Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase.
Substrate specificity of the heparan sulfate hexuronic acid 2-O-sulfotransferase.
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DOI：
10.1021/bi002926p
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发表时间：
2001-05
期刊：
影响因子：
2.9
通讯作者：
J. Rong;H. Habuchi;K. Kimata;U. Lindahl;M. Kusche‐Gullberg
中科院分区：
文献类型：
--
作者：
J. Rong;H. Habuchi;K. Kimata;U. Lindahl;M. Kusche‐Gullberg
硫酸乙酰肝素与不同配体蛋白的相互作用取决于多糖链中O-硫酸基团的精确位置。我们先前已经表明，小鼠肥大细胞瘤2-O-磺基转移酶（2-OST）在人肾293细胞中的过表达优先增加D-葡糖醛酸基单元的2-O-硫酸化水平[Rong，J.，Habuchi，H.，Kimata，K.，林达尔大学，和Kusche-Gullberg，M.（2000）Biochem.J.346，463-468]。在本研究中，我们进一步研究了小鼠肥大细胞瘤2-OST的底物特异性。将不同的多糖受体底物与来自2-OST转染的293细胞的细胞提取物以及硫酸盐供体3 '-磷酸腺苷5'-磷酸[（35）S]硫酸盐一起孵育。与主要由[（4）alphaIdoA（1）-（4）alphaGlcNSO（3）（1）-]（n）（）组成的O-乙酰肝素孵育，导致艾杜糖醛酸的2-O-硫酸化。另一方面，当使用来自大肠杆菌K5的具有结构[（4）betaGlcA（1）-（4）alphaGlcNSO（3）（1）-]（n）（）的N-硫酸化荚膜多糖作为受体时，硫酸盐几乎仅转移到葡萄糖醛酸的C2。以大约相等比例含有艾杜糖醛酸和葡萄糖醛酸残基的底物强烈有利于艾杜糖醛酸的硫酸化。与这些结果一致，发现2-OST对含艾杜糖醛酸的底物二糖单元的亲和力（K（m）约为3.7 μ M）比对含葡萄糖醛酸的底物二糖单元的亲和力（K（m）约为19.3 μ M）高约5倍。
The interaction of heparan sulfate with different ligand proteins depends on the precise location of O-sulfate groups in the polysaccharide chain. We have previously shown that overexpression in human kidney 293 cells of a mouse mastocytoma 2-O-sulfotransferase (2-OST), previously thought to catalyze the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to C2 of L-iduronyl residues, preferentially increases the level of 2-O-sulfation of D-glucuronyl units [Rong, J., Habuchi, H., Kimata, K., Lindahl, U., and Kusche-Gullberg, M. (2000) Biochem. J. 346, 463-468]. In the study presented here, we further investigated the substrate specificity of the mouse mastocytoma 2-OST. Different polysaccharide acceptor substrates were incubated with cell extracts from 2-OST-transfected 293 cells together with the sulfate donor 3'-phosphoadenosine 5'-phospho[(35)S]sulfate. Incubations with O-desulfated heparin, predominantly composed of [(4)alphaIdoA(1)-(4)alphaGlcNSO(3)(1)-](n)(), resulted in 2-O-sulfation of iduronic acid. When, on the other hand, an N-sulfated capsular polysaccharide from Escherichia coli K5, with the structure [(4)betaGlcA(1)-(4)alphaGlcNSO(3)(1)-](n)(), was used as an acceptor, sulfate was transferred almost exclusively to C2 of glucuronic acid. Substrates containing both iduronic and glucuronic acid residues in about equal proportions strongly favored sulfation of iduronic acid. In agreement with these results, the 2-OST was found to have a approximately 5-fold higher affinity for iduronic acid-containing substrate disaccharide units (K(m) approximately 3.7 microM) than for glucuronic acid-containing substrate disaccharide units (K(m) approximately 19.3 microM).