Tài liệu Báo cáo khoa học: 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 docx

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 Takeshita
1,
*, Toshihiro Yasuda
2,
*, Tamiko Nakajima
1
, Kouichi Mogi
1
, Yasushi Kaneko
1
,
Reiko Iida
3
and Koichiro Kishi
1
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
DNasesIhaveaSer205insertioninaCa
2+
-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: cDNA cloning; deoxyribonuclease I; molecular
evolution; snake; thermal stability.
Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an enzyme
that preferentially attacks, by Ca
2+
-and Mg
2+

-dependent
endonucleolytic cleavage, double-stranded DNA to
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 DNA metabolism in vivo is merely
digestion. Recently, DNase I was postulated to be
responsible for the removal of DNA from 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 Ca
2+
-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:
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) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03387.x
and amphibian [14] DNases I. As the primary structures of
amphibian DNases I differ considerably from those of
other vertebrate DNases I, comprehensive characterization,
including determination of thermal stabilities, of reptilian
DNases I is required, not only to elucidate the molecular
evolutional aspect of the DNase I family but also to address

the queries about how the higher vertebrate DNases I
acquired their thermal stabilities described above. In this
study, we purified DNases I from the pancreases of three
snake species, Elaphe quadrivirgata (Shima-hebi in Japan-
ese) and Elaphe climacophora (Aodaisho) of the Colubridae
OPPEL and Agkistrodon blomhoffii (Nihon-mamushi) of
the Viperidae Laurenti, which are widely distributed in
Japan, and cloned the cDNA of each. A single amino acid
(aa) substitution was confirmed to affect the thermal
stabilities of vertebrate DNases I and, furthermore, one of
the postulated mechanisms whereby thermal stability is
acquired by a DNase I family at the evolutional step from
cold-blooded vertebrates, such as snakes, to warm-blooded
ones, such as mammals, is discussed.
Materials and methods
Materials and biological samples
Three different species of snake, E. quadrivirgata,
E. climacophora and A. blomhoffii weighing about 210 g
(110 cm long), 270 g (130 cm long) and 120 g (70 cm long),
respectively, were obtained from the Japan Snake Institute,
Gunma, Japan. Phenyl Sepharose CL-4B, DEAE Seph-
arose CL-6B and Superdex 75 were purchased from
Amersham Pharmacia Biotech; Concanavalin A (Con A)-
agarose was from Seikagaku Kogyo (Tokyo, Japan); rabbit
muscle G-actin and salmon testis DNA were from Sigma;
Superscript II reverse transcriptase (RT), all the oligonu-
cleotide primers used, and the RACE systems were from
Life Technologies; the Expanded High Fidelity PCR system
was from Roche Diagnostics. All the other chemicals used
were of reagent grade and available commercially. The

snakes and Japanese white rabbits were acquired, main-
tained and used in accordance with the Guidelines for the
Care and Use of Laboratory Animals (NIH, USA; revised
1985).
Analytical methods
DNase I activity was assayed by the previously described
test tube [15] or single radial enzyme diffusion (SRED) [2]
methods, except that 50 m
M
Tris/HCl buffer, pH 7.5,
containing 10 m
M
MgCl
2
and 1 m
M
CaCl
2
was substi-
tuted for the reaction buffer. Protein concentrations were
measured using a protein assay kit (Bio-Rad) with BSA as
the standard. The enzymological properties of the snake
enzymes and the inhibitory effects of specific antibodies
on their activities were examined as described previously
[4,15,20]. Samples of 15 different tissues were obtained
from each snake as soon as possible after it had been
killed by exsanguination under general anesthesia with
diethyl ether. Preparation of the samples for the assays
and determination of enzyme activity were performed as
described previously [7,21–22]. The N-terminal aa se-

quences of the purified enzymes were determined by
Edman degradation [4]. The presence of DNase I-specific
mRNA was verified by RT-PCR amplification of the total
RNA extracted from each snake tissue using sets of
primers corresponding to the N- and C-terminal aa
sequences of the respective enzymes [23].
Purification of DNases I from snake pancreases
All the procedures described below were carried out at
0–4 °C. Pancreas samples, weighing approximately 3.4, 1.2,
and 0.7 g, were obtained from five individuals each of the
species E. quadrivirgata, E. climacophora and A. blomhoffii,
respectively. The samples were minced separately and
homogenized in 5–10 mL 25 m
M
Tris/HCl buffer, pH 7.5
(buffer I), containing 1
M
ammonium sulfate and 1 m
M
phenylmethane sulfonyl fluoride. After centrifugation
(10 000 g, 20 min), the supernatant (crude extract) was
applied to a first phenyl Sepharose CL-4B column
(1.6 · 15 cm) pre-equilibrated with the same buffer and
the adsorbed materials were eluted with a 300-mL linear
reverse ammonium sulfate concentration gradient (1.0–0
M
)
in buffer I. The active fractions eluted with about 500 m
M
ammonium sulfate were collected and solid ammonium

