Hashimoto Y, Orellana A, Gil G, Hirschberg C B
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655.
J Biol Chem. 1992 Aug 5;267(22):15744-50.
N-Heparan sulfate sulfotransferase catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the nitrogen of glucosamine in heparan sulfate. The enzyme has been previously purified to apparent homogeneity from rat liver (Brandan, E., and Hirschberg, C. B. (1988) J. Biol. Chem. 263, 2417-2422). We have now cloned the rat liver enzyme using the following strategy: (a) the amino acid sequence was obtained from tryptic peptides of the purified protein, (b) mixed oligonucleotides were generated based on the sequence of the tryptic peptides, (c) a polymerase chain reaction fragment was obtained using mixed oligonucleotide interprimer amplification of cDNA, and (d) this fragment was used to screen rat liver lambda gt 10 and lambda ZAP libraries. Three clones were obtained, one of which seems to contain the complete coding sequence of the N-heparan sulfate sulfotransferase (N-HSST). Evidence that the cDNA clone corresponds to the previously purified and characterized N-HSST was the following: (a) the predicted sequence of the N-HSST contains all of the 11 tryptic peptides obtained from the purified protein, (b) when a cDNA containing the sequence coding for the N-HSST was introduced in a eukaryotic expression vector and transfected in COS-1 cells, the enzyme activity was expressed 9-fold over controls, and (c) the characteristic of the predicted protein fits with the purified protein in terms of molecular weight, membrane localization, and its being an N-linked glycoprotein. The size of the longest cDNA isolated is 4.1 kilobases, which is in close agreement with the 4.2-kilobase size of one of the mRNA observed in Northern analyses. In addition, messages of 7.0 and 8.5 kilobases were also observed, suggesting that a large portion is untranslated. The latter messages were the major mRNA species detected.
N-硫酸乙酰肝素磺基转移酶催化硫酸基团从3'-磷酸腺苷5'-磷酸硫酸转移至硫酸乙酰肝素中葡糖胺的氮原子上。该酶先前已从大鼠肝脏中纯化至表观均一状态（布兰丹，E.，和赫希伯格，C. B.（1988年）《生物化学杂志》263，2417 - 2422）。我们现在采用以下策略克隆了大鼠肝脏中的该酶：（a）从纯化蛋白的胰蛋白酶肽段中获得氨基酸序列，（b）根据胰蛋白酶肽段的序列生成混合寡核苷酸，（c）使用混合寡核苷酸对cDNA进行引物间扩增获得聚合酶链反应片段，（d）用该片段筛选大鼠肝脏λgt 10和λZAP文库。获得了三个克隆，其中一个似乎包含N-硫酸乙酰肝素磺基转移酶（N-HSST）的完整编码序列。该cDNA克隆与先前纯化和鉴定的N-HSST相对应的证据如下：（a）N-HSST的预测序列包含从纯化蛋白中获得的所有11个胰蛋白酶肽段，（b）当将包含N-HSST编码序列的cDNA引入真核表达载体并转染到COS-1细胞中时，酶活性比对照高9倍表达，（c）预测蛋白的特性在分子量、膜定位以及其为N-连接糖蛋白方面与纯化蛋白相符。分离出的最长cDNA大小为4.1千碱基，这与Northern分析中观察到的一种mRNA的4.2千碱基大小非常一致。此外，还观察到7.0和8.5千碱基的mRNA，表明大部分是未翻译的。后一种mRNA是检测到的主要mRNA种类。
