Molecular cloning and characterization of two related and interferon-induced 56-kDa and 30-kDa proteins highly similar to 2′-5′ oligoadenylate synthetase
Molecular cloning and characterization of two related and interferon-induced 56-kDa and 30-kDa proteins highly similar to 2′-5′ oligoadenylate synthetase
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DOI：
10.1046/j.1432-1327.1998.2570319.x
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发表时间：
1998-10-01
期刊：
影响因子：
--
通讯作者：
Hovanessian, AG
中科院分区：
文献类型：
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作者：
Rebouillat, D;Marié, I;Hovanessian, AG
2‘-5’寡腺苷合成酶(OAS)是一类干扰素诱导的蛋白质家族，被双链(DS)RNA激活后可将ATP聚合成2‘-5’连接的低聚物，通式为PPPA(2‘P5’A)(N)，n大于或等于1。人类OAS有三种形式，分别对应于40/46、69/71和100 kDa的蛋白质(p40/p46、p69/p71和p100)。针对p100交叉的多克隆抗体可与其他形式的p40/p46、p69/p71反应，也可与干扰素诱导的56 kDa蛋白(P56)反应。通过筛选表达文库，这些多克隆抗体选择了编码p56的cDNA.利用该基因的RACE程序进一步研究，获得了一条30 kDa的p56相关蛋白(P30)。在干扰素处理的细胞中，p56和p30mRNA分别以2kb和1.8kb的转录本表达。1.8kb的信使核糖核酸与2kb的信使核糖核酸同源，但在1011位有243个核苷酸缺失，导致移码。因此，P56和P30的前219个氨基酸残基相同，但在C末端不同。在体外转录翻译的p56和p30cDNAs分别产生56和30 kDa的蛋白质。推导的p56 kDa氨基酸序列与已克隆的OASs有很强的相似性，并含有以p40/p46和p69/p71形式保守的亚区。在HeLa细胞中的瞬时表达表明，p30具有胞浆定位，而p56具有胞浆和核仁定位。从转基因细胞中分离到的p56能与dsRNA和DNA结合，但它缺乏这种酶的三个已知农场典型的2‘-5’OAS活性。因此，P56和P30是2‘-5’OA家族之外的两个相关的干扰素诱导蛋白，可能具有未知的催化活性或功能。
The 2'-5' oligoadenylate synthetase (OAS) represents a family of interferon-induced proteins which when activated by double-stranded (ds) RNA polymerizes ATP into 2'-5' linked oligomers with the general formula pppA(2'p5'A)(n), n greater than or equal to 1. Three forms of human OASs have been described corresponding to proteins of 40/46, 69/71 and 100 kDa (p40/p46, p69/p71 and p100). Polyclonal antibodies raised against p100 cross reacted with the other forms p40/p46, p69/p71 but also with an interferon-induced 56-kDa protein (p56). By screening a cDNA expression library, these polyclonal antibodies selected a cDNA encoding p56. Further studies by the RACE procedure using primers corresponding to this cDNA, a p56-related protein of 30 kDa (p30) was isolated. Both p56 and p30 mRNA are expressed in interferon-treated cells as transcripts of 2 kb and 1.8 kb, respectively. The 1.8-kb mRNA is homologous to the 2-kb mRNA but with a 243-nucleotide deletion at position 1011, which results in a frameshift. Consequently, the p56 and p30 have their first 219 amino acid residues identical but differ at their C-termini. In vitro transcription-translation of p56 and p30 cDNAs generated proteins of 56 and 30 kDa, respectively. The deduced amino acid sequence of p56 kDa shares strong similarity with the previously cloned OASs, and contains the subdomains conserved in p40/p46 and p69/p71 forms. Transient expression in HeLa cells indicated that p30 has a cytoplasmic localization, whereas p56 has cytoplasmic and nucleolar localizations. The p56 isolated from transfected cells was shown to bind dsRNA and DNA, but it was devoid of 2'-5'OAS activity typical of the three known farms of this enzyme. Thus, p56 and p30 are two related and interferon-induced proteins outside the family of 2'-5'OAS, which might have as yet unidentified catalytic activities or functions.