sulfate was added to give a concentration of 1.0
M
.Thiswas
then applied to a second phenyl Sepharose CL-4B column
(1 · 10 cm) pre-equilibrated with the same buffer. The
DNase I was eluted with 100 mL of the same gradient. The
active fractions were collected, dialyzed against buffer I,
applied to a DEAE Sepharose CL-6B column (1 · 15 cm)
pre-equilibrated with buffer I and the adsorbed materials
were eluted with a 100-mL linear NaCl concentration
gradient (0–1.0
M
) in buffer I. The active fractions eluted
over the NaCl concentration range of 250–300 m
M
were
concentrated using polyethylene glycol 6,000, then subjec-
tedtogelfiltrationusingtheA
¨
KTA FPLC system
(Amersham Pharmacia Biotech) equipped with a Superdex
75 column (1.6 · 60 cm) with buffer I containing 150 m
M
NaCl as the eluent. The active fractions were collected and
then applied to a Con A-agarose column (1 · 2cm)pre-
equilibrated with buffer I containing 150 m
M
NaCl. The
column was washed well with the same buffer and then
DNase I was eluted with 300 m

M
methyl-a-
D
-mannopyr-
anoside in the same buffer. The active fractions were
collected and used as the purified enzymes for the
subsequent experiments. A specific rabbit antibody against
purified DNase I from E. quadrivirgata was prepared as
described previously [15].
Construction of the cDNA species encoding
E. quadrivirgata
,
E. climacophora
and
A. blomhoffii
DNases I
Total RNA was isolated from each snake pancreas by the
acid guanidinium thiocyanate/phenol/chloroform method
[24] and any DNA contamination was removed by treat-
ment with RNase-free DNase I (Stratagene). The 3¢-end
region of cDNAs of all the snakes were obtained by
3¢-RACE method using two degenerate primers based on aa
sequences that are highly conserved in vertebrate DNase I,
the Tyr97–Cys104 and Met166–Cys173 sequences of
human DNase I [25]. Next, the 5¢-end regions of the
cDNAs were amplified by the 5¢-RACE method using
308 H. Takeshita et al. (Eur. J. Biochem. 270) Ó FEBS 2003
gene-specific primers based on the nucleotide (nt) sequences
determined in this study. These RACE procedures were
carried out using the 3¢-and5¢-RACE systems described

above, according to the manufacturer’s instructions. The
RACE products were subcloned into the pCR II TA
cloning vector (Invitrogen, San Diego, CA, USA) and
sequenced. The nt sequences were determined by the
dideoxy chain-termination method using a Dye Terminator
Cycle sequencing kit (Applied Biosystems, Urayasu, Japan).
The sequencing run was performed on a Genetic Analyzer
(model 310, Applied Biosystems) and all the DNA
sequences were confirmed by reading both strands.
Construction of expression vectors and transient
expression of the constructs in COS-7 cells
A DNA fragment containing the entire coding sequence
of E. quadrivirgata DNase I cDNA was prepared from
the total RNA derived from the pancreas by RT-PCR
amplification using an Expanded High Fidelity PCR
system with a set of two primers, 5¢-GAATTCGAGGCC
ATGAAGACCATCTTG-3¢ (sense) and 5¢-CTCGAGG
GGCTCAGGTGGATTTTAGG-3¢ (antisense), corres-
ponding to the nt sequences of the cDNA from positions
28–48 and 867–885, respectively. The amplified fragment
was ligated into the pcDNA3.1 (+) vector (Invitrogen) to
construct the expression vector. Six other expression
vectors with cDNA inserts encoding E. climacophora,
A. blomhoffii, Xenopus laevis, human, rat and mouse
DNases I, were prepared in the same manner. Two
substitution mutants for surveying the G-actin binding site
at aa position 67, E. quadrivirgata (Ile67Val) and
A. blomhoffii (Val67Ile), in which an Ile or Val residue
was substituted with Val or Ile, respectively, at aa position
67 in E. quadrivirgata and A. blomhoffii DNases I,

respectively, were constructed using the splicing by overlap
extension method [26] with the corresponding wild-type
construct as a template. Ten mutants for surveying the
aa sites responsible for thermal stability at positions 130
and 166, E. quadrivirgata (Leu130Ile), A. blomhoffii
(Leu130Ile), human (Ile130Leu), rat (Ile130Leu), mouse
(Ile130Leu), E. quadrivirgata (Leu166Met), A. blomhoffii
(Leu166Met), human (Met166Leu), rat (Met166Leu) and
mouse (Met166Leu), and four double substitution
mutants, E. quadrivirgata (Leu130Ile/Leu166Met), rat
(Ile130Leu/Met166Leu), mouse (Ile130Leu/Met166Leu)
and human (Ile130Leu/Met166Leu), were prepared in
the same manner. All the constructs had their sequences
confirmed and were purified for transfection using the
Plasmid Midi kit (Qiagen). Transient expression of
the constructs in COS-7 cells followed by analysis of the
enzyme was performed as described previously [27]. All
transfections were performed in triplicate with at least two
different plasmid preparations.
Phylogenetic analysis
The nt sequences of the DNase I cDNA of human [28],
mouse [17], rat [29], rabbit [5], pig [16], fish (Oreochromis
mossambicus) [30], cow [18], chicken [19], two frogs, toad
and newt [14] with the following respective database
accession numbers EMBL M55983, EMBL U00478,
EMBL X56060, EMBL D82875, EMBL AB048832,
EMBL AJ001305, EMBL AJ001538, EMBL AB013751,
EMBL AB030958, EMBL AB038776, EMBL AB045037
and EMBL AB041732 were obtained. Phylogenetic trees
with the nt sequence of the open reading frame (ORF) of

