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 Yasuda
1
, Reiko Iida
2
, Misuzu Ueki
1
, Yoshihiko Kominato
3
, Tamiko Nakajima
3
, Haruo Takeshita
3
,
Takanori Kobayashi
4
and Koichiro Kishi
3
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 (DN ase 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 w as
unique in being a b asic protein. Full-length cDNAs enco ding
the DNases I of two s hark 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 C ys 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 r egion 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 r egion 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 p rotein 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 h ave been
reported to be lower in SLE patients than i n 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 w ere
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 t hat DNase I or D Nase I-like e ndo-
nucleases may be responsible for internucleosomal DNA
degradation during apoptosis [9,10], have focused attention
on the potential physiological r oles of DNase I. In this
context, we have attempted to elucidate the intrinsic intra-
and extracellular f unction(s) of DNase I, as well a s 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 d ifferent 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 t o a ddress this question, a systematic survey of
Chondrichthye DNases I i s 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 , Departm ent of Legal Me dicine,
Gunma University Graduate School of Medicine, Maebashi,
Gunma 371-8511, Japan. Fax: +81 27 220 8035,
E-mail:
Abbreviations: SLE, systemic lupus eryth ematosus; SRED, s ingle
radial enzyme diffusion; UTR, u ntr anslated region.
Enzyme: DNase I (EC 3.1.21.1 ).
Note: The nucleotide sequenc e data rep orted will app ear in DDBJ,
EMBL an d GenBank N uc leotide Sequence Database und er accession
numbers AB126699 and AB126700.
(Received 3 August 2004, revised 15 September 2004,
accepted 28 September 2004)
Eur. J. Biochem. 271, 4428–4435 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04381.x
greater extent than the enzymes from other vertebrate

classes. Comprehensive characterization of Chondrichthye
DNases I will thus allow us t o elucidate the m olecular
evolutionary aspect of the vertebrate DNase I family.
In this s tudy, we cloned cDNAs encoding DN ases I
from two Chondrichthyes, Heterodontus japonicus and
Triakis scyllia, species of shark which are widely
distributed in the seas around Japan, and purified the
former’s enzyme. The expression of a series of m utant
constructs was a lso e xamined in mammalian cells,
allowing several common structural and functional char-
acteristics o f shark DNas es I to b e confirmed. The
molecular evolutionary aspect of the vertebrate DNase I
family is also discussed.
Materials and methods
Materials and biological samples
Two different species of shark, H. japonicus and T. scyllia,
weighing approximately 5 .0 kg (1.2 m long) and 4.7 kg
(1.0 m long), respectively, were obtained from T oba
Aquarium, Mie, Japan. LipofectaminPlus, all oligonucle-
otide primers, and the 3¢-and5¢-RACE systems were
obtained from Invitrogen; CM-Sepharose CL-6B, Mono
S 5/50 GL and Superdex 75 were from Amersham
Pharmacia Biotech; the Expanded High Fidelity PCR
system was from Boehringer Mannheim GmbH. Anti-
bodies s pecific to human, hen, Ra na catesbeiana (frog),
Elaphe quadrivirgata (snake) and Cyprin us carpio (carp)
DNases I were prepared using previously described
methods [11,17–19,21]. All other chemicals used were of
reagent grade and commercially available.
Analytical methods

DNase I activity was assayed using the single r adial enzyme
diffusion (SRED) method [2,22] or test tube method [11] as
described previously, except that 50 m
M
Hepes/NaOH
buffer pH 8.0, containing 20 m
M
MgCl
2
and 2 m
M
CaCl
2
was substituted for the reaction buffer. The enzymatic [23],
proteochemical [11] and thermal stability [18,19] character-
istics of the enzymes were examined as described previously.
Proteins were determined using a protein assay kit (Bio-
Rad) with BSA as a standard. SDS/PAGE was performed
in 12.5% (w/v) gels according to the method of Laemmli
[24], and the proteins thus separated were visualized by the
silver-staining method. Activity-staining for DNase I was
performed using a DNA casting-PAGE method [25],
and conventional methods were used for the assay of
b-galactosidase [26].
Purification of shark DNase I from pancreatic tissue
All procedures were carried out at 0–4 °C. Pancreatic tissue
obtained from H. japonicus, weighing approximately 4 g,
was minced and homogenized in 50 m
M
Mes/NaOH,

