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: kkoichi@med.gunma-u.ac.jp 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 (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 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. 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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. Molecular evolution of shark and other vertebrate DNases I Toshihiro Yasuda 1 , Reiko Iida 2 , Misuzu Ueki 1 , Yoshihiko Kominato 3 , Tamiko Nakajima 3 ,