Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3)
Comparative characterization of rat deoxyribonuclease 1 (Dnase1) and murine deoxyribonuclease 1-like 3 (Dnase1l3)
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DOI：
10.1042/bj20042124
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发表时间：
2005-07-15
影响因子：
4.1
通讯作者：
Kloeckl, T
中科院分区：
文献类型：
--
作者：
Napirei, M;Wulf, S;Kloeckl, T
脱氧核糖核酸酶1(DNase1，DNase1)和类脱氧核糖核酸酶I-L3(DNase-L3，DNase-Gamma，DNase Y，LS-DNase)是DNase1蛋白家族的成员，具有相似的生化特性，如钙/镁依赖性，最适pH约7.0，以及它们在核酸和氨基酸序列上的高度相似性。在本研究中，我们描述了大鼠DNase1和小鼠DNasc113作为C端标记的绿色荧光蛋白的融合蛋白在NIH-3T3成纤维细胞和牛晶状体上皮细胞中的重组表达。这两种酶都被转运到粗面内质网，沿着整个分泌途径运输，最后分泌到细胞培养液中。没有检测到核酸酶的核出现。然而，两种核酸酶的N-末端信号肽的缺失导致了两种融合蛋白的胞质和核分布。DNase1优先降解裸质粒DNA，而DNase113则高活性地切割核DNA。Dnase113能够在没有蛋白质降解帮助的情况下以核小体间的方式切割染色质。相比之下，DNase1只有在降解染色质结合蛋白的蛋白酶存在的情况下才能实现这种切割模式。对来自DNase1基因敲除小鼠的小鼠血清的详细分析表明，除了主要的血清核酸酶DNase1外，血清还含有额外的DNase113样核溶解活性，与DNase1合作，可能有助于通过降解死亡细胞释放的核染色质来抑制抗DNA自身免疫。
Deoxyribonuclease 1 (DNASE1, DNase 1) and deoxyribonuclease I-like 3 (DNASE1L3, DNase gamma, DNase Y, LS-DNase) are members of a DNASE1 protein family that is defined by similar biochemical properties such as Ca2+/Mg2+-dependency and an optimal pH of about 7.0 as well as by a high similarity in their nucleic acid and amino acid sequences. In the present study we describe the recombinant expression of rat Dnase1 and murine Dnasc113 as fusion proteins tagged by their C-terminus to green fluorescent protein in NIH-3T3 fibroblasts and bovine lens epithelial cells. Both enzymes were translocated into the rough endoplasmic reticulum, transported along the entire secretory pathway and finally secreted into the cell culture medium. No nuclear occurrence of the nucleases was detectable. However, deletion of the N-terminal signal peptide of both nucleases resulted in a cytoplasmic and nuclear distribution of both fusion proteins. Dnase1 preferentially hydrolysed 'naked' plasmid DNA, whereas Dnase113 cleaved nuclear DNA with high activity. Dnase113 was able to cleave chromatin in an internucleosomal manner without proteolytic help. By contrast, Dnase1 was only able to achieve this cleavage pattern in the presence of proteases that hydrolysed chromatin-bound proteins. Detailed analysis of murine sera derived from Dnase1 knockout mice revealed that serum contains, besides the major serum nuclease Dnase1, an additional Dnase113-like nucleolytic activity, which, in co-operation with Dnase1, might help to suppress anti-DNA autoimmunity by degrading nuclear chromatin released from dying cells.