their cDNAs and the corresponding aa sequences, in
Table 1. Summary of the purification of DNases I from the pancreases of three species of snake. The results of the sequential enzyme purification
procedure, using pancreases obtained from five individuals of each species as starting material, are summarized.
Species and purification step Protein (mg) Total activity (U) Specific activity (UÆmg
)1
) Purification (fold) Yield (%)
E. quadrivirgata
Crude extract 600 1100 1.8 1.0 100
Phenyl Sepharose CL-4B 120 970 8.1 4.5 88
28 950 34 20 86
DEAE Sepharose CL-6B 5.4 780 140 80 71
Superdex 75 0.55 770 1400 780 70
Con-A agarose 0.3 750 2500 1400 68
A. blomhoffii
Crude extract 220 400 1.8 1.0 100
Phenyl Sepharose CL-4B 45 350 7.8 4.3 88
10 330 33 18 83
DEAE Sepharose CL-6B 2.0 210 110 58 53
Superdex 75 0.23 200 870 480 50
Con-A agarose 0.05 190 3800 2100 48
E. climacophora
Crude extract 110 290 2.6 1 100
Phenyl Sepharose CL-4B 18 280 16 6 97
5 270 51 20 88
DEAE Sepharose CL-6B 0.5 160 320 122 55
Superdex 75 0.1 120 1200 460 41
Con-A agarose 0.02 100 5000 1900 34
Ó FEBS 2003 A mechanism from cold-blooded to warm-blooded vertebrates (Eur. J. Biochem. 270) 309
which the region of the putative signal peptide was not
included, aligned by the neighbor-joining algorithm using

the
CLUSTALW
program were constructed [31,32].
Results and discussion
Purification and characterization of snake pancreatic
DNases I
The purification results for each snake pancreatic DNase I
are summarized in Table 1. This procedure, using four
different types of column chromatography, allowed all
three snake DNases I to be easily and reproducibly
isolated and purified to electrophoretic homogeneity
(Fig. 1, A1), representing 1400- to 2100-fold purification.
Tandem phenyl Sepharose chromatographies without loss
of total enzyme activity were a particularly effective step
for purifying each snake enzyme. Both gel filtration and
SDS/PAGE analysis showed that E. quadrivirgata
DNaseIhadamolecularmassof35kDa.ItspH
optimum of 7.5 was higher than those of mammalian
enzymes [4,5,7,15–17] and lower than those of amphibia
(pH 8.0) [14]. Edman degradation of the purified E. quad-
rivirgata and A. blomhoffii enzymes revealed the same
N-terminal aa sequences over the first 10 cycles: Leu1-
Arg-Ile-Gly-Ala-Phe-Asn-Ile-Arg-Ala10.
Although G-actin is known to be a potent inhibitor of
human [15], cow [33] and mouse [17] DNases I, the
activities of rat [4], porcine [16], chicken [19] and
amphibian [14] DNases I were unaffected by G-actin.
G-actin (3.2 n
M
) abolished the enzyme activity of human

DNase I (0.3 units) and reduced that of A. blomhoffii
wild-type DNase I to 50% of its initial level, but did not
affect the activities of E. quadrivirgata or E. climacophora
wild-type DNases I. It has been suggested that two aa
residues (Tyr65 and Val67) are mainly responsible for
actin binding in human and bovine DNases I [34,35]. The
latter residue (Ile67) in both E. quadrivirgata and
E. climacophora DNase I was substituted. In comparison
with the susceptibility of each wild-type enzyme to
G-actin, the susceptibility of the E. quadrivirgata (Ile67-
Val) enzyme was increased to the level of the wild-type
A. blomhoffii enzyme, whereas the A. blomhoffii (Val67Ile)
enzyme and E. quadrivirgata wild-type enzymes were
equally susceptible. These findings show that the presence
of Val67 is one of the essential factors responsible for the
actin-binding capacity of these snake DNases I.
Tissue distribution of snake DNases I
The DNase I activities in the 15 different tissues from
each snake were determined. The activity detected in the
pancreas of each snake was over three orders of magni-
tude greater than that in the small intestine. However,
other tissues listed in Fig. 1 exhibited no DNase I activity
under our assay conditions. These enzyme activities were
abolished by 20 m
M
EDTA, 5 m
M
EGTA and the
appropriate specific anti-DNase I Ig, which confirmed
they were due to DNase I. The presence of DNase

