Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain
Characterization of the Human Sulfatase Sulf1 and Its High Affinity Heparin/Heparan Sulfate Interaction Domain
复制标题
DOI：
10.1074/jbc.m109.035808
复制
发表时间：
2009-10-09
影响因子：
4.8
通讯作者：
Dierks, Thomas
中科院分区：
文献类型：
--
作者：
Frese, Marc-Andre;Milz, Fabian;Dierks, Thomas
细胞外磺化酶Sulf1和Sulf2重塑细胞表面硫酸肝素蛋白聚糖的60-磺化状态，从而调节生长因子信号。与所有其他磺化酶不同，硫酶含有一个独特的，带正电的亲水结构域（HD），大约有320个氨基酸残基。利用多种HD缺失突变体和谷胱甘肽s -转移酶(GST)-HD融合蛋白，本研究表明HD是酶活性所必需的，并作为高亲和力的肝素/硫酸肝素相互作用结构域。HD与细胞表面的关联对肝素酶处理敏感，强调对硫酸肝素链的特异性。相应地，分离的GST-HD在体外与肝素和硫酸肝素以及活细胞都有很强的结合。表面等离子体共振研究表明GST-HD对固定化肝素具有纳米级亲和力。不同突变体的比较表明，特别是HD的外部区域介导硫酸肝素结合，可能涉及“串联”相互作用。有趣的是，与硫酸肝素的结合取决于60个硫酸底物基团的存在，这表明底物的周转促进了酶从底物中的释放。HD内部不太保守的区域的缺失会显著增加Sulf1的分泌，而不会影响酶活性或底物特异性，从而为体外调节hs依赖性信号通路提供了一种工具，如本文所示的成纤维细胞生长因子2的信号转导。综上所述，本研究表明，HD的特定区域影响HS结合、细胞定位和酶功能的不同方面。
The extracellular sulfartases Sulf1 and Sulf2 remodel the 60-sulfation state of heparan sulfate proteoglycans on the cell surface, thereby modulating growth factor signaling. Different from all other sulfartases, the Sulfs contain a unique, positively charged hydrophilic domain (HD) of about 320 amino acid residues. Using various HD deletion mutants and glutathione S-transferase (GST)-HD fusion proteins, this study demonstrates that the HD is required for enzymatic activity and acts as a high affinity heparin/heparan sulfate interaction domain. Association of the HD with the cell surface is sensitive to heparinase treatment, underlining specificity toward heparan sulfate chains. Correspondingly, isolated GST-HD binds strongly to both heparin and heparan sulfate in vitro and also to living cells. Surface plasmon resonance studies indicate nanomolar affinity of GST-HD toward immobilized heparin. The comparison of different mutants reveals that especially the outer regions of the HD mediate heparan sulfate binding, probably involving "tandem" interactions. Interestingly, binding to heparan sulfate depends on the presence of 60-sulfate substrate groups, suggesting that substrate turnover facilitates release of the enzyme from its substrate. Deletion of the inner, less conserved region of the HD drastically increases Sulf1 secretion without affecting enzymatic activity or substrate specificity, thus providing a tool for the in vitro modulation of HS-dependent signaling as demonstrated here for the signal transduction of fibroblast growth factor 2. Taken together, the present study shows that specific regions of the HD influence different aspects of HS binding, cellular localization, and enzyme function.