- Email: [email protected]
cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata
Biochimie 85 (2003) 1033–1039 www.elsevier.com/locate/biochi
cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata Yanzhen Yang, Shujian Cun, Lisheng Peng, Xiaojin Xie, Jianwen Wei, Wenli Yang, Anlong Xu * Department of Biochemistry, The Open Laboratory for Marine Functional Genomics of National High-Tech Development Program, Key Laboratory of Gene Engineering of Ministry of Education, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, 135 W. Xin Gang Rd, Guangzhou 510275, PR China Received 20 June 2003; accepted 16 July 2003
Abstract Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser97-Trp98, which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately Mr = 12,800 displayed an obvious inhibition of papain (Ki value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4–11 at 4 °C, but unstable at neutral pH at >50 °C. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cystatin; Jellyfish; Tentacle; Inhibitory activity; Stability
1. Introduction Jellyfish is known to be a rich source of variant polypeptide toxins [1–3], hemolysins [4,5] and neuropeptides [6,7], but little is known about the presence of proteolytic enzymes and their inhibitors. Cystatins, which have not yet been found in jellyfish before, play an important regulatory role not only in the protection of cells from unfavorable proteolysis by intracellular and external cysteine proteinase, but also in biological defense systems against invaders [8,9]. The known proteins of the cystatin superfamily can be grouped into three protein families as defined by Ni et al.
Abbreviations: BAEE, Na-benzoyl-L-arginine ethyl ester hydrochloride; b-ME, b-Mercaptoethanol; cDNA, Complementary DNA; EST, Expressed sequence tag; PB, Phosphate buffer. * Corresponding author. Tel.: +86-20-8411-3655; fax: +86-20-8403-8377. E-mail address: [email protected] (A. Xu). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0300-9084(03)00132-9
[10]. Family 1 cystatins (also called stefins), cystatins A and B, which contain approximately 100 amino acid residues (Mr~11,000), lack disulfide bonds and carbohydrate. Family 2 cystatins are secreted inhibitors of about 120 amino acid residues (Mr~13,000), which have two intramolecular disulfide bonds and lack carbohydrate, containing cystatin C, D, E, S and SN. Family 3 cystatins, the kininogens, are single chain plasma proteins, whose N-terminal heavy chain is composed of three glycosylated cystatin-like domains with inhibitory activity. All of the three protein family members are reversible, tight-binding enzyme inhibitors with specificity against papain-like cysteine proteinases [10]. For cystatin, the inhibition of cysteine peptidases is due to the conserved residues forming a tripartite wedge quite complementary to the active site clefts of the enzyme, and slotting into the active site [11]. The conserved enzyme-binding domains include a Gly at the N-terminal, a central Gln–Xaa–Val– Xaa–Gly motif, and a Pro-Trp pair at the C-terminal [12]. The cystatin-enzyme interaction is dominated by hydrophobic contacts [11].
1034
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
In this article, we described the cDNA cloning and identification of a novel cystatin from jellyfish, Cyanea capillata, which showed only 26% identities to Family 2 cystatins, and had a Ser97-Trp98 pair at the C-terminal, which differed from the conserved active site in all inhibitory cystatins. We reported the kinetic properties of the novel cystatin’s interaction with papain, and its stability under a certain condition. 2. Materials and methods 2.1. cDNA library Total RNA was extracted from tentacle of C. capillata by the TRIZOL® LS Reagent (Gibcol BRL), and cDNA library was constructed using the SMART™ cDNA Construction Kit (Clontech).
Fig. 1. Construction of pETTrx/cystatin designed for the expression of Trx–cystatin fusion protein. The cDNA fragment of cystatin J was amplified by PCR and ligated into Kpn I/Not I sites of pETTrx. The enterokinase cleavage sites are shown.
