Tài liệu Báo cáo khoa học: Molecular evolution of shark and other vertebrate DNases I pptx

Molecular evolution of shark and other vertebrate DNases I

Toshihiro Yasuda1

, Reiko Iida2

, Misuzu Ueki1

, Yoshihiko Kominato3

, Tamiko Nakajima3

, Haruo Takeshita3

,

Takanori Kobayashi4 and Koichiro Kishi3

1

Division of Medical Genetics and Biochemistry and 2

Division of Legal Medicine, Faculty of Medical Sciences, University of Fukui,

Japan; 3

Department of Legal Medicine, Gunma University Graduate School of Medicine, Japan; 4

National Research Institute of

Fisheries Science, Japan

We purified pancreatic deoxyribonuclease I (DNase I) from

the shark Heterodontus japonicus using three-step column

chromatography. Although its enzymatic properties resem￾bled 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 Ca2+-

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 a-helical structure content) was

replaced with its amphibian, fish and shark counterparts

(which have low a-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.

Keywords: cDNA cloning; deoxyribonuclease I; molecular

evolution; shark; signal sequence.

Deoxyribonuclease I (DNase I, EC 3.1.21.1) is present

principally in organs associated with the digestive system,

such as the pancreas and parotid glands, from which it is

secreted into the alimentary tract to hydrolyse exogenous

DNA [1–3]. Recently, it has been demonstrated that

DNase I-deficient mice have an increased incidence of

systemic lupus erythematosus (SLE), with classical findings

including the presence of autoreactive antibodies and

glomerulonephritis occurring in a DNase I-level-dependent

manner; this suggests that DNase I may protect against

autoimmunity by digesting extracellular nucleoprotein [4].

Furthermore, serum DNase I activity levels have been

reported to be lower in SLE patients than in healthy

subjects, resulting in expansion of the autoreactive lympho￾cytes that react with nucleosomal antigens [5,6]. Thus, it is

plausible that DNase I activity must be maintained at a

certain level in the serum to prevent the initiation of SLE.

We have also found that serum DNase I activity levels were

transiently reduced by somatostatin through an effect on

gene expression [7], and were elevated at the onset of acute

myocardial infarction [8]. These, together with other

findings suggesting that DNase I or DNase I-like endo￾nucleases may be responsible for internucleosomal DNA

degradation during apoptosis [9,10], have focused attention

on the potential physiological roles of DNase I. In this

context, we have attempted to elucidate the intrinsic intra￾and extracellular function(s) of DNase I, as well as the

phylogenetic origins of the vertebrate DNase I family, by

carrying out comprehensive comparisons of the enzymes

from lower and higher vertebrates: the biochemical

and molecular characterizations of mammalian [11–16],

avian [17], reptilian [18] and amphibian [19] DNases I

have already been reported. Previous studies on piscine

DNases I, from Oreochromis mossambica (tilapia) [20] and

five different species of the Osteichthye class [21], have

demonstrated that these enzymes possess some unique

features compared with those of other vertebrates: a

relatively high pH for optimum activity and greater

structural diversity. However, as all these species of fish

belong to the Osteichthyes, it remains unknown whether

these features are shared by species of Chondrichthyes. In

order to address this question, a systematic survey of

Chondrichthye DNases I is required. Chondrichthyes,

including sharks, separated from other vertebrates at the

most distant evolutionary stage on the phylogenetic tree. It

could therefore be expected that Chondrichthye DNase I

may conserve biochemical and molecular features inherent

in a postulated ancestral form of vertebrate DNase I to a

Correspondence to K. Kishi, Department of Legal Medicine,

Gunma University Graduate School of Medicine, Maebashi,

Gunma 371-8511, Japan. Fax: +81 27 220 8035,

E-mail: [email protected]

Abbreviations: SLE, systemic lupus erythematosus; SRED, single

radial enzyme diffusion; UTR, untranslated region.

Enzyme: DNase I (EC 3.1.21.1).

Note: The nucleotide sequence data reported will appear in DDBJ,

EMBL and GenBank Nucleotide Sequence Database under accession

numbers AB126699 and AB126700.

(Received 3 August 2004, revised 15 September 2004,

accepted 28 September 2004)

Eur. J. Biochem. 271, 4428–4435 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04381.x