酪氨酸硫化修饰对酪氨酸硫化转移酶活性的调节

Regulation of Tyrosylprotein Sulfotransferases Activity by Sulfotyrosine

目的研究酪氨酸硫化修饰对酪氨酸硫化转移酶(TPST)1和TPST2的调节作用。方法以人cDNA为模板,采用聚合酶链反应方法扩增人TPST1和TPST2基因野生型的全长cDNA序列,并融合入人IgG1的Fc编码区域;采用定点突变方法将TPST1和TPST2蛋白中可硫化的酪氨酸突变为苯丙氨酸。针对TPST1(259～275位核苷酸)和TPST2(73～94位核苷酸)基因,分别构建小干扰RNA(shRNA)1和shRNA2重组质粒。测定所有构建质粒开放读框序列。证实序列正确后,将上述质粒分别转染人肾胚胎上皮细胞(HEK)293T。24h后将细胞分成3份:1份用于35S-蛋氨酸/半胱氨酸或35S-亚硫酸钠标记,48h后再进行免疫沉淀反应;1份用于125I标记配基结合;1份用于流式细胞仪检测受体的表达水平。结果TPST1326位和TPST2325位酪氨酸在翻译后被硫化修饰,这一翻译后修饰被特异性的硫化抑制剂shRNA1和shRNA2及非特异性的硫化抑制剂NaClO3显著抑制。shRNA1和shRNA2可显著抑制补体3a和5a受体的硫化,并进一步降低这两种受体与特异性配基的结合效率。结论TPST1和TPST2翻译后受酪氨酸硫化修饰的调节,硫化抑制剂shRNA可以显著抑制TPST的活性,并进一步抑制补体3a和5a受体结合其特异性配基的作用。

Objective To investigate the role of sulfated tyrosine in regulating the activity of tyrosylprotein sulfotransferases （TPST） 1 and TPST2. Methods Constructs of TPST 1 and TPST2 were amplified by polymerase chain reaction （PCR） , then fused into immunoglobulin G1 Fc region. All the variants in which sulfated tyrosines were mutated to phenylalanine were made by the PCR-based Quick Change method and confirmed by sequencing the entire reading frame. Small hairpin RNA （shRNA） constructs-targeting nucleotides 259-275 of TPST1 and nucleotides 73-94 of TPST2 were generated and subcloned into pBluescript. Human embryonic kidney （HEK） 293T cells were transfected with these plasmids. One day later, cells were split： one part was labeled with 35S-cysteine and methionine or ^35S-Na2SO3 overnight, the second part was used for ^125I labeled binding experiment, and the third part was retained for binding and flow cytometry. Results Tyrosines at position 326 of TPST1 and position 325 of TPST2 were sulfated posttranslationally. Tyrosine sulfation of TPSTs was effectively inhibited by sulfation inhibitors, including specific shRNAs and non-specific NaClO3. shRNAs reduced the sulfation of C3 a receptor and C5 a receptor, and partially blocked the binding of these two receptors to their respective ligands. Conclusions The activities of TPSTs were regulated by tyrosine sulfation. Inhibition of sulfotyrosine decreases the binding ability of C3 a receptor and C5 a receptor to their respective ligands.