pH 6.0, containing 1 m
M
phenylmethanesulfonyl fluoride
(buffer I). After centrifugation, the supernatant (crude
extract) was applied to a CM-Sepharose CL-6B column
(1.6 · 8 cm) pre-equilibrated w ith t he same buffer. The
adsorbed materials were eluted with a 200-mL linear
gradient of 0–1
M
NaCl in buffer I. The DNase I-active
fractions eluted with 0.2
M
NaCl were colle cted and dialysed
against buffer I containing 10 m
M
CaCl
2
. The dialysate was
subjected to cation exchange chromatography using t he
A
¨
KTAFPLC
1
system (Amersham Pharmacia Biotech)
equipped with a Mono S 5/50 GL column ( 0.46 · 10 cm).
The adsorbed materials were eluted with a 100-mL linear
gradient of 0–1
M
NaCl in buffer I. The active fractions
eluted with 0.2

M
NaCl were collected, concentrated and
subjected to gel filtration using the A
¨
KTAFPLC system
equipped with a Superdex 75 column (1.6 · 60 cm) pre-
equilibrated with buffer I containing 150 m
M
NaCl. The
active fractions were collected, concentrated by ultrafiltra-
tion and used as the purified enzyme for subsequent
experiments.
Construction of cDNAs encoding the
H. japonicus
and
T. scyllia
DNases I
Total RNA was isolated from the pancreas of each shark
using Sepasol-RNA I (Nacalai tesque, Kyoto, Japan). The
3¢-end region of the DNase I cDNA was obtained by
3¢-RACE using two degenerate primers based on two amino
acid (aa) sequences which are highly conserved in piscine
DNases I, the Ala15–Asp23 and Gln38–Leu44 sequences
[21]: 5¢-GCITT(C/T)AA(C/T)ATCAG(A/G)GCITT(T/
C)GGIGA-3¢ and 5¢-CA(A/G)GA(A/G)GTICGIGA(C/
T)GCIGA(C/T)CT-3¢. Next, the 5¢-end region of the
cDNA was amplified by 5¢-RACE using gene-specific
primers based on the nucleotide sequences determined in
this study. These RACE procedures were carried out using
the 3¢-and5¢-RACE systems, according to the manufac-

turer’s instructions. The RACE products were subcloned
into the pCR 2.1 TA cloning vector (Invitrogen) and
sequenced. The nucleotide sequences were determined by
the dideoxy chain-termination method using a Dye Termi-
nator Cycle Sequencing kit (Applied Biosystems). 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 mammalian cells
A DNA fragment containing the entire coding sequence of
H. japonicus DNase I cDNA, corresponding to both the
signal sequence and mature enzyme regions, was prepared
from the total RNA derived from the pancreas by reverse
transcription/PCR amplification using an Expanded High
Fidelity PCR system with a set of two primers correspond-
ing to the nucleotide sequences of the cDNA at positions
48–69 and 881–901, respectively. The amplified fragment
was ligated into a p cDNA3.1(+) vector (Invitrogen) to
construct the wild-type expression vector. Expression vec-
tors for wild-type human, frog, Anguilla japonica (eel) and
T. scyllia DNases I were constructed in the same manner.
A chimeric mutant, H. japonicus-chi(human:sig), in which
the signal sequence region of H. japonicus DNase I was
replaced by its counterpart from the human enzyme, was
constructed by splicing using the overlap extension method
[27] with each of the wild-type constructs as a template.
Seven other chimeric mutants, human-chi(H. japonicus:sig),
Ó FEBS 2004 Molecular evolution of vertebrate DNase I family (Eur. J. Biochem. 271) 4429
-chi(T. scyllia:sig), -chi(eel:sig), -chi(frog:sig), frog-

