Yasuda Toshihiro, Iida Reiko, Ueki Misuzu, Kominato Yoshihiko, Nakajima Tamiko, Takeshita Haruo, Kobayashi Takanori, Kishi Koichiro
Division of Medical Genetics and Biochemistry, Faculty of Medical Sciences, University of Fukui, Japan.
Eur J Biochem. 2004 Nov;271(22):4428-35. doi: 10.1111/j.1432-1033.2004.04381.x.
We purified pancreatic deoxyribonuclease I (DNase I) from the shark Heterodontus japonicus using three-step column chromatography. Although its enzymatic properties resembled those of other vertebrate DNases I, shark DNase I was unique in being a basic protein. Full-length cDNAs encoding the DNases I of two shark species, H. japonicus and Triakis scyllia, were constructed from their total pancreatic RNAs using RACE. Nucleotide sequence analyses revealed two structural alterations unique to shark enzymes: substitution of two Cys residues at positions 101 and 104 (which are well conserved in all other vertebrate DNases I) and insertion of an additional Thr or Asn residue into an essential Ca(2+)-binding site. Site-directed mutagenesis of shark DNase I indicated that both of these alterations reduced the stability of the enzyme. When the signal sequence region of human DNase I (which has a high alpha-helical structure content) was replaced with its amphibian, fish and shark counterparts (which have low alpha-helical structure contents), the activity expressed by the chimeric mutant constructs in transfected mammalian cells was approximately half that of the wild-type enzyme. In contrast, substitution of the human signal sequence region into the amphibian, fish and shark enzymes produced higher activity compared with the wild-types. The vertebrate DNase I family may have acquired high stability and effective expression of the enzyme protein through structural alterations in both the mature protein and its signal sequence regions during molecular evolution.
我们采用三步柱层析法从日本异齿鲛（Heterodontus japonicus）中纯化了胰腺脱氧核糖核酸酶I（DNase I）。尽管其酶学性质与其他脊椎动物的DNase I相似，但鲨鱼DNase I的独特之处在于它是一种碱性蛋白质。利用RACE技术，从两种鲨鱼——日本异齿鲛和条纹斑竹鲨（Triakis scyllia）的胰腺总RNA构建了编码其DNase I的全长cDNA。核苷酸序列分析揭示了鲨鱼酶特有的两种结构改变：101位和104位的两个半胱氨酸残基被替换（这两个残基在所有其他脊椎动物的DNase I中高度保守），以及在一个必需的钙离子结合位点额外插入了一个苏氨酸或天冬酰胺残基。鲨鱼DNase I的定点诱变表明，这两种改变都降低了酶的稳定性。当将人DNase I的信号序列区域（具有高α-螺旋结构含量）替换为两栖动物、鱼类和鲨鱼的对应区域（具有低α-螺旋结构含量）时，转染哺乳动物细胞中的嵌合突变体构建体所表达的活性约为野生型酶的一半。相反，将人信号序列区域替换到两栖动物、鱼类和鲨鱼的酶中，与野生型相比产生了更高的活性。在分子进化过程中，脊椎动物DNase I家族可能通过成熟蛋白及其信号序列区域的结构改变，获得了该酶蛋白的高稳定性和有效表达。