I-specific mRNA was verified by RT-PCR analysis of the
total RNA extracted from each snake tissue (Fig. 1B).
Specific PCR products were amplified only from the
pancreatic and small intestinal total RNAs of the three
snakes. No amplified products were obtained from the
RNAs of the other tissues. The restriction of DNase I
gene expression to only two tissues, the pancreas and
small intestine, in snakes and amphibia [14] contrasts with
the situation in mammals and birds, in which more
widespread expression has been observed in various
tissues, including the kidney, liver and stomach, as well
as the pancreas and small intestine [2,7,19].
cDNA structures encoding three snake DNases I
and expression of the DNase I cDNAs in COS-7 cells
The total RNAs isolated from the pancreases of the three
snake species were amplified separately by the 3¢-and
5¢-RACE methods to construct cDNAs encoding
DNases I. The use of two primers based on aa sequences
Fig. 1. Electrophoretic patterns of snake purified DNases I and
recombinant enzymes (A), and RT-PCR analysis of the total RNA from
several tissues of E. quadrivirgata (B). (A1) Purified DNase I (about
1 lg) from human urine (lane 1), E. quadrivirgata pancreas (lane 2),
E. climacophora pancreas (lane 3) and A. blomhoffii pancreas (lane 4)
were subjected to SDS/PAGE using a 12.5% gel [20], followed by silver
staining. (A2) Recombinant E. quadrivirgata, E. climacophora and
A. blomhoffii DNases I expressed in COS-7 cells were subjected to
activity staining for DNase I using a DNA-cast PAGE method [21,22]:
aliquots containing about 0.2 units of activity of the purified (lanes 1, 3
and 5) and recombinant (lanes 2, 4 and 6) enzymes were used. Lanes 1
and 2, E. quadrivirgata enzyme; lanes 3 and 4, E. climacophora

enzyme; lanes 5 and 6, A. blomhoffii enzyme. The cathode is at the top.
(B) The total RNA isolated from several tissues, including the pancreas
(lane 2), small intestine (lane 3), liver (lane 4), kidney (lane 5), large
intestine (lane 6), stomach (lane 7) and parotid gland (lane 8) of
E. quadrivirgata was reverse-transcribed and PCR-amplified with a set
of specific primers. A unique 850-bp fragment corresponding to the
region encoding E. quadrivirgata DNase I was amplified from only the
pancreas and small intestine: the cerebrum, heart, lung, spleen, skin,
muscle, esophagus and Harder’s gland gave no amplified fragment.
Also, RT-PCR analysis of the corresponding set of tissues from
E. climacophora and A. blomhoffii exhibited the same results as
E. quadrivirgata (data not shown). Lane 1 contains a DNA marker
derived from /X174 DNA digested with HaeIII.
310 H. Takeshita et al. (Eur. J. Biochem. 270) Ó FEBS 2003
that are highly conserved in vertebrate DNases I allowed
successful amplification of specific RACE products from
the total RNA of each species. The full-length cDNA
encoding E. quadrivirgata DNase I (accession number
AB046545) comprised 1071 bp, including an ORF of
849 bp coding for 283 aas, a 33-bp 5¢-untranslated region
(UTR) and a 189-bp 3¢-UTR. The sequence flanking the
first ATG (positions 34 –36) was in accordance with the
Kozak consensus for a translation start site [36]. We also
cloned and sequenced the cDNA species encoding the
DNases I of A. blomhoffii (accession number AB050701)
and E. climacophora (accession number AB058784) and
found full-length sequences of 1050 and 1071 bp, respect-
ively. The entire nt sequences of both the ORF and
5¢-UTR regions of E. climacophora DNase I cDNA were
identical to those of E. quadrivirgata, but 8.4% (16/189) of

the entire nt sequence in the 3¢-UTR of the former was
different from that of the latter. The N-terminal aa
sequences determined chemically from the purified
enzymes exactly matched those deduced from the cDNA
data of E. quadrivirgata and A. blomhoffii, indicating that
each putative upstream signal sequence containing the first
Met residue was 20 aa residues long. About 12% (33/283)
of the aa residues in the entire sequence of A. blomhoffii
deduced from its cDNA data differ from those of
E. quadrivirgata and E. climacophora.
Each expression vector containing the entire coding
region of E. quadrivirgata, E. climacophora or A. blomhoffii
DNase I cDNA was transiently transfected into COS-7
cells. The snake enzyme activities expressed in the COS-7
cells were abolished by the appropriate specific antibodies
and, furthermore, they migrated to positions corresponding
to the purified pancreatic enzymes on the DNA-cast PAGE
gel (Fig. 1A2), confirming that the isolated cDNAs did
indeed encode the expected snake DNases I. Comparison of
the predicted primary structure (Fig. 2) with human,
chicken and frog sequences allowed us to demonstrate
several common structural features unique to the snake
enzymes. The four residues responsible for the catalytic
activity of the other vertebrate DNases I, Glu78, His134,
Asp212 and His252 [5,14–19,25,28,33], were conserved in all
the snake enzymes. Cys173 and Cys209, which form the
disulfide bond responsible for the stability of the enzyme
[37], and also Arg41 and Tyr76, that mediate DNase I–
DNA contact in the other vertebrate DNases I and
orientate the scissile phosphate relative to the enzyme [38],