chi(human:sig), eel-chi(human:sig) and T. scyllia-chi(hu-
man:sig), were prepared in the same manner. Four other
mutants including two substitution mutants, human-
sub(Cys101Ala/Cys104Thr) and H. japonicus-sub(Ala100-
Cys/Thr103Cys), one deletion mutant, H. japonicus-
del(Thr206), and one insert ion mutant, human- ins(Thr206),
were constructed u sing the human wild-type DNase I and
H. japonicus-chi(human:sig) mutant constructs as a tem-
plate. All constructs were sequence-confirmed and purified
using the CONCERT High Purity Plasmid Midi kit
(Invitrogen) for transfection.
COS-7 cells were maintained in Dulbecco’s modified
Eagle’s medium containing 1 m
ML
-glutamine, 50 UÆmL
)1
penicillin, 50 lgÆmL
)1
streptomycin and 1 0% (v/v) fetal
bovine serum (Invitrogen) at 37 °C under 5% (v/v) CO
2
in
air. The cells were transiently tran sfected using Lipofecta-
minPlus reagent according to a previously described method
[28]. A mixture containing 2 lg of the relevant expression
vector and 0.6 lg of the pSV-b-galactosidase vector
(Promega; for es timation of transfection efficiency) was
introduced into the cells. Two days later, the medium and
cells were recovered for analysis. DNase I activity in the
medium was determined by the SRED method and the cell

lysates were assayed for b-
D
-galactosidase. All transfections
were performed in triplicate w ith a t least two different
plasmid preparations.
Results and Discussion
Purification and characterization of shark pancreatic
DNase I
Among various tissue samples collected from H. japonicus,
the pancreas s howed the h ighest DNase I activity
(1.3 ± 0.31 UÆmg
)1
protein); moderate activity was also
detected in the small intestine (0.010 ± 0.0013 UÆmg
)1
protein). Thus, the pancreas was u sed as the starting
material for the purification of shark DNase I.
The results of purification are summarized in Table 1.
The purification procedure, using three different kinds of
column chromatography, allowed the enzyme to be repro-
ducibly isolated and purified approximately 2000-fold to
electrophoretic homogeneity (Fig. 1). Although anion
exchange chromatography using resins such as DEAE–
Sepharose CL-6B has generally been found useful for the
purification of vertebrate DNases I, including the human
[11], rat [12], rabbit [13], amphibian [19] and reptile [18]
enzymes, shark DNase I was retained on a cation exchange
resin but not on an anion exchange one. As shown below,
H. japonicus DNase I consisted of 262 amino acid residues;
however, it was found to contain more basic amino acids

(32 residues) than acidic ones (27 residues), whereas the
human enzyme has 2 4 b asic and 31 a cidic amino acid
residues. This makes H. japonicus DNase I unique among
the verteb rate DNase I family in that it is less acidic than all
the other vertebrate enzymes studied so far. The purified
shark DNase I had a molecular mass of  33 kDa, as
determined by both gel-filtration and SDS/PAGE. This
value is similar to those for the DNases I of other
vertebrates except amphibians. The N-terminal amino acid
sequence of the purified shark DNase I as determined by
Edman degradation up to the tenth residue was IHISAIN-
RA(1–10). When the thermal stability of t he shark DNase I
was examined by preincubating the enzyme at 45 °C for up
to 80 min (Fig. 2), i ts ac tivity c ould be detec ted for o nly the
first 30 min of incubation, whereas t hat o f t he human
Table 1. Summary of the purification of DNase I from 4 g of pancreas of H. japonicus.
Purification step
Protein
(mg)
Total activity
(U)
Volume
(ml)
Specific activity
(UÆmg
)1
)
Purification
(fold)
Yield

(%)
Crude extract 42 65 22 1.5 1.0 100
CM-Sepharose CL-6B 4.7 57 65 12 8.0 88
Mono S 5 /50 GL 0.88 48 5.0 55 36 74
Superdex75 0.014 40 3.5 2800 1900 62
Fig. 1. Electrophoretic patterns of purified shark DNase I and the
recombinant enzyme revealed by silver-staining and activity-staining.
The final DNase I preparation recover ed from the gel- filtrat ion step
was concentrated and used for SDS/PAGE analysis. The purified
enzyme (approx. 0.5 lg) from H. japonicus (lane 1) was dissolved
in 10 m
M
Tris/HCl pH 6.8, containing glycerol (10%, v/v), SDS ( 2%,
w/v) and 25 m
M
dithiothreitol, heated at 100 °Cfor5minandsub-
jected to SDS/PAGE using a 12.5% gel, followed by silver-staining.
An expression ve ctor, H . japonicus-chi(human:sig), co ntaining an
H. japonicus DNase I cDNA insert (lane 2) was transfected into
COS-7 cells by the lipofection method. The recombinant DNase I
secreted into the medium was subjected to DNA-casting PAGE, fol-
lowed by activity-staining [25]. The purified enzyme (lane 3) was
analysed in the same m anner. The cathode is at the top. The positions
of the molecular mass markers are indicated on the left.
4430 T. Yasuda et al. (Eur. J. Biochem. 271) Ó FEBS 2004
enzyme remained almost unchanged. Therefore, shark
DNase I is more unstable than the mammalian enzymes.
The catalytic properties such as the effects of pH, ionic
strength and m etal ions on the activity, o f the purified shark
DNase I resembled those of the other vertebrate DNases I.