2.2. Identification of cDNA for cystatin J More than 2000 EST sequences obtained from the cDNA library of C. capillata tentacle were analyzed for possible identification of encoded function proteins. Sequence homology comparisons of each EST were performed against the GenBank database using the BLASTX algorithms, followed by BLASTP searches of the open reading frames [13]. ESTs having homology to previously identified sequences were given a tentative name based on the name of the sequence to which it was homologous. By this method, cystatin ESTs were identified. The cDNAs of cystatin were completely sequenced on each DNA strand and were found to be full-length. 2.3. Construction of cystatin J expression vector pETTrx/cystatin J The open reading frame of this cDNA sequence encoding the mature cystatin was amplified by PCR using a upstream primer (with tailing KpnI site and recognition site, DDDDK, for enterokinase underlined), 5'-GCT GAA TTC GGT ACC GAC GAC GAC GAC AAA CTA CTT CCT GGC GGA ATA AGA CG–3'; and a downstream primer (with NotI site), 5'-CGT GCG GCC GCT TAT CAA GCT CGG AGG CAT TTT GT–3'. The PCR product was digested with Kpn I and Not I, followed by ligation to pETTrx vector (a self-made expression vector in this laboratory, Fig. 1), which was previously linearized with Kpn I and Not I, resulting in the expression plasmid pETTrx/cystatin. The positive clones were sequenced with a BigDye™ DNA Sequencing Kit (Applied Biosystems) to ensure that it contained no mutations introduced by PCR. 2.4. Production and purification of recombinant Trx–cystatin J The original vector (pETTrx) as well as the recombinant plasmid (pETTrx/ cystatin) was transformed into E. coli
strain BL21 (DE3) bacteria and propagated in luria broth (LB) in the presence of 100 µg/ml ampicillin for selection of the cells transformed with the pETTrx/cystatin expression plasmid. The expression of recombinant protein was induced in exponentially growing bacteria (A600 = 0.6–0.8) with 0.1 mM isopropyl-1-thio–b-D-galactopyranoside for 8 h at 25 °C with vigorous agitation. The bacteria were harvested by centrifugation, washed with TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and resuspended in lysis buffer, 50 mM Tris-HCl, pH 8.0, containing 100 mM NaCl. Cells were lysed on ice by mild sonication, and the suspension was centrifuged at 5000 rpm for 30 min. All subsequent purification steps were carried out at 25 °C. The lysis supernatant was applied to a Ni2+ chelate column (Pharmacia Biotech), equilibrated with lysis buffer. The column was washed with the same buffer and bound protein was then eluted by 50 mM Tris-HCl, pH 7.0, containing 100 mM NaCl and a series of increasing imidazole concentrations. Analysis of the fractions was carried out on SDS-PAGE. Gels were stained with Coomassie Blue R-250. Fractions containing fusion proteins were pooled and applied to a Sephadex G-25 column (Pharmacia Biotech) equilibrated with 50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl. Protein concentrations were determined by the Lowry Protein Quantitation Kit (Biocolor Co. Ltd.) using a standard curve generated with BSA. The Trx carrier was proteolytically cleaved from the fusion protein with enterokinase (produced by recombinant expression in this laboratory). The reaction was carried out at an enzyme to protein volume ratio of 1:500 for 4 h at 4 °C in 50 mM Tris-HCl, 100 mM NaCl, pH 7.5. Cleaved protein was applied to a SP-Sepharose column (Pharmacia Biotech), equilibrated with PB, pH 6.3. The column was washed with the same buffer and bound protein was then eluted by PB, pH 7.0, containing 150 mM NaCl. The purity of cystatin was assessed by SDS-PAGE.