were also found in all the snake enzymes. The unique
cysteine-rich-terminus and inserted Ser in the Ca
2+
-binding
site of amphibian DNases I [14], were not observed in the
snake enzymes. As in mammalian, but not amphibian
DNases I [14–19,25,28,33], two potential N-linked glycosy-
lation sites, Asn18 (Asn-Gln-Thr) and Asn106 (Asn-Gly/
Thr-Thr), were well present in the snake enzymes, which
also showed high affinity for Con A–lectin. These findings
indicate that, with respect to their structural relationships,
snake DNases I are far from amphibian enzymes, but close
to mammalian and avian DNases I.
Thermal stabilities of wild-type and substitution
mutant snake DNases I
The thermal stabilities of the wild-type and mutant
enzymes of the snakes were examined by measuring the
activities remaining after incubation for 40 min at various
temperatures (Fig. 3). Wild-type snake and amphibian
DNases I are more thermally unstable than those of higher
vertebrates, such as the human, rabbit, rat, mouse and
chicken [4,5,15,17,19]. When the primary structures of the
three snake enzymes were compared with those of other
vertebrates, only two aa residues, Leu130 and Leu166, of
Fig. 2. Alignment of the amino acid sequences of snake DNases I with those of human, chicken and X. laevis DNases I. The aa sequences of the snake
DNases I were deduced from their respective cDNAs and compared with those published for human, chicken and X. laevis DNases I. Position 1 aa
was assigned by comparison with the N-terminal aa sequences determined chemically from the purified enzymes. The aa sequences of
E. climacophora DNase I are not shown because they were identical to those of the E. quadrivirgata enzyme. Alignment of the sequences was
performed using the Genedoc program (available at />Ó FEBS 2003 A mechanism from cold-blooded to warm-blooded vertebrates (Eur. J. Biochem. 270) 311
the former were observed in all the lower vertebrates

studied, i.e. four amphibia [14] and one fish [37], which are
all classified as cold-blooded vertebrates. These two
residues were replaced by Ile130 and Met166, respectively,
in warm-blooded vertebrates, i.e. the human [28], cow [18],
pig [16], rabbit [5], rat [4], mouse [17] and chicken [19].
These findings furnished us with a clue to a mechanism
whereby DNases I have evolved, i.e. Leu130Ile and/or
Leu166Met might convert the thermally unstable DNases I
of cold-blooded vertebrates to the thermally stable ones of
warm-blooded vertebrates. Therefore, we constructed a
series of substitution mutant enzymes (Fig. 3). In brief,
E. quadrivirgata, A. blomhoffii and X. laevis wild-type
DNases I were all less thermally stable than human, rat
and mouse wild-type enzymes. The mutant enzymes
E. quadrivirgata (Leu130Ile), E. quadrivirgata (Leu130Ile/
Leu166Met) and A. blomhoffii (Leu130Ile) were all more
thermally stable than the corresponding wild-type
DNases I, whereas E. quadrivirgata (Leu166Met) and
A. blomhoffii (Leu166Met) were as thermally unstable as
their wild-type counterparts. These results suggest that
Leu130Ile conferred increased thermal stabilities to the
snake enzymes, but Leu166Met did not. Conversely, the
human (Ile130Leu) and human (Ile130Leu/ Met166Leu)
mutants were more thermally unstable than their wild-type
counterparts, whereas human (Met166Leu) was not. The
same was true for these mutants of the rat and mouse
DNase I (Fig. 3). These findings demonstrate that the
nature of the amino acid at position 130 may generally and
markedly affect the thermal stabilities of vertebrate
DNases I. The 3D structure of DNase I based on X-ray

structure analysis of the bovine enzyme has demonstrated
that the central core of DNase I is formed by two tightly
packed six-stranded b-sheets and that the extended
hydrophobic core is mainly responsible for the structural
stability and rigidity of DNase I [37,39]. The aa residue at
position 130 is located in the central core, whereas that at
position 166 is not. Accordingly, it could be predicted that
a substitution of the former residue might induce some
alterations in the structural stability of DNase I, whereas
that of the latter would not. These predictions were found
to be compatible with the experimental results described
above. Therefore, these facts suggest that the aa residue at
position 130 may be responsible for the thermal stability of
DNase I.
We have reported another mechanism of generating a
thermally stable enzyme form from a thermally unstable
one: frog, toad and newt DNases I all have a Ser205
insertion in a domain that contains an essential Ca
2+
-
binding site in the mammalian enzymes and are thermally
Fig. 3. Comparison of the thermal stabilities of wild-type and mutant
DNases I derived from snakes (A), human (B) and other vertebrates (C).
Each wild-type and mutant DNase I sample (1.0 unit) was incubated
in 50 m
M
Tris/HCl buffer, pH 7.5, for 40 min at various temperatures,
as indicated in the figure, using a Dry Thermo Unit DTU-2B (TAI-
TEC, Saitama, Japan), and then its residual activity was measured by
the SRED method (2). The temperature of thermal denaturation (T

