EXPRESSION AND REGULATION BY INTERFERON OF A DOUBLE-STRANDED-RNA-SPECIFIC ADENOSINE-DEAMINASE FROM HUMAN-CELLS - EVIDENCE FOR 2 FORMS OF THE DEAMINASE
EXPRESSION AND REGULATION BY INTERFERON OF A DOUBLE-STRANDED-RNA-SPECIFIC ADENOSINE-DEAMINASE FROM HUMAN-CELLS - EVIDENCE FOR 2 FORMS OF THE DEAMINASE
复制标题
DOI：
10.1128/mcb.15.10.5376
复制
发表时间：
1995-10-01
影响因子：
5.3
通讯作者：
SAMUEL, CE
中科院分区：
文献类型：
--
作者：
PATTERSON, JB;SAMUEL, CE
在干扰素（IFN）调控的cDNA筛选中，分离出一个编码双链RNA （dsRNA）特异性腺苷脱氨酶的6474个核苷酸的人cDNA克隆K88。Northern (RNA) blot分析显示，K88 cDNA在人细胞中杂交成一个约6.7 kb的主要转录本，经IFN处理后，该转录本增加了约5倍。以谷胱甘肽s -转移酶（GST）融合蛋白表达的K88 cDNA产物制备的多克隆抗血清经免疫印迹分析识别出两种蛋白。在人羊膜U和神经母细胞瘤SH-SY5Y细胞系中检测到IFN诱导的150-kDa蛋白和组成性表达的110-kDa蛋白，其水平不受IFN处理的影响。在小鼠成纤维细胞中只检测到150-kDa蛋白，并对重组人蛋白提出了抗血清；小鼠150-kDa蛋白是IFN诱导的。免疫荧光显微镜和细胞分离分析表明，110-kDa蛋白只存在于细胞核中，而150-kDa蛋白在人细胞的细胞质和细胞核中都存在。从K88 cDNA中推断出的氨基酸序列包括三个高度保守的R基序，通常在dsrna结合蛋白中发现。从人核提取物中制备的150-kDa和110-kDa蛋白均与双链RNA亲和柱结合，而不与单链RNA亲和柱结合。此外，大肠杆菌表达的包含R基序的GST K88融合蛋白具有dsrna结合活性。从转染K88 cdna的细胞或从ifn处理的细胞中提取的提取物都含有增加的dsrna特异性腺苷脱氨酶活性。这些结果证实K88编码ifn诱导的dsrna特异性腺苷脱氨酶，并表明至少有两种形式的dsrna特异性腺苷脱氨酶存在于人类细胞中。
A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of similar to 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.