Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate
Characterization of the D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate
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参与肝素和硫酸肝素生物合成的 D-葡萄糖醛酸 C5-酰亚胺酶的特征
DOI：
10.1074/jbc.m011783200
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发表时间：
2001-06-08
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;Gong, F;Lindahl, U
使用先前分离的牛肺cDNA片段（Li，J. - P.，Hagner-McWhirter，A.克塞林湖Palgi，J.，Jalkanen，M.，Lindahl，U.（1997）J.Biol.Chem.272，28158 - 28163）作为探针。小鼠11-乙酰胆碱酯酶对基因包含从第一个ATG到终止密码子的3个外显子，定位于9号染色体。Southern分析的基因组DNA和染色体定位表明发生一个单一的差向异构酶基因。基于基因组序列，分离出小鼠肝脏cDNA，其编码618个氨基酸残基的蛋白质，从而将从（不完整的）牛cDNA推导的序列延伸174个N末端残基。小鼠、牛和人差向异构酶cDNA结构的比较表明在氨基酸水平上具有96 - 99%的同一性。小鼠肝脏物种相同的cDNA被证明在小鼠肥大细胞致力于肝素生物合成。这些发现表明，艾杜糖醛酸残基肝素和硫酸乙酰肝素，尽管不同的结构背景下，产生相同的C5-差向异构酶。发现在昆虫细胞中表达的重组全长小鼠肝差向异构酶的催化活性比先前克隆的较小的牛重组蛋白高> 2个数量级。类似于52-kDa，类似地高活性的酶最初从牛肝纯化（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.269，26953 - 26958）被发现与通过完整大小的蛋白质的单次蛋白水解切割产生的22-kDa肽相关。
The murine gene for the glucuronyl C5-epimerase involved in heparan sulfate biosynthesis was cloned, using a previously isolated bovine lung cDNA fragment (Li, J.-P., Hagner-McWhirter, A. Kjellen, L., Palgi, J., Jalkanen, M., and Lindahl, U. (1997) J. Biol. Chem. 272, 28158-28163) as probe. The similar to 11-kilobase pair mouse gene contains 3 exons from the first ATG to stop codon and is localized to chromosome 9. Southern analysis of the genomic DNA and chromosome mapping suggested the occurrence of a single epimerase gene. Based on the genomic sequence, a mouse liver cDNA was isolated that encodes a 618-amino acid residue protein, thus extending by 174 N terminal residues the sequence deduced from the (incomplete) bovine cDNA. Comparison of murine, bovine, and human epimerase cDNA structures indicated 96-99% identity at the amino acid level. A cDNA identical to the mouse liver species was demonstrated in mouse mast cells committed to heparin biosynthesis. These findings suggest that the iduronic acid residues in heparin and heparan sulfate, despite different structural contexts, are generated by the same C5-epimerase enzyme. The catalytic activity of the recombinant full-length mouse liver epimerase, expressed in insect cells, was found to be >2 orders of magnitude higher than that of the previously cloned, smaller bovine recombinant protein. The similar to 52-kDa, similarly highly active, enzyme originally purified from bovine liver (Campbell, P., Hannesson, H. H., Sandback, D., Roden, L., Lindahl, U., and Li, J.-P. (1994) J. Biol. Chem. 269, 26953-26958) was found to be associated with an 22-kDa peptide generated by a single proteolytic cleavage of the full-sized protein.