Molecular evolution of shark and other vertebrate DNases I.
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.
Yasuda
Toshihiro
T
Division of Medical Genetics and Biochemistry, Faculty of Medical Sciences, University of Fukui, Japan.
Iida
Reiko
R
Ueki
Misuzu
M
Kominato
Yoshihiko
Y
Nakajima
Tamiko
T
Takeshita
Haruo
H
Kobayashi
Takanori
T
Kishi
Koichiro
K
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003850:Deoxyribonuclease I / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology; D021382:Protein Sorting Signals; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D012754:Sharks; D014714:Vertebrates
D018076:DNA, Complementary; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I