1/2
)
is defined as that at which the DNase I activity is halved and shown in
the figure. The value for each enzyme represents triplicate determina-
tions and the assay precision was estimated to be within 10%. (A)
E. quadrivirgata wild-type (s), E. quadrivirgata (Leu130Ile) (d),
E. quadrivirgata (Leu130Ile/Leu166Met) (j)andE. quadrivirgata
(Leu166Met) (h) DNases I. The thermal stabilities of A. blomhoffii
wild-type, A. blomhoffii (Leu130Ile) and A. blomhoffii (Leu166Met)
enzymes were similar to those of the corresponding E. quadrivirgata
enzymes. (B) Human wild-type (s), human (Ile130Leu) (d), human
(Ile130Leu/Met166Leu) (j) and human (Met166Leu) (h)DNasesI.
(C) Rat wild-type (s), rat (Met166Leu) (h), rat (Ile130Leu) (d), rat
(Ile130Leu/Met166Leu) (j)andX. laevis wild-type (m)DNasesI.
The thermal stabilities of the mouse wild-type, mouse (Ile130Leu),
mouse (Ile130Leu/Met166Leu) and mouse (Met166Leu) enzymes were
very similar to those of the corresponding rat enzymes.
312 H. Takeshita et al. (Eur. J. Biochem. 270) Ó FEBS 2003
unstable [14]. Insertion of a corresponding Ser residue
between Ala204 and Thr205 of human and rat DNases I
reduced their thermal stabilities to levels similar to those of
amphibian enzymes. These findings led us to conclude that
there are at least two mechanisms that might be involved in
changing the thermally stable characteristics of vertebrate
DNases I, substitution of Ile130Leu in snakes and insertion
of Ser205 in amphibia. It is interesting that DNases I of
warm-blooded vertebrates, such as humans, pigs, rabbits,
rats and chicken, are all thermally stable, while those of
cold-blooded vertebrates, such as snakes, frogs, toads and
newts, are all thermally unstable. It could be postulated that

the thermally stable DNases I of the higher vertebrates must
have been produced principally by the Leu130Ile substitu-
tion first in avian enzymes at the evolutionary stage from
reptiles to birds after deletion of Ser205 from the enzymes of
amphibians as they evolved into reptiles. Thermal stability
of the enzyme might be evaluated as one of the factors that
reflect the DNase I activity levels in vivo [13].Asalackof,or
decrease in, DNase I activity has been suggested to be a
critical factor in the initiation of human and rat SLE [12,40],
the acquisition of thermally stable characteristics during
DNase I evolution may provide a clue to the etiology of
SLE in humans and mice, which are classified as warm-
blooded vertebrates.
Phylogenetic analyses of interspecies variations
in the DNase I family
Based on both the aa and nt sequences of 14 vertebrate
DNases I, the phylogenetic trees for the DNase I family
were constructed (Fig. 4). The bootstrap values calculated
in the tree based on the aa sequence were lower than those in
the latter tree, and an alignment of nt sequences was found
to be more adequate for molecular evolutionary analysis of
the DNase I family. The mammalian group formed a
relatively tight cluster, while the snake (E. quadrivirgata,
E. climacophora and A. blomhoffii), amphibian (X. laevis,
Rana catesbeiana, Bufo vulgaris japonicus and Cynops
pyrrhogaster), avian (chicken) and fish (O. mossambicus)
DNases I were individually situated at independent posi-
tions far from the mammalian DNase I cluster. The snake
enzymes are placed closer to the avian than the amphibian
forms. With regard to the evolutionary origin of birds,

conflicting results of phylogenetic analysis supporting a
bird–mammal or bird–reptile relationship have been repor-
ted [41,42]. However, our data based on the nt sequences of
DNase I molecules may provide evidence of a bird–reptile
rather than bird–mammal relationship.
Acknowledgements
We thank Dr Atsushi Sakai, The Japan Snake Institute, Gunma,
Japan, for providing us three kinds of snake. We thank Mrs Masako
Itoi and Miss Emiko Nakazato for their excellent technical
assistance. This work was supported in part by Grants-in-Aid from
Japan Society for the Promotion of Science (12770216 to H. T.,
12307011, 14657111 to K. K. and 12357003 to T. Y.) and grants
from the Japan Science Society 2001 to K. M. (JSS-12-153), Uehara
Memorial Foundation 2002 to H. T. and Daiwa Securities Health
Foundation to K. K.
References
1. Moore, S. (1981) Chapter 15. Pancreatic DNase. In The Enzymes
(Boyer, P.D., ed), Vol. 14, 3rd edn. pp. 281–296. Academic Press,
New York, USA.
2.Nadano,D.,Yasuda,T.&Kishi,K.(1993)Measurementof
deoxyribonuclease I activity in human tissues and body fluids by a
single radial enzyme-diffusion method. Clin. Chem. 39, 448–452.
3. Takeshita, H., Yasuda, T., Nadano, D., Tenjo, E., Sawazaki, K.,
Iida, R. & Kishi, K. (1994) Detection of deoxyribonuclease I and
II (DNases I and II) activities in reproductive organs of male
rabbits. Int. J. Biochem. 26, 1025–1031.
4. Takeshita, H., Yasuda, T., Nadano, D., Iida, R. & Kishi, K.
(1995) Deoxyribonuclease I from rat urine: affinity purification,
characterization, and immunochemical studies. J. Biochem. 118,
932–938.