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
1035
2.5. Active site titration Papain (3 nM final concentration) was incubated with increasing amounts of cystatin (0–9 nM final concentration) [14] in 500 µl of 20 mM PB, pH 6.2, containing 0.5 mM EDTA, 0.06 mM b-ME and 2 mM cysteine. After 10 min of incubation, added to 7.5 ml BAEE (Sigma) solution, pH 6.2, containing 1 M NaCl, 0.4 mM EDTA and 2 mM cysteine. The residual activity of papain was calculated by the volume of acid hydrolyzed by BAEE, as described by Stellmach [15]. 2.6. Inhibition kinetics of papain by cystatin J The kinetics of the reaction between cystatin and papain was analyzed under various concentrations of BAEE (i.e. 1.5, 3, 6 mM). The residual activity of papain was monitored as described above. The inhibition constant, Ki value, of cystatin J was obtained from a Dixon plot [16,17]. All the experiments were repeated at least three times. 2.7. Stability of cystatin J The thermal stability of cystatin J inhibitory activity in 20 mM PB, pH 6.2 was investigated by two methods: one was by incubating aliquots of inhibitor for 10 min at selected temperatures between 20 and 90 °C, then cooled on ice prior to determination of residual activity towards papain [9]; The other was by incubating at 4, 30, 60 and 90 °C for various times from 5 min to 72 h, the remaining inhibitor activity was determined by papain assay [16,17]. The pH stability of cystatin J inhibitory activity was investigated from pH 3–13 at 4 °C for 24 h. Two micromolar sodium phosphate buffer was used for pH 3–9 adjusting pH with 1 N HCl or NaOH, 2 mM Tris–HCl buffer was used for pH 10–13. The remaining inhibitory activity of cystatin J was analyzed with BAEE as a substrate after adjusting the pH to 6.2 with 200 mM PB [16,17]. 3. Results and discussion 3.1. Discovery of a cDNA encoding a novel cystatin On analysis of EST sequences obtained from cDNA library of C. capillata tentacle, several clones with low but significant homology to the Family 2 cystatin sequence were identified. The full-length cDNA clone contained an open reading frame encoding a 131-residue preprotein (Fig. 2), whose first 18 amino acids probably constituted the signal peptide were analyzed by SignalP V2.0 program. The cDNA started translation at the first ATG codon according to an alignment with Cyprinus carpio and Oncorhynchus keta cystatin sequence and had a purine (A) at -3 position [18]. The stop codon was followed by a 3'-untranslated sequence of 86 nucleotides with a polyadenylation signal, AATAAA [19], and a poly (A) tail (Fig. 2). The deduced mature protein sequence was just 26% identical to that of O. keta Family 2 cystatin, showed similar
Fig. 2. Nucleotide and deduced amino acid sequence of cDNA encoding cystatin J. Complete amino acid sequence of cystatin J inferred from the cDNA is given in one-letter code below the first nucleotide of each codon. The polyadenylation signal is underlined. The cleavage site of the signal peptide is shown by an arrow. Residues involved in enzyme-binding for cystatins are boxed.
resemblance (23–26% identity) to the sequences of the other known human Family 2 cystatins C, D, E/M, S, SN (Fig. 3) and cystatins from different species including carp, rat, chicken, salmon, and puff adder. The similarity to cystatin domain 1 of human kininogen was relatively high (34% identity), whereas it showed no significant similarity to the domain 2 and 3, as well as to Family 1 cystatins, A and B. The mature cystatin J contained a Gly4 residue at the active site near the N terminals, liked the Gly11 of cystatin C and Gly9 of chicken cystatin [20,21]. The novel cystatin had exactly the same central Gln–Xaa–Val–Xaa–Gly motif as the other cystatin sequences, and also a Trp98 residue toward the C-terminal end of the translation product, conserved in Family 2 or 3 cystatins. The sequence also contained four Cys residues towards the C-terminal end, corresponding to form two disulfide bridges in cystatin C and chicken cystatin, thereby stabilizing the cystatin structure [21,22]. Since the novel protein showed significantly similarity in overall sequence to known cystatins, and especially in the portion essential for structure and function, it should belong to the cystatin superfamily. But according to the significantly low sequence similarities, this novel protein should be considered as a new member in the superfamily. Therefore, this novel cystatin was designated as cystatin J, just for the first letter of jellyfish. The GenBank accession number of cystatin J gene is AY167572. The novel cystatin contained some notable characteristics, including some insertion and deletion of residues among the sequence, which did not locate in the enzyme-binding site [11] and seemed not to affect the inhibitory function. But, it was remarkable that the Pro-Trp pair at the C-terminal, which
1036
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
Fig. 3. Relationship between jellyfish cystatin and other cystatins. (A) Alignment of cystatin J sequence with other Family 2 cystatins. Numbering of amino acid sequence begins at the first residue of the mature protein. Dashes indicate gaps inserted for an optimal alignment. Vertical lines indicate residues identical in cystatin J and the other Family 2 cystatins. Boxes indicate residues that are involved in the cystatin inhibitory activity of the Family 2 cystatins and cystatin J. The four Cys residues shown to form two disulfide bridges in Cystatin C are marked with solid brackets. (B) The phylogenetic relationship between cystatin J and other cystatins. The phylogenetic tree was constructed using program on the web site (clustalw. genome. ad. jp). The tree indicated that cystatin J may belong to a new family.
