约46 kDa）之间明显的差异。这两种3 - OST与所有先前鉴定的硫酸乙酰肝素生物合成酶N - 脱乙酰酶/N - 磺基转移酶形式也表现出约50%的相似性，这表明硫酸乙酰肝素生物合成酶共享一个共同的磺基转移酶结构域。