5. Yasuda, T., Takeshita, H., Nakajima, T., Hosomi, O., Nakashima,
Y. & Kishi, K. (1997) Rabbit DNase I: purification from urine,
immunological and proteochemical characterization, nucleotide
sequence, expression in tissues, relationships with other mamma-
lian DNases I and phylogenetic analysis. Biochem. J. 325, 465–473.
6. Tsutsumi, S., Kaneko, Y., Asao, T., Kuwano, H., Kudo, S.,
Takeshita, H., Yasuda, T. & Kishi, K. (2001) DNase I is present
in the chief cells of human and rat stomachs. Histochem. J. 33,
531–535.
7. Takeshita, H., Mogi, K., Yasuda, T., Nakajima, T., Nakashima,
Y., Mori, S., Hoshino, T. & Kishi, K. (2000) Mammalian deoxy-
ribonucleases I are classified into three types: pancreas, parotid,
Fig. 4. Phylogenetic analysis of the vertebrate DNase I family based on their aa (A) and nt (B) sequences. The nt and aa sequences of the DNase I
proteins of various species were subjected to phylogenetic analysis. The numbers in figure represent bootstrap values calculated using the
CLUSTALW
program. Frog, Rana catesbeiana; toad, Bufo vulgaris japonicus;newt,Cynops pyrrhogaster;fish(tilapia),Oreochromis mossambicus.
Ó FEBS 2003 A mechanism from cold-blooded to warm-blooded vertebrates (Eur. J. Biochem. 270) 313
and pancreas-parotid (mixed), based on differences in their tissue
concentrations. Biochem. Biophys. Res. Commun. 269, 481–484.
8. Zhivotovsky, B., Wade, D., Nicotera, P. & Orrenius, S. (1994)
Role of nucleases in apoptosis. Int. Arch. Allergy Immunol. 105,
333–338.
9. Yasuda, T., Sawazaki, K., Nadano, D., Takeshita, H., Nakanaga,
M. & Kishi, K. (1993) Human seminal deoxyribonuclease I
(DNase I): purification, enzymological and immunological char-
acterization and origin. Clin. Chim. Acta 218, 5–16.
10. Polzar, B., Zanotti, S., Stephan, H., Rauch, F., Peitsch, M.C.,
Irmler, M., Tschopp, J. & Mannherz, H.G. (1994) Distribution of
deoxyribonuclease I in rat tissues and its correlation to cellular
turnover and apoptosis (programmed cell death). Eur. J. Cell Biol.

64, 200–210.
11. Peitsch, M.C., Irmler, M., French, L.E. & Tschopp, J. (1995)
Genomic organisation and expression of mouse deoxy-
ribonuclease I. Biochem. Biophys. Res. Commun. 207, 62–68.
12. Napirei, M., Karsunky, H., Zevnik, B., Stephan, H., Mannherz,
H.G. & Mo
¨
ro
¨
y, T. (2000) Features of systemic lupus
erythematosus in DNase1-deficient mice. Nat. Genet. 25, 177–181.
13. Yasuda, T., Takeshita, H., Iida, R., Kogure, S. & Kishi, K. (1999)
A new allele, DNASE1*6, of human deoxyribonuclease I poly-
morphism encodes an Arg to Cys substitution responsible for its
instability. Biochem. Biophys. Res. Commun. 260, 280–283.
14. Takeshita, H., Yasuda, T., Iida, R., Nakajima, T., Mori, S., Mogi,
K., Kaneko, Y. & Kishi, K. (2001) Amphibian DNases I are
characterized by a C-terminal end with a unique, cysteine-rich
stretch and by the insertion of a serine residue into the Ca
2+
-
binding site. Biochem. J. 357, 473–480.
15. Yasuda,T.,Awazu,S.,Sato,W.,Iida,R.,Tanaka,Y.&Kishi,K.
(1990) Human genetically polymorphic deoxyribonuclease: pur-
ification, characterization, and multiplicity of urine deoxy-
ribonuclease I. J. Biochem. 108, 393–398.
16. Mori, S., Yasuda, T., Takeshita, H., Nakajima, T., Nakazato, E.,
Mogi, K., Kaneko, Y. & Kishi, K. (2001) Molecular, biochemical
and immunological analyses of porcine pancreatic DNase I.
Biochim. Biophys. Acta 1547, 275–287.

17. Takeshita, H., Yasuda, T., Nakajima, T., Hosomi, O.,
Nakashima, Y. & Kishi, K. (1997) Mouse deoxyribonuclease I
(DNase I): biochemical and immunological characterizations,
cDNA structure and tissue distribution. Biochem. Mol. Biol. Int.
42, 65–75.
18. Chen,C Y.,Lu,S C.&Liao.T H.(1998)Cloning,sequencing
and expression of a cDNA encoding bovine pancreatic deoxy-
ribonuclease I in Escherichia coli: purification and characterization
of the recombinant enzyme. Gene 206, 181–184.
19. Nakashima, Y., Yasuda, T., Takeshita, H., Nakajima, T.,
Hosomi, O., Mori, S. & Kishi, K. (1999) Molecular, biochemical
and immunological studies of hen pancreatic deoxyribonuclease I.
Int. J. Biochem. Cell Biol. 31, 1315–1326.
20. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T
4
. Nature (London) 227,
680–685.
21. Nakajima, T., Yasuda, T., Nakashima, Y., Hosomi, O., Takesh-
ita, H. & Kishi, K. (1998) Two novel screening methods for
selecting monoclonal antibodies which specifically inhibit DNase I
enzyme activity. Immunol. Invest. 27, 145–152.
22. Nakajima, T., Yasuda, T., Takeshita, H., Nakashima, Y., Mori,
S., Mogi, K. & Kishi, K. (2000) Rapid purification of human
DNase I using mouse monoclonal anti-DNase I antibodies and
characterization of the antibodies. Exp. Clin. Immunogenet. 17,
71–76.
23. Yasuda, T., Takeshita, H., Iida, R., Nakajima, T., Hosomi, O.,
Nakashima, Y. & Kishi, K. (1998) Molecular cloning of the
cDNA encoding human deoxyribonuclease II. J. Biol. Chem. 273,

