Tài liệu Báo cáo khoa học: A single amino acid substitution of Leu130Ile in snake DNases I

A single amino acid substitution of Leu130Ile in snake DNases I

contributes to the acquisition of thermal stability

A clue to the molecular evolutionary mechanism from cold-blooded

to warm-blooded vertebrates

Haruo Takeshita1,*, Toshihiro Yasuda2,*, Tamiko Nakajima1

, Kouichi Mogi1

, Yasushi Kaneko1

,

Reiko Iida3 and Koichiro Kishi1

1

Department of Legal Medicine, Gunma University School of Medicine, Maebashi, Japan; 2

Department of Biology and 3

Department of Legal Medicine, Fukui Medical University, Matsuoka, Japan

We purified pancreatic deoxyribonucleases I (DNases I)

from three snakes, Elaphe quadrivirgata, Elaphe climaco￾phora and Agkistrodon blomhoffii, and cloned their cDNAs.

Each mature snake DNase I protein comprised 262 amino

acids. Wild-type snake DNases I with Leu130 were more

thermally unstable than wild-type mammalian and avian

DNases I with Ile130. After substitution of Leu130Ile, the

thermal stabilities of the snake enzymes were higher than

those of their wild-type counterparts and similar to mam￾malian wild-type enzyme levels. Conversely, substituting

Ile130Leu of mammalian DNases I made them more

thermally unstable than their wild-type counterparts.

Therefore, a single amino acid substitution, Leu130Ile,

might be involved in an evolutionally critical change in the

thermal stabilities of vertebrate DNases I. Amphibian

DNases I have a Ser205 insertion in a Ca2+-binding site of

mammalian and avian enzymes that reduces their thermal

stabilities [Takeshita, H., Yasuda, T., Iida, R., Nakajima, T.,

Mori, S., Mogi, K., Kaneko, Y. & Kishi, K. (2001) Biochem.

J. 357, 473–480]. Thus, it is plausible that the thermally stable

wild-type DNases I of the higher vertebrates, such as

mammals and birds, have been generated by a single

Leu130Ile substitution of reptilian enzymes through

molecular evolution following Ser205 deletion from

amphibian enzymes. This mechanism may reflect one of the

evolutionary changes from cold-blooded to warm-blooded

vertebrates.

Keywords: cDNAcloning; deoxyribonuclease I; molecular

evolution; snake; thermal stability.

Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an enzyme

that preferentially attacks, by Ca2+-and Mg2+-dependent

endonucleolytic cleavage, double-stranded DNAto

produce oligonucleotides with 5¢-phospho and 3¢-hydroxy

termini [1]. It is considered to play a major role in

digestion in the alimentary canal, because, in mammals, it

is secreted by exocrine glands such as the pancreas and/or

parotid gland [2–7]. However, DNase I also exists outside

the alimentary tract [8–11], raising a doubt as to whether

its major role in DNAmetabolism in vivo is merely

digestion. Recently, DNase I was postulated to be

responsible for the removal of DNAfrom nuclear

antigens at sites of high cell turnover and thus for the

prevention of systemic lupus erythematosus (SLE) [12].

The gene product of human DNASE1*6 was more

thermally unstable than that of the other alleles and

subjects who were heterozygous for this allele had

significantly low serum DNase I activity levels [13]. These

findings indicate that the thermal stabilities of DNase I

in vitro might reflect the enzyme activities in vivo. We

found that amphibian DNases I are characterized by a C￾terminal end with a unique cysteine-rich stretch and by

insertion of a Ser residue into the Ca2+-binding site,

resulting in thermal instability compared with DNases I

from mammals and birds [14]. Fish DNase I also

exhibited similar low thermal stability relative to amphi￾bian DNases I (K. Mogi, H. Takeshita, T. Yasuda,

T. Nakajima, E. Nakazato, Y. Kaneko, M. Itoi &

K. Kishi, personal communication). In these contexts, it

would be very interesting how the higher vertebrates, such

as mammals and birds, which are also classified as warm￾blooded vertebrates, have acquired thermal stability of

their DNase I molecules through the evolutionary steps

from the lower, cold-blooded, vertebrates, such as amphi￾bia and fish.

We have already reported the purification and biochemi￾cal characterization of mammalian [4,5,7,14–18], avian [19]

Correspondence to K. Kishi, Department of Legal Medicine,

Gunma University School of, Medicine, Maebashi,

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

E-mail: kkoichi@med.gunma-u.ac.jp

Abbreviations: aa, amino acid; Con A, Concanavalin A;

nt, nucleotide; SLE, systemic lupus erythematosus;

SRED, single radial enzyme diffusion.

Enzymes: DNase I, (EC 3.1.21.1).

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

EMBL and GenBank Nucleotide Sequence Databases under accession

nos. AB046545, AB050701 and AB058784.

*Note: These authors contributed equally to this research and listed

in alphabetical order.

(Received 23 September 2002, revised 18 November 2002,

accepted 25 November 2002)

Eur. J. Biochem. 270, 307–314 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03387.x