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
Fig. 4. SDS-PAGE electrophoresis of cystatin J. Purification and enterokinase cleavage of recombinant Trx–cystatin fusion protein were analyzed by 15% SDS-PAGE at reducing conditions and stained with Coomassie Brilliant Blue R-250. Lane 1, recombinant Trx-cystatin fusion protein purified by Ni2+ chelate column; Lane 2, enterokinase cleavage of recombinant Trx-cystatin fusion protein. These step caused some unspecific degradation; Lane 3, SP-Sepharose column washed with the equilibrious buffer. The fraction contained fusion protein and Trx; Lane 4, purified cystatin J; Lane M, molecular weight markers with sizes of relevant bands indicated to the left.
was rather conserved in cystatin superfamily [11,23], was replaced by Ser97-Trp98 pair that was not found previously in anyone of cystatin superfamily. The Pro-Trp pair had been shown to be involved in a hydrophobic patch important for the integrity of the enzyme-binding surface of the inhibitor [23]. Since Ser residue had polarity and hydrophilicity, the substitution of Ser for Pro in cystatin J probably destroyed the hydrophobic contact between cystatin and enzyme, and then influenced the enzyme-binding property of the protein. The phylogenetic relationship between cystatin J and other cystatins (Fig. 3B) together with the low sequence similarities and unusual enzyme-binding domain indicated that the novel cystatin probably belongs to a new cystatin family, which differed from classic cystatin, named Family 4.
1037
Fig. 5. Active site titration of cystatin J with papain. Inhibition of 3 nM papain with increasing concentration of cystatin J. When the ratio of cystatin to papain reached 1:1, the activity of the enzyme was completely inhibited. This indicated that the cystatin bound with 1:1 stoichiometry to papain.
papain, and the residual enzyme activity was measured at various ratios of cystatin to papain. When papain incubated with increasing concentrations of cystatin, there was an obvious decrease in the activity of the enzyme (Fig. 5), which indicated that the novel cystatin is able to completely inhibit papain activity against BAEE by forming a 1:1 complex with the enzyme. The Ki value of cystatin J against papain was studied under various concentration of BAEE (Fig. 6). The Dixon plot indicated the cystatin was a competitive inhibitor against papain, with a Ki below 0.5 nM [16]. The Ki value of the novel cystatin was in similar level with cystatins extracted from chum salmon and sea anemone [14,24], which suggested that the substitution of hydrophilic Ser for hydrophobic Pro was not affecting the reaction between cystatin and
3.2. Production and characterization of recombinant cystatin J The cDNA encoding the mature cystatin J was amplified using a pair of specific primers. The PCR product, which did not contain the signal peptide, was cloned into the pETTrx expression vector and expressed in E. coli as a Trx-cystatin fusion protein. The fusion protein was purified by Ni2+ chelate column, resulting in a >95% pure protein band according to SDS-PAGE. This band indicated that the protein’s Mr was about 25,700 (Fig. 4, lane 1) and did not present in control extracts (not shown). The Trx carrier was cleaved from the purified fusion protein by proteolytic digestion at the enterokinase site (Fig. 4, lane 2). Cleaved protein was further purified by SP-Sepharose column and eluted as a single band with a relative molecular mass of 12,800. Since unspecific degradation occurred in the proteolytic reaction, the cleaved cystatin J showed a relatively low yield (Fig. 4, lane 4). 3.3. Cysteine proteinase inhibitory activity of cystatin J To investigate a potential protease inhibitory function of cystatin J, the recombinant protein was allowed to react with
Fig. 6. Dixon plots of papain inhibition with cystatin J using various concentration of BAEE. The concentration of BAEE was 1.5 mM ( ), 3 mM ("), 6 mM (m), respectively. The plot indicated the cystatin was a competitive inhibitor against papain, with a Ki below 0.5 nM.
