Molecular evolution of shark and other vertebrate DNases IToshihiro Yasuda1, Reiko Iida2, Misuzu Ueki1, Yoshihiko Kominato3, Tamiko Nakajima3, Haruo Takeshita3, Takanori Kobayashi4and Ko
Trang 1Molecular evolution of shark and other vertebrate DNases I
Toshihiro Yasuda1, Reiko Iida2, Misuzu Ueki1, Yoshihiko Kominato3, Tamiko Nakajima3, Haruo Takeshita3, Takanori Kobayashi4and 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: kkoichi@med.gunma-u.ac.jp
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)
Trang 2greater extent than the enzymes from other vertebrate
classes Comprehensive characterization of Chondrichthye
DNases I will thus allow us to elucidate the molecular
evolutionary aspect of the vertebrate DNase I family
In this study, we cloned cDNAs encoding DNases 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 mutant
constructs was also examined in mammalian cells,
allowing several common structural and functional
char-acteristics of shark DNases I to be 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 Toba
Aquarium, Mie, Japan LipofectaminPlus, all
oligonucle-otide primers, and the 3¢- and 5¢-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 specific to human, hen, Rana catesbeiana (frog),
Elaphe quadrivirgata (snake) and Cyprinus 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 radial enzyme
diffusion (SRED) method [2,22] or test tube method [11] as
described previously, except that 50 mM Hepes/NaOH
buffer pH 8.0, containing 20 mMMgCl2and 2 mMCaCl2
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 0–4C Pancreatic tissue
obtained from H japonicus, weighing approximately 4 g,
was minced and homogenized in 50 mM Mes/NaOH,
pH 6.0, containing 1 mM 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 with the same buffer The
adsorbed materials were eluted with a 200-mL linear
gradient of 0–1M NaCl in buffer I The DNase I-active fractions eluted with 0.2MNaCl were collected and dialysed against buffer I containing 10 mMCaCl2 The dialysate was subjected to cation exchange chromatography using the A¨KTAFPLC
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 0–1M NaCl in buffer I The active fractions eluted with 0.2M 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 mM NaCl The active fractions were collected, concentrated by ultrafiltra-tion and used as the purified enzyme for subsequent experiments
Construction of cDNAs encoding theH 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¢- and 5¢-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 pcDNA3.1(+) vector (Invitrogen) to construct the wild-type expression vector Expression vec-tors for wild-type human, frog, Anguilla japonica (eel) and
T scylliaDNases 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),
Trang 3-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 insertion mutant, human-ins(Thr206),
were constructed using 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 mM L-glutamine, 50 UÆmL)1
penicillin, 50 lgÆmL)1 streptomycin and 10% (v/v) fetal
bovine serum (Invitrogen) at 37C under 5% (v/v) CO2in
air The cells were transiently transfected 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 estimation 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 with at 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 showed the highest 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 used 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 japonicusDNase 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 24 basic and 31 acidic amino acid residues This makes H japonicus DNase I unique among the vertebrate 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 the shark DNase I was examined by preincubating the enzyme at 45C for up
to 80 min (Fig 2), its activity could be detected for only the first 30 min of incubation, whereas that of the 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 (%)
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 recovered from the gel-filtration 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 C for 5 min and sub-jected to SDS/PAGE using a 12.5% gel, followed by silver-staining.
An expression vector, H japonicus-chi(human:sig), containing 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 manner The cathode is at the top The positions
of the molecular mass markers are indicated on the left.
Trang 4enzyme 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 metal ions on the activity, of 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 were 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 resemblance 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 amplified by RACE methods to construct 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 lysate or 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 the COS-7 cells, unambiguous activity levels were expressed This activity was completely abolished by 1 mM EGTA Furthermore, the enzyme activity expressed in the cells migrated to the position corresponding to the purified DNase I on the DNA-casting PAGE gels [25] (Fig 1), confirming that the cloned cDNA did indeed encode the expected
H japonicusDNase I
In order to elucidate any common features unique to shark DNases I, we also cloned and 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 other vertebrates (200 ± 89 bp), including the human [29], rabbit [13], mouse [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 therefore 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 two shark DNases I predicted by each 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 and 263 residues, respectively Comparison of the primary structures of these shark DNases I with those of the 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 conserved in both of the shark enzymes Two Cys residues at positions 173 and 209, which form a disulfide bond involved in the structural stability of the enzyme [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 are well conserved in vertebrate DNases I, these residues were replaced by Ala100 and Thr103 in H japonicus DNase I and by Ser101 and Ser104
in T scyllia These Cys residues 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 mutant
H japonicus-sub(Ala100Cys/Thr103Cys) (j), and (B) human
wild-type DNase I (d) and its substitution mutants human-sub(Cys101Ala/
Cys104Thr) (s) and human-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 used for the incubation.
