Hortin G L, Farries T C, Graham J P, Atkinson J P
Department of Pediatrics, Washington University School of Medicine, Saint Louis, MO 63110.
Proc Natl Acad Sci U S A. 1989 Feb;86(4):1338-42. doi: 10.1073/pnas.86.4.1338.
Sulfation of tyrosine residues recently has been recognized as a biosynthetic modification of many plasma proteins and other secretory proteins. Effects of this site-specific modification on protein function are not known, but the activity of several peptides such as cholecystokinin is greatly augmented by sulfation. Here, we examine the role of sulfation in the processing and activity of C4 (the fourth component of complement), one of the few proteins in which sites and stoichiometry of tyrosine sulfation have been characterized. Our results, with C4 as a paradigm, suggest that sulfation of tyrosine residues can have major effects on the activity of proteins participating in protein-protein interactions. Sulfation of C4 synthesized by Hep G2 cells was blocked by incubating the cells with NaClO3 and guaiacol. These sulfation inhibitors did not alter secretion or other steps in the processing of C4. However, hemolytic activity of C4 was decreased more than 50%. The inhibitors' effect on C4 activity was prevented by adding Na2SO4 to restore sulfation of C4. Activity of C3, a complement component homologous to C4 but lacking tyrosine sulfate residues, was minimally reduced (19%) by the inhibitors. Decreased hemolytic activity of nonsulfated C4 apparently resulted from impaired interaction with complement subcomponent C1s (EC 3.4.21.42), the protease that physiologically activates C4. Purified C1s was able to cleave nonsulfated C4, but approximately 10-fold higher concentrations of C1s were required for that cleavage than to yield equivalent cleavage of sulfated C4. Our results suggest that activation of C4, a central component in the classical pathway of complement activation, is influenced by the level of sulfation of the protein. Thus, sulfation of C4 provides a potential locus for physiological or pharmacological modulation of complement-mediated opsonization and inflammation.
酪氨酸残基的硫酸化最近已被确认为许多血浆蛋白和其他分泌蛋白的生物合成修饰。这种位点特异性修饰对蛋白质功能的影响尚不清楚，但硫酸化可大大增强胆囊收缩素等几种肽的活性。在这里，我们研究了硫酸化在补体C4（补体的第四成分）的加工和活性中的作用，C4是少数已确定酪氨酸硫酸化位点和化学计量的蛋白质之一。我们以C4为范例的研究结果表明，酪氨酸残基的硫酸化可对参与蛋白质-蛋白质相互作用的蛋白质活性产生重大影响。用NaClO3和愈创木酚孵育Hep G2细胞可阻断其合成的C4的硫酸化。这些硫酸化抑制剂不会改变C4加工过程中的分泌或其他步骤。然而，C4的溶血活性降低了50%以上。通过添加Na2SO4恢复C4的硫酸化可防止抑制剂对C4活性的影响。与C4同源但缺乏酪氨酸硫酸化残基的补体成分C3的活性仅被抑制剂轻微降低（19%）。未硫酸化的C4溶血活性降低显然是由于与补体亚成分C1s（EC 3.4.21.42）的相互作用受损所致，C1s是生理上激活C4的蛋白酶。纯化的C1s能够切割未硫酸化的C4，但与切割硫酸化的C4产生同等切割效果相比，所需的C1s浓度要高约10倍。我们的结果表明，补体经典激活途径中的核心成分C4的激活受该蛋白硫酸化水平的影响。因此，C4的硫酸化为补体介导的调理作用和炎症的生理或药理调节提供了一个潜在位点。