1038
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
Fig. 9. pH stability of cystatin J inhibitory activity. The cystatin was incubated in pH 3–13 for 24 h at 4 °C. The residual inhibitory activity was tested. Fig. 7. Effect of temperature on the stability of cystatin J. Cystatin was incubated at various temperatures for 10 min and was immediately put on ice. The inhibitory activity towards papain was tested.
papain. To identify whether the mutation of cystatin J would bring higher inhibitory activity, the recombinant protein with Ser-97 was mutated to Pro-97 and expressed in the prokaryotic system. However, the mutant protein was degraded by a proteolytic reaction. The probable reason is that the mutation might affect the stable folding of the protein. Hence, it became difficult to investigate the protease inhibitory function of the mutant. The inhibitory activity of recombinant Trx–cystatin fusion protein, of which the N-terminal of cystatin is not dissociative, was also studied. The Ki value of the fusion protein was >20 nM and the protein–papain affinity was about 50–fold lower than cystatin, which indicated that the correct folding of the N-terminal of cystatin was important for papain inhibition. 3.4. Stability of cystatin J Heat stability of cystatin J was investigated by two methods. The cystatin was incubated between 20 and 90 °C for 10 min, its activity was stable at 20–40 °C and had an obvious decrease to 82% at 50 °C, then reduced to 27% when the temperature reached 90 °C (Fig. 7). The investigation was also performed by incubating the cystatin at 4, 30, 60 and
90 °C for various times from 5 min to 72 h. The activity was very stable at 4 °C, and still retained 82% after incubation for 72 h at 30 °C. Whereas, the activity decreased significantly when incubating at 60 and 90 °C, and remained about 9% after incubation for 12 h at 60 °C and for 1 h at 90 °C (Fig. 8A). The inactive constants (Kinact) of cystatin J were determined to be 0 for 4 °C, 4.17 × 10–5/min for 30 °C, 1.26 × 10–3/min for 60 °C, 1.52 × 10–2/min for 90 °C. The relationship between temperature (Kelvin) and Kinact was shown by Arrhenius plots (Fig. 8B) [16]. Activation energy, Ea, is a property of the given reaction. The larger the Ea, the greater is the effect the temperature has on the reaction. Ea of heat denaturation for the novel cystatin was determined from the slope of the plot as 10.28 kcal/mol (slope = –Ea/R) [25]. The heat sensibility of cystatin J showed a little higher than human cystatin C [26] and cystatin extract from chum salmon [9], which had remarkable stability at 70 and 90 °C, respectively. The inhibitory activity of cystatin J against papain was very stable in pH 5–8 for 24 h at 4 °C and was still maintained above 80% in pH 4 and pH 9–11. But, in pH 3 and pH 13 the activity was obviously reduced to 45% and 55%, respectively (Fig. 9). Cystatin J seemed to have no significant difference from the cystatins extract from fishes and plants, which showed high stability at pH 4–10 [16].
Fig. 8. Heat stability of cystatin J inhibitory activity. (A) Cystatin incubate at 4 (m), 30 (×), 60 ("), 90 ( ) °C for various times. The residual inhibitory activity was tested. (B) Arrhenius plots of the inactivation rate constants of cystatin J. Ea of heat denaturation for the novel cystatin was determined from the slope of the plot as 10.28 kcal/mol.
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
4. Conclusion A novel jellyfish cystatin, cystatin J, had been characterized and included a nature mutation in the critical position of enzyme-binding site, but shared a similar inhibitory activity and stability to other cystatins. Due to its unique feature, this novel cystatin should belong to a new family, the Family 4.
Acknowledgements We thank Xiaofeng Zhong for providing us help in protein expression and purification. This work was supported by the National High-Tech Development Program (863 Program) of China (2001AA626010), (819-06-06), and the key project from the National Natural Science Foundation of China (69935020).
References [1]
G. Rottini, L. Gusmani, E. Parovel, M. Avian, P. Patriarca, Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis, Toxicon 33 (1995) 315–326.
[2]
C.J. Cao, M.E. Eldefrawi, A.T. Eldefrawi, J.W. Burnett, R.J. Mioduszewski, D.E. Menking, J.J. Valdes, Toxicity of sea nettle toxin to human hepatocytes and the protective effects of phosphorylating and alkylating agents, Toxicon 36 (1998) 269–281.
[3]
L.K. Sun, Y. Yoshii, A. Hyodo, H. Tsurushima, A. Saito, T. Harakuni, Y.P. Li, M. Nozaki, N. Morine, Apoptosis induced by box jellyfish (Chiropsalmus quadrigatus) toxin in glioma and vascular endothelial cell lines, Toxicon 40 (2002) 441–446.
[4]
J.J. Chung, L.A. Ratnapala, I.M. Cooke, A.A. Yanagihara, Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom, Toxicon 39 (2001) 981–990.