Trang 5(Cys101Ala/Cys104Thr), in which the two Cys residues of
human DNase I were substituted by their counterparts
from H japonicus, exhibited lower thermal stability than the
corresponding wild-type, whereas double substitution of
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 residues is also seen
in some species of the Osteichthye class, such as tilapia and
eel [21] Taken together, these findings suggest that the
formation of the disulfide bond 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 around this location has been postulated
to form an essential Ca2+-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 compared 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 Ca2+-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 residues are also found in the DNases I of amphibians [19] As in amphibian DNases I, the insertion 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 residues and the insertion of an additional Thr/ Asn residue We 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 enzyme present in higher vertebrates such as humans during the course of evolution, it could be postulated that the DNase I protein has acquired increasing 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 activity was detectable in the medium or 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 shark enzymes with that derived 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 of activity, we constructed a series of chimeric
Fig 3 Alignment of the amino acid sequences of the two shark DNase I molecules with those of the human, amphibian, reptilian and piscine enzymes The amino acid sequences of the shark DNases I were deduced from their respective cDNAs and compared with published sequences for the human [29], snake [18], frog [19] and eel [21] The amino acids of each mature protein are numbered from the N terminus The dots indicate residues that are the same as those in H japonicus, while the horizontal bars indicate deleted amino acid residues.
Trang 6mutant 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 corresponding regions of the frog, eel,
T scylliaand 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 with
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 that the signal sequence for each vertebrate DNase I
extensively affects the expression level of the enzyme; the
DNase I signal sequences of lower vertebrates such as
amphibians, Chondrichthyes and Osteichthyes exerted a
relatively small effect on expression of the enzyme, whereas those of higher vertebrates such as mammals contributed 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 the presence of an a-helical structure
in the signal sequence region of the protein [40–42] Analysis usingDNASIS PROsoftware revealed no distinct differences in 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
2Program (http://www.ddbj.nig.ac.jp/search/ssthread) 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 of the human enzyme (Table 3) The lower the a-helical structure content in the signal sequence region of 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 discrepancy in the efficiency of enzyme expression by the cells transfected with each of the expression vectors Based on the DNase 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 (n¼ 2), 34%; Osteichthyes (n ¼ 5), 24 ± 16%; Amphibia (n¼ 4), 10 ± 12%; Reptilia (n¼ 2), 66%; Aves (n ¼ 1), 75%; Mammalia (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 vertebrate 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 text The enzyme activities
secreted into the medium by cells transfected with each of the mutant
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 corresponding wild-type enzyme 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 the method of Ito et al [43] The portions of the signal sequence regions of each vertebrate DNase I with an a-helical structure are underlined The content is expressed as the ratio of the number 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 MetHisArgLeuIleThrAlaLeuThrLeuThrCysLeuMetGlyAlaAlaSerSer 42
T scyllia MetArgGlnLeuIleThrValLeuThrLeuAlaCysValProSerThrValHisSer 26
Eel MetLysIleIleGlyAlaPheLeuLeuIleLeuAlaPheValGluLeuSerThrGlySer 45
Frog MetLysSerLeuLeuLeuValThrLeuAlaAlaCysPheLeuHisAlaGlySerAla 0
Human MetArgGlyMetLysLeuLeuGlyAlaleuLeuAlaLeuAlaAlaLeuLeuGlnGlyAlaValSer 70
Trang 7increased during the 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 caused 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 acquired 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 to conclude that this molecular evolution
may permit higher vertebrates such as the Mammalia to
maintain higher DNase I activity levels in vivo A lack of, 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 Promotion of Science (15209023 to TY, 16209023 to KK
and 15590575 to YK).
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