However, when specific antibodies against the mammalian
(human), amphibian (frog), avian (hen), reptilian (snake)
and Osteichthyes ( carp) enzymes we re tested for cross-
inhibition of activity, all five antibodies were ineffective
against the shark DNase I, indicating that, from an
immunological standpoint, shark DNase I bears little or
no resemblan ce to the mammalian, avian, reptilian,
amphibian or Osteichthye enzymes.
cDNA constructs encoding shark DNases I
The total RNA isolated from the pancreas of H. japonicus
was a mplified by RACE methods to c onstruct cDNA
encoding the species DNase I. The use of degenerate
primers based on an amino acid sequence highly conserved
in Osteichthye DNases I allowed the successful amplifica-
tion of specific RACE products from the total RNA of
shark pancreases.
The full-length cDNA encoding H. japonicus DNase I
(accession number AB126699) was composed of 996 bp,
including an ORF of 846 bp coding for 281 amino acid
residues, a 53 bp 5 ¢-untranslated region (UTR) and a 97 bp
3¢-UTR. The ORF started with an ATG start codon at
position 54 and ended with a TAA stop codon at position
899. The N-terminal amino acid sequence deduced from
cDNA data exactly matched that determined chemically
from the purified enzyme by Edman degradation, and
indicated a 19 amino acid long putative upstream signal
sequence.
An expression vector containing the entire coding region
of H. japonicus DNase I cDNA was transiently transfected
into COS-7 cells; however, no DNase I activity could be

detected in either the cell l ysate o r the medium of the
transfected cells. When we constructed a chimeric mutant,
H. japonicus-chi(human:sig), in which the signal sequence
region of the shark enzyme was substituted by its human
counterpart, and transfected this into th e COS-7 cells,
unambiguous activity levels were expressed. This activity
was completely abolished by 1 m
M
EGTA. Furthermore,
the enzyme activity expressed in the cells migrated to the
position corresponding to the purified D Nase I on the
DNA-casting PAGE gels [25] (Fig. 1), confirming that
the cloned cDNA did indeed encode the expected
H. japonicus DNas e I.
In order to elucidate any common features unique to
shark DNases I, we also cloned a nd sequenced cDNA
encoding the DNase I of another shark, T. scyllia (acces-
sion number AB126700), and found it to contain 998 bp.
This cDNA was composed of a 48 bp 5¢-UTR, an 855 bp
ORF and a 95 bp 3¢-UTR. Thus, shark DNase I cDNAs
appear to be characterized by a shorter 3¢-UTR (average of
96 bp) than those cloned from most o ther vertebrates
(200 ± 89 bp), including the human [29], rabbit [13], m ouse
[14], rat [30], cow [31], hen [17], pig [15] and snake [18]
enzymes. In this respect, shark DNases I resemble the
amphibian (89 ± 22 bp) [19] and Osteichtye (112 ± 20 bp)
[20,21] enzymes. It could t herefore be postulated that the 3¢-
UTR of vertebrate DNase I cDNA lengthened about
twofold during the evolutionary stage between amphibians
and reptiles.