[5]
M. Torres, M.B. Aguilar, A. Falcon, L. Sanchez, F.F. Radwan, J.W. Burnett, E.P. Heimer-de la Cotera, R.O. Arellano, Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana, Toxicon 39 (2001) 1297–1307.
[6]
C.J. Grimmelikhuijzen, M. Hahn, K.L. Rinehart, A.N. Spencer, Isolation of pyroGlu–Leu–Leu–Gly–Gly–Arg–Phe–NH2 (Pol-RFamide), a novel neuropeptide from hydromedusae, Brain Res. 475 (1988) 198–203.
[7]
A. Moosler, K.L. Rinehart, C.J. Grimmelikhuijzen, Isolation of three novel neuropeptides, the Cyanea-RFamides I–III, from Scyphomedusae, Biochem. Biophys. Res. Commun. 236 (1997) 743–749.
[8]
S.L. Olsson, B. Ek, I. Bjork, The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C, Biochim. Biophys. Acta 1432 (1999) 73–81.
[9]
M. Synnes, Purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.), Comp. Biochem. Physiol. B Biochem. Mol. Biol. 121 (1998) 257–264.
1039
[10] J. Ni, M. Abrahamson, M. Zhang, M.A. Fernandez, A. Grubb, J. Su, G.L. Yu, Y. Li, D. Parmelee, L. Xing, T.A. Coleman, S. Gentz, R. Thotakura, N. Nguyen, M. Hesselberg, R. Gentz, Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins, J. Biol. Chem. 272 (1997) 10853–10858. [11] M.T. Stubbs, B. Laber, W. Bode, R. Huber, R. Jerala, B. Lenarcic, V. Turk, The refined 2.4 A X–ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction, EMBO J. 9 (1990) 1939–1947. [12] M. Alvarez-Fernandez, A.J. Barrett, B. Gerhartz, P.M. Dando, J. Ni, M. Abrahamson, Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site, J. Biol. Chem. 274 (1999) 19195–19203. [13] S. Halfon, J. Ford, J. Foster, L. Dowling, L. Lucian, M. Sterling,Y. Xu, M. Weiss, M. Ikeda, D. Liggett, A. Helms, C. Caux, S. Lebecque, C. Hannum, S. Menon, T. McClanahan, D. Gorman, G. Zurawski, Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells, J. Biol. Chem. 273 (1998) 16400–16408. [14] B. Lenarcic, A. Ritonja, B. Strukelj, B. Turk, V. Turk, Equistatin, a new inhibitor of cysteine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain, J. Biol. Chem. 272 (1997) 13899–13903. [15] B. Stellmach (Ed.), The Mensuration of Enzyme, The People Publishers of China, Beijing, 1988, pp. 243–255. [16] J. Wu, N.F. Haard, Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 127 (2000) 209–220. [17] G. Chen, R.Q. Chou (Eds.), Enzymology, The Science and Technology Publishers, Hunan, 1987, pp. 311–350. [18] M. Kozak, Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs, Nucleic Acids Res. 12 (1984) 857–872. [19] N.J. Proudfoot, G.G. Brownlee, 3' non-coding region sequences in eukaryotic messenger RNA, Nature 263 (1976) 211–214. [20] L. Johansson, A. Grubb, M. Abrahamson, F. Kasprzykowski, R. Kasprzykowska, Z. Grzonka, U.H. Lerner, A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro, Bone 26 (2000) 451–459. [21] G. Sotiropoulou, A. Anisowicz, R. Sager, Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer, J. Biol. Chem. 272 (1997) 903–910. [22] J. Ni, M.A. Fernandez, L. Danielsson, R.A. Chillakuru, J. Zhang, A. Grubb, J. Su, R. Gentz, M. Abrahamson, Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor, J. Biol. Chem. 273 (1998) 24797–24804. [23] W. Bode, R. Engh, D. Musil, U. Thiele, R. Huber, A. Karshikov, J. Brzin, J. Kos, V. Turk, The 2.0 A X–ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases, EMBO J. 7 (1988) 2593–2599. [24] M. Yamashita, S. Konagaya, A novel cysteine protease inhibitor of the egg of chum salmon, containing a cysteine-rich thyroglobulin-like motif, J. Biol. Chem. 271 (1996) 1282–1284. [25] N.N. Ulusu, M.S. Kus, N.L. Acan, E.F. Tezcan, A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, Int. J. Biochem. Cell Biol. 31 (1999) 787–796. [26] A. Hall, K. Hakansson, R.W. Mason, A. Grubb, M. Abrahamson, Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N–terminal binding region as a selectivityconferring residue in the inhibition of mammalian cysteine peptidases, J. Biol. Chem. 270 (1995) 5115–5121.
cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata Yanzhen Yang, Shujian Cun, Lisheng Peng, Xiaojin Xie, Jianwen Wei, Wenli Yang, Anlong Xu * Department of Biochemistry, The Open Laboratory for Marine Functional Genomics of National High-Tech Development Program, Key Laboratory of Gene Engineering of Ministry of Education, College of Life Sciences, Sun Yat-Sen (Zhongshan) University, 135 W. Xin Gang Rd, Guangzhou 510275, PR China Received 20 June 2003; accepted 16 July 2003
Abstract Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser97-Trp98, which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately Mr = 12,800 displayed an obvious inhibition of papain (Ki value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4–11 at 4 °C, but unstable at neutral pH at >50 °C. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Cystatin; Jellyfish; Tentacle; Inhibitory activity; Stability
1. Introduction Jellyfish is known to be a rich source of variant polypeptide toxins [1–3], hemolysins [4,5] and neuropeptides [6,7], but little is known about the presence of proteolytic enzymes and their inhibitors. Cystatins, which have not yet been found in jellyfish before, play an important regulatory role not only in the protection of cells from unfavorable proteolysis by intracellular and external cysteine proteinase, but also in biological defense systems against invaders [8,9]. The known proteins of the cystatin superfamily can be grouped into three protein families as defined by Ni et al.
Abbreviations: BAEE, Na-benzoyl-L-arginine ethyl ester hydrochloride; b-ME, b-Mercaptoethanol; cDNA, Complementary DNA; EST, Expressed sequence tag; PB, Phosphate buffer. * Corresponding author. Tel.: +86-20-8411-3655; fax: +86-20-8403-8377. E-mail address: [email protected] (A. Xu). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/S0300-9084(03)00132-9
[10]. Family 1 cystatins (also called stefins), cystatins A and B, which contain approximately 100 amino acid residues (Mr~11,000), lack disulfide bonds and carbohydrate. Family 2 cystatins are secreted inhibitors of about 120 amino acid residues (Mr~13,000), which have two intramolecular disulfide bonds and lack carbohydrate, containing cystatin C, D, E, S and SN. Family 3 cystatins, the kininogens, are single chain plasma proteins, whose N-terminal heavy chain is composed of three glycosylated cystatin-like domains with inhibitory activity. All of the three protein family members are reversible, tight-binding enzyme inhibitors with specificity against papain-like cysteine proteinases [10]. For cystatin, the inhibition of cysteine peptidases is due to the conserved residues forming a tripartite wedge quite complementary to the active site clefts of the enzyme, and slotting into the active site [11]. The conserved enzyme-binding domains include a Gly at the N-terminal, a central Gln–Xaa–Val– Xaa–Gly motif, and a Pro-Trp pair at the C-terminal [12]. The cystatin-enzyme interaction is dominated by hydrophobic contacts [11].
1034
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
In this article, we described the cDNA cloning and identification of a novel cystatin from jellyfish, Cyanea capillata, which showed only 26% identities to Family 2 cystatins, and had a Ser97-Trp98 pair at the C-terminal, which differed from the conserved active site in all inhibitory cystatins. We reported the kinetic properties of the novel cystatin’s interaction with papain, and its stability under a certain condition. 2. Materials and methods 2.1. cDNA library Total RNA was extracted from tentacle of C. capillata by the TRIZOL® LS Reagent (Gibcol BRL), and cDNA library was constructed using the SMART™ cDNA Construction Kit (Clontech).
Fig. 1. Construction of pETTrx/cystatin designed for the expression of Trx–cystatin fusion protein. The cDNA fragment of cystatin J was amplified by PCR and ligated into Kpn I/Not I sites of pETTrx. The enterokinase cleavage sites are shown.
2.2. Identification of cDNA for cystatin J More than 2000 EST sequences obtained from the cDNA library of C. capillata tentacle were analyzed for possible identification of encoded function proteins. Sequence homology comparisons of each EST were performed against