Mechanism of The Activation of 2'-5'Oligoadenylate Synthetase by Double-Stranded RNA

1.小鼠2‘-5’寡腺苷合成酶的性质小鼠2-5AS基因编码区编码367个氨基酸残基(42kD)，并分离到两个亚型(S^+和S^-)，它们在14个氨基酸上存在差异。S^-的酶活力仅为S^+的1/1000左右，而突变株S^-的176Trp转变为176Glu，其活力是S^-的30倍。S^+酶与双链核糖核酸的结合活性高于S^-，而S^-突变体的结合活性与S^+相近。这些结果表明，酶的中心部分的结构既与酶的活性有关，也与双链RNA2的结合有关。2-5AS基因在小鼠肠道中的生理表达正常小鼠，未经任何干扰素诱导剂的外源性处理，2-5AS活性水平已被证明是增强的。在正常小鼠的脏器中，肠道组织中2-5AS基因的表达水平最高，此外，42kD2-5AS基因在无菌小鼠和胚胎的肠道中也有表达。用抗42kD2-5AS的单抗进行免疫印迹分析表明，在正常小鼠的器官中产生了42kD的酶和30kD的交叉反应蛋白。

1. Nature of mouse 2'-5'oligoadenylate synthetase (2-5AS)The ORF of mouse 2-5AScDNA encodes 367 amino acid residues (42kD) , and two subtypes (S^+ and S^-) of the cDNA have been isolated, which are different in 14 amino acids. The enzymatic activity of S^- is only about 1/1000 of that of S^+, while the S^-mutant, whose 176Trp is converted to 176Glu, has an increase of activity by 30 times of that of S^-. The S^+ enzyme had a higher binding activity to double stranded RNA comparing with S^-, but the S^- mutant had a similar activity to S^+. These results indicate that the structure of the central part of the enzyme is responsible both to the enzyme activity and to the binding to double stranded RNA.2. Physiological expression of the 2-5AS gene in mouse intestineThe normal mouse, not treated exogenously with any IFN of IFN inducers, has been shown to have an enhanced level of 2-5AS activity. Among the organs of normal mouse, intestinal tissues had the highest levels of 2-5AS mRNA.Furthermore, the 42kD 2-5AS mRNA was expressed in intestines from germ-free mice and fetuses. Immunoblotting analysis using a monoclonal antibody against the 42kD 2-5AS revealed that the 42kD enzyme as well as a cross-reactive protein of 30kD were produced in the organs of normal mouse.