Structural features of shark DNases I
The amino acid sequences of the t wo shark DNases I
predicted b y e ach of their nucleotide sequences are shown in
Fig. 3. Considering the N-terminal amino acid sequences
of the purified enzymes, the mature forms of H. japonicus
and T. scyllia DNases I were estimated to be composed of
262 a nd 263 residues, respectively. Comparison of the
primary structures o f these shark DNases I with those of t he
other vertebrate enzymes available [13–21,32,33] allowed
us to identify several structural features unique to shark
DNases I. All four residues responsible for the catalytic
activity of DNase I, Glu78, His134, Asp212 and His252
[34], were c onserved in both o f t he shark e nzymes. Two Cys
residues at positions 173 and 209, which form a disulfide
bond involved in the structural stability of the e nzyme [35]
were found. Osteichthye DNases I all possess a specific but
variable region in the enzyme protein between positions 56
and 64, in which insertion or deletion of one amino acid
residue occurs; they also show deletion of one residue
corresponding to Ala214 in the human enzyme [21]. The
shark enzymes did not share these features. Therefore,
despite all belonging to the piscine DNase I family, the
Osteichthye DNases I could be distinguished easily from the
Chondrichthye enzymes.
The shark DNases I had two unique structural altera-
tions in common. First, although the two Cys residues at
positions 101 and 104 a re well conserved in v ertebrate
DNases I, these residues were replaced by Ala100 and
Thr103 in H. japonicus DNase I and by Ser101 and Ser104
in T. scyllia. T hese Cys r esidues form a disulfide bond which

contributes to the structural stability of the enzyme protein,
in addition to another disulfide bond formed by Cys173
and Cys209 [36,37]. The substitution mutant human-sub
Fig. 2. Heat stability of shark (A) and human (B) DNases I and their
mutant constructs. The medium from COS-7 cells transfected with
(A) H. japonicus-chi(human:sig) (d) and its substitution mu tant
H. japonicus-sub(Ala100Cys/Thr103Cys) (j), and (B) human wild-
type DNase I (d) and it s substitution m utants hum an-sub (Cys101Ala/
Cys104Thr) (s) and hu man-ins (Thr204) ( j), was incubated at 45 °C
for the durations indicated, and residual DNase I activities were
determined by the SRED method. The same amounts ( 1 · 10
)7
unit) of each enzyme were u sed for the inc ubatio n.
Ó FEBS 2004 Molecular evolution of vertebrate DNase I family (Eur. J. Biochem. 271) 4431
(Cys101Ala/Cys104Thr), in which the two Cys residues of
human DNase I were substituted by their counterparts
from H. japonicus, e xhibited lower t hermal stability than the
corresponding wil d-type, whereas double substitution o f
Ala100 and Thr103 by Cys residues in the H. japonicus
DNase I, H. japonicus-sub(Ala100Cys/Thr103Cys), made
the enzyme more thermally stable compared with the wild-
type (Fig. 2 ). Deletion of these two Cys r esidues is also seen
in some species of the Osteichthye class, such as tilapia and
eel [21]. T aken together, t hese findings s uggest that the
formation of t he disulfide bo nd between Cys101 and Cys104
may have been acquired during the evolutionary stage in
Osteichthyes, resulting in the production of a more stable
form of the enzyme. Recently, Chen et al. have reported
that the corresponding disulfide bond is important for the
structural integrity of bovine DNase I [38].

The second structural alteration in T. scyllia and
H. japonicus DNases I was the insertion of Asn205 and
Thr206, respectively, in their mid-regions, corresponding to
the position between Ala204 and Thr205 in the human
DNase I. The area a round this location has b een postulated
to form an essential Ca
2+
-binding site responsible for the
stability of the enzyme [36]. Two mutants, human-
ins(Thr205) and H. japonicus-del(Thr206), in which a Thr
residue was inserted or deleted in the human and H. japon-
icus enzymes, respectively, were constructed and their
thermal stability was c ompared with that of the corres-
ponding wild-types. The insertion of a Thr residue into the
human enzyme rendered it thermally labile, maybe due to
the structural alteration caused by insertion of the residue
into the Ca
2+
-binding site (Fig. 2B), whereas deletion of
the additional Thr residue from the H. japonicus enzyme
reduced its activity to undetectable levels. Such additional
amino acid r esidues are also fou nd in t he DNases I of
amphibians [19]. As in a mphibian DNases I, the insertio n of
an additional amino acid residue into the shark enzymes
may be essential for the generation of an active enzyme,
irrespective of whether this induces instability of the
enzyme protein.
As shown above, shark DNases I share two structural
alterations that reduce the stability of the enzyme protein
compared with those of other vertebrates: the deletion of

