Biosynthesis of heparin/heparan sulfate - DNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung
Biosynthesis of heparin/heparan sulfate - DNA cloning and expression of D-glucuronyl C5-epimerase from bovine lung
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DOI：
10.1074/jbc.272.44.28158
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发表时间：
1997-10-31
影响因子：
4.8
通讯作者：
Lindahl, U
中科院分区：
文献类型：
--
作者：
Li, JP;HagnerMcWhirter, A;Lindahl, U
葡糖醛酸C5-差向异构酶在糖胺聚糖的生物合成期间催化D-葡糖醛酸（GlcUA）转化为L-艾杜糖醛酸（IdceA）单元。与肝素/硫酸乙酰肝素的产生有关的差向异构酶先前已从牛肝脏中纯化至同质（坎贝尔，P.，Hannesson，H. H、Sandback，D.，罗登湖，林达尔大学，和Li，J. - p，（1994）J.Biol.Chem.269，2695-26958）。本文报道了肺酶的分子克隆和功能表达，克隆的酶含有444个氨基酸残基，分子量为49，905 Da，分离的肝酶的N-末端序列分析表明，该物种是一个截短的形式，缺乏73个残基的N-将编码cDNA插入片段克隆到杆状病毒表达载体中并在Sf 9昆虫细胞中表达，用重组差向异构酶感染的细胞显示酶活性增加20-30倍，测量为从含有C5-H-3标记的己糖醛酸单元的多糖底物释放（H2O）-H-3。此外，将表达的蛋白质与适当的载体孵育。（GlcUA-GlcNSO（3））（n）底物导致类似于20%的GlcUA单位转化为IdceA残基，北方分析表明牛肺和肝组织中存在两种差向异构酶转录物，一种与9-腺苷酸酶（kb）mRNA类似的显性转录物和一种与5-kb物质类似的次要转录物，小鼠肥大细胞瘤细胞仅显示与5-kb转录本相似的转录本。克隆的差向异构酶与催化藻酸盐生物合成中类似反应的酶的比较显示没有明显的氨基酸序列相似性。
Glucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H. H., Sandback, D., Roden, L., Lindahl, U., and Li, J.-p, (1994) J. Biol. Chem. 269, 2695-26958). The present report describes the molecular cloning and functional expression of the lung enzyme, The cloned enzyme contains 444 amino acid residues and has a molecular mass of 49,905 Da, N-terminal sequence analysis of the isolated liver enzyme showed this species to be a truncated form lacking a 73-residue N-terminal domain of the deduced amino acid sequence.The coding cDNA insert was cloned into a baculovirus expression vector and expressed in Sf9 insect cells, Cells infected with recombinant epimerase showed a 20-30-fold increase in enzyme activity, measured as release of (H2O)-H-3 from a polysaccharide substrate containing C5-H-3-labeled hexuronic acid units. Furthermore, incubation of the expressed protein with the appropriate (GlcUA-GlcNSO(3))(n) substrate resulted in conversion of similar to 20% of the GlcUA units into IdceA residues, Northern analysis implicated two epimerase transcripts in both bovine lung and liver tissues, a dominant similar to 9-kilobase (kb) mRNA and a minor similar to 5-kb species, Mouse mastocytoma cells showed only the similar to 5-kb transcript. A comparison of the cloned epimerase with the enzymes catalyzing an analogous reaction in alginate biosynthesis revealed no apparent amino acid sequence similarity.