Lamanna William C, Frese Marc-André, Balleininger Martina, Dierks Thomas
Fakultät für Chemie, Biochemie I, Universität Bielefeld, 33615 Bielefeld, Germany.
Zentrum für Biochemie und Molekulare Zellbiologie, Abteilung Biochemie II, Universität Göttingen, 37073 Göttingen, Germany.
J Biol Chem. 2008 Oct 10;283(41):27724-27735. doi: 10.1074/jbc.M802130200. Epub 2008 Aug 6.
Sulf1 and Sulf2 are two heparan sulfate 6-O-endosulfatases that regulate the activity of multiple growth factors, such as fibroblast growth factor and Wnt, and are essential for mammalian development and survival. In this study, the mammalian Sulfs were functionally characterized using overexpressing cell lines, in vitro enzyme assays, and in vivo Sulf knock-out cell models. Analysis of subcellular Sulf localization revealed significant differences in enzyme secretion and detergent solubility between the human isoforms and their previously characterized quail orthologs. Further, the activity of the Sulfs toward their native heparan sulfate substrates was determined in vitro, demonstrating restricted specificity for S-domain-associated 6S disaccharides and an inability to modify transition zone-associated UA-GlcNAc(6S). Analysis of heparan sulfate composition from different cell surface, shed, glycosylphosphatidylinositol-anchored and extracellular matrix proteoglycan fractions of Sulf knock-out cell lines established differential effects of Sulf1 and/or Sulf2 loss on nonsubstrate N-, 2-O-, and 6-O-sulfate groups. These findings indicate a dynamic influence of Sulf deficiency on the HS biosynthetic machinery. Real time PCR analysis substantiated differential expression of the Hs2st and Hs6st heparan sulfate sulfotransferase enzymes in the Sulf knock-out cell lines. Functionally, the changes in heparan sulfate sulfation resulting from Sulf loss were shown to elicit significant effects on fibroblast growth factor signaling. Taken together, this study implicates that the Sulfs are involved in a potential cellular feed-back mechanism, in which they edit the sulfation of multiple heparan sulfate proteoglycans, thereby regulating cellular signaling and modulating the expression of heparan sulfate biosynthetic enzymes.
硫酸乙酰肝素6 - O - 内硫酸酯酶1（Sulf1）和硫酸乙酰肝素6 - O - 内硫酸酯酶2（Sulf2）是两种硫酸乙酰肝素6 - O - 内硫酸酯酶，可调节多种生长因子（如成纤维细胞生长因子和Wnt）的活性，对哺乳动物的发育和生存至关重要。在本研究中，利用过表达细胞系、体外酶活性测定和体内Sulf基因敲除细胞模型对哺乳动物Sulfs进行了功能表征。亚细胞Sulf定位分析显示，人类异构体与其先前表征的鹌鹑直系同源物在酶分泌和去污剂溶解性方面存在显著差异。此外，在体外测定了Sulfs对其天然硫酸乙酰肝素底物的活性，结果表明其对S结构域相关的6 - O - 硫酸化二糖具有受限的特异性，并且无法修饰过渡区相关的葡萄糖醛酸 - N - 乙酰氨基葡萄糖（6 - O - 硫酸化）。对Sulf基因敲除细胞系不同细胞表面、脱落、糖基磷脂酰肌醇锚定和细胞外基质蛋白聚糖组分的硫酸乙酰肝素组成分析，确定了Sulf1和/或Sulf2缺失对非底物N - 、2 - O - 和6 - O - 硫酸基团的不同影响。这些发现表明Sulf缺乏对硫酸乙酰肝素生物合成机制具有动态影响。实时PCR分析证实了硫酸乙酰肝素硫酸转移酶Hs2st和Hs6st在Sulf基因敲除细胞系中的差异表达。在功能上，Sulf缺失导致的硫酸乙酰肝素硫酸化变化对成纤维细胞生长因子信号传导产生了显著影响。综上所述，本研究表明Sulfs参与了一种潜在的细胞反馈机制，在该机制中，它们编辑多种硫酸乙酰肝素蛋白聚糖的硫酸化，从而调节细胞信号传导并调节硫酸乙酰肝素生物合成酶的表达。