two cysteine r esidues and the insertion of an additional Thr/
Asn residue. W e have previously reported that a single
Leu130Ile substitution in reptilian DNases I may produce
the thermally stable form of the higher vertebrates [18].
Therefore, with regard to the genesis of the DNase I e nzyme
present in higher vertebrates such as humans during the
course of evolution, it could be postulated that the DN ase I
protein has acquired incre asing structural stability through
the introduction of the two Cys residues and deletion of the
additional residue, followed by Leu130Ile substitution.
Effect of the signal sequence regions of vertebrate
DNases I on their expression in transfected cells
Although no a ctivity w as detectable in the medium o r
lysates of cells that had been transfected with expression
vectors containing the entire coding regions of the wild-type
shark DNases I, substitution of the signal sequence regions
of each of the s hark enzymes w ith that d erived from human
DNase I gave rise to expression of activity. In order to
examine the possible effect of the signal sequence region on
the expression o f activity, we constructed a series of chimeric
Fig. 3. Alignment of the amino acid sequences of the t wo shark DNase I molecules wi th those of the human, amphibian, re ptilian and p iscine enzymes.
The amino ac id seq ue nces o f t he shark D N ases I were deduced f rom t heir respective cDN As a nd co mpared with published s eque nces for the hum an
[29], snake [18], frog [19] and eel [21]. The amino acids of each mature prot ein are nu mbe red fro m the N terminus. The dots in dicate residue s that a re
the same as those in H. japonicus, while the horizontal bars indicate deleted amino acid residues.
4432 T. Yasuda et al. (Eur. J. Biochem. 271) Ó FEBS 2004
mutant enzymes, and compared the activity secreted into
the medium from the transfected COS-7 cells (Table 2).
When the signal sequence region of human DNase I was
replaced by the c orresponding r egions of the frog, eel,
T. scyllia and H. japonicus enzymes, the activities detected

in the medium were definitely reduced to 0.15-, 0.67-, 0.30-
and 0.67-fold that of the wild-type enzyme, respectively. In
contrast, substitution of the signal sequence regions of frog
and eel DNases I with their human counterpart resulted in
an approximate doubling of the activity level compared w ith
each of the wild-type enzymes. Lower activity of the shark
DNase I in the medium compared to that of the human
enzyme may be due to low stability and/or specific activity
inherent to shark enzymes. Similar results were obtained
when these expression vectors were transfected into human
hepatoma HepG2 cells (data not shown). These findings
suggest t hat the signal sequence for each vertebrate D Nase I
extensively affects the expression level of the enzyme; the
DNase I signal sequences of lower vertebrates such as
amphibians, Chondrichthyes and Osteichthyes e xerted a
relatively small effect on expression of the enzyme, whereas
those of higher v ertebrates such as m ammals co ntributed to
more effective expression of the enzyme.
Secretory proteins such as DNase I contain a signal
sequence that directs the emerging polypeptide and ribo-
some to the endoplasmic reticulum by cotranslational
protein targeting. Cotranslational targeting of a protein to
the endoplasmic reticulum is initiated when a signal
recognition particle binds to a hydrophobic signal sequence
present at the N-terminus of the nascent chain, and the
common physicochemical properties of this sequence,
irrespective of the lack of any specific consensus amino
acid sequence, are essential for its function [39]. Two
particular features appear to be necessary for entry into the
cotranslational protein targeting pathway: hydrophobicity

of the central core and t he presence of an a-helical stru cture
in the signal s equence r egion of the protein [40–42]. A nalysis
using
DNASIS PRO
software revealed no distinct differences i n
the hydrophobicity profiles of the signal sequence regions
of human, eel, frog, T. scyllia and H. japonicus DNases I.
However, prediction of the secondary structure of the
corresponding part of the enzyme using the
SSTHREAD
2
Program ( />according to the method of Ito et al. [43] revealed that
the a-helical structure contents of the T. scyllia, H. japon-
icus, eel and frog DNases I were significantly lower than
that o f the human enzyme (Table 3). The lower the a-helic al
structure c ontent i n t he signal sequence r egion o f each
DNase I, the lower the expression level each enzyme
exhibits; replacement of the human DNase I signal
sequence by the counterpart of the frog enzyme having
the lowest a-helical structure content had the greatest effect
on reducing the expression levels. It seems reasonable to
assume that the low a-helical structure contents of the signal
sequence regions of the T. scyllia, H. japonicus, eel and frog
DNases I may reduce their ability to function as cotrans-
lational targeting signals compared with the latter, resulting
in the observed d iscrepancy in the efficiency of enz yme
expression by the cells transfected with each of the
expression v ector s. Base d on t he D Nase I cDNA data
available from databases, the average a-helical structure
contents of the signal sequence regions of DNase I proteins