2610–2616.
24. Chomczynski, P. & Sacchi, N. (1987) Single-step method of RNA
isolation by acid guanidinium thiocyanate-phenol-chloroform
extraction. Anal. Biochem. 162, 156–159.
25. Yasuda, T., Kishi, K., Yanagawa, Y. & Yoshida, A. (1995)
Structure of the human deoxyribonuclease I (DNase I) gene:
identification of the nucleotide substitution that generates its
classical genetic polymorphism. Ann. Hum. Genet. 59, 1–15.
26. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R.
(1989) Site-directed mutagenesis by overlap extension using the
polymerase chain reaction. Gene 77, 51–59.
27. Takeshita, H., Yasuda, T., Nakazato, E., Nakajima, T., Mori, S.,
Mogi, K., Kaneko, Y., Iida, R. & Kishi, K. (2001) Use of human
recombinant DNase I expressed in COS-7 cells as an immunogen
to produce a specific anti-DNase I antibody. Exp. Clin.
Immunogenet. 18, 226–232.
28. Shak, S., Capon, D.J., Hellmiss, R., Marsters, S.A. & Baker, C.L.
(1990) Recombinant human DNase I reduces the viscosity of
cystic fibrosis sputum. Proc. Natl Acad. Sci. USA 87, 9188–9192.
29. Polzar, B. & Mannherz, H.G. (1990) Nucleotide sequence of a full
length cDNA clone encoding the deoxyribonuclease I from the rat
parotid gland. Nucleic Acids Res. 18, 7151.
30. Hsiao, Y M., Ho, H C., Wang, W Y., Tam, M.F. & Liao, T H.
(1997) Purification and characterization of tilapia (Oreochromis
mossambicus) deoxyribonuclease I–primary structure and cDNA
sequence. Eur. J. Biochem. 249, 786–791.
31. Saitou, N. & Nei, M. (1987) The neighbor-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,
406–425.
32. Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL

W: improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–
4680.
33. Paudel, H.K. & Liao, T H. (1986) Comparison of the three pri-
mary structures of deoxyribonuclease isolated from bovine, ovine,
and porcine pancreas. Derivation of the amino acid sequence of
ovine DNase and revision of the previously published amino acid
sequence of bovine DNase. J. Biol. Chem. 261, 16012–16017.
34. Mannherz, H.G. (1992) Crystallization of actin in complex with
actin-binding proteins. J. Biol. Chem. 267, 11661–11664.
35. Ulmer, J.S., Herzka, A., Toy, K.J., Baker, D.L., Dodge, A.H.,
Sinicropi,D.,Shak,S.&Lazarus,R.A.(1996)Engineeringactin-
resistant human DNase I for treatment of cystic fibrosis. Proc.
NatlAcad.Sci.USA93, 8225–8229.
36. Kozak, M. (1987) At least six nucleotides preceding the AUG
initiator codon enhance translation in mammalian cells. J. Mol.
Biol. 196, 947–950.
37. Oefner, C. & Suck, D. (1986) Crystallographic refinement and
structure of DNase I at 2 A
˚
resolution. J. Mol. Biol. 192, 605–
632.
38.Pan,C.Q.,Ulmer,J.S.,Herzka,A.&Lazarus,R.A.(1998)
Mutational analysis of human DNase I at the DNA binding
interface: implications for DNA recognition, catalysis, and metal
ion dependence. Protein Sci. 7, 628–636.
39. Suck, D., Oefner, C. & Kabsch, W. (1984) Three-dimensional
structure of bovine pancreatic DNase I. EMBO J. 3, 2423–2430.
40.Yasutomo,K.,Horiuchi,T.,Kagami,S.,Tsukamoto,H.,

Hashimura, C., Urushihara, M. & Kuroda, Y. (2001) Mutation of
DNASE1 in people with systemic lupus erythematosus. Nat.
Genet. 28, 313–314.
41. Hedges, S.B., Moberg, K.D. & Maxson, L.R. (1990) Tetrapod
phylogeny inferred from18S and 28S ribosomal RNA sequences
and a review of the evidence for amniote relatioships. Mol. Biol.
Evol. 7, 607–633.
42. Hedges, S.B. (1994) Molecular evidence for the origin of birds.
Proc.NatlAcad.Sci.USA91, 2621–2624.
314 H. Takeshita et al. (Eur. J. Biochem. 270) Ó FEBS 2003