derived from each class of vertebrates were estimated as
follows: Chondrichthyes (n ¼ 2), 34%; Osteichthyes (n ¼
5), 24 ± 16%; Amphibia (n ¼ 4), 10 ± 12% ; Reptilia
(n ¼ 2), 66%; Aves (n ¼ 1), 75%; M ammalia (n ¼ 6),
62 ± 16%. These findings strongly indicate that the
a-helical structure contents of the signal sequence regions
Table 2. Effect of the signal sequences for each vert ebrate DNase I
on expression of the enzyme. Chimeric mutants, in which the signal
sequence of each vertebrate DNase I was replaced with counterparts
from the other vertebrate enzymes, were constructed and transiently
expressed in COS-7 cells, as described in the t ext. The enzyme activities
secreted into the medium b y cells tr ansfectedwitheachofthemutant
DNases I were measured using the SRED method. Values represent
the mean ± S.D. (n ¼ 5). The activity of each chimeric mutant was
compared with that of the c orrespond ing wild- type e nzym e. n.d., Not
detected.
Mature protein
from
Signal sequence
from
Activity
(UÆml
)1
) Ratio
Human Human 1.5 ± 0.28 · 10
-3
–
Frog 2.2 ± 0.20 · 10
-4
0.15

Eel 9.9 ± 0.24 · 10
-4
0.67
T. scyllia 4.5 ± 0.41 · 10
-4
0.30
H. japonicus 1.0 ± 0.24 · 10
-3
0.67
Frog Frog 3.9 ± 0.39 · 10
-4
–
Human 9.9 ± 0.86 · 10
-4
2.5
Eel Eel 1.6 ± 0.40 · 10
-4
Human 2.6 ± 0.23 · 10
-4
1.6
T. scyllia T. scyllia n.d. –
Human 1.0 ± 0.51 · 10
-5
–
H. japonicus H. japonicus n.d. –
Human 3.7 ± 0.82 · 10
-6
–
Table 3. a-Helical structure contents of the signal sequence regions of the vertebrate DNases I used in expression analysis. a-Helical structure contents
were estimated by t he m ethod o f Ito et al. [43]. The portions of the signal sequence regions of each vertebrate DNase I with an a-helical s t ructu re a re

underlined. The content is expressed as the ratio of the numbe r of amino acid residues forming the a-helical structure to the total number of
residues.
Species Amino acid sequence of signal sequence Content (%)
H. japonicus MetHisArgLeuIleThr
AlaLeuThrLeuThrCysLeuMetGlyAlaAlaSerSer 42
T. scyllia Met
ArgGlnLeuIleThrValLeuThrLeuAlaCysValProSerThrValHisSer 26
Eel MetLysIleIleGlyAlaPheLeu
LeuIleLeuAlaPheValGluLeuSerThrGlySer 45
Frog MetLysSerLeuLeuLeuValThrLeuAlaAlaCysPheLeuHisAlaGlySerAla 0
Human MetArg
GlyMetLysLeuLeuGlyAlaleuLeuAlaLeuAlaAlaLeuLeuGlnGlyAlaValSer 70
Ó FEBS 2004 Molecular evolution of vertebrate DNase I family (Eur. J. Biochem. 271) 4433
increased during t he evolutionary stage between amphibians
and reptiles. Therefore, it could be postulated that DNase I
expression levels in vertebrates increased due to improve-
ments in the efficiency of cotranslational targeting of
secretory DNase I, perhaps c aused by the structural alter-
ations in the signal sequence region of the enzyme during the
course of its molecular evolution.
In conclusion, these findings demonstrate that the
vertebrate DNase I family has acqu ired high structural
stability and effective expression of the enzyme through
structural alterations in both the mature protein and signal
sequence regions during the course of its molecular evolu-
tion. It is plausible t o c onclude that this molecular evolution
may permit higher vertebrates such as the Mammalia to
maintain higher DNase I activity l evels in vivo.Alackof,or
decrease in, DNase I activity has been suggested to be a
critical factor in the initiation of human and rat SLE [5,6].

Acknowledgements
This work was supported in part by Grants-in-Aid from the Japan
Society for the Prom otion o f S cience (1520 9023 to T Y, 162 09023 to KK
and 15590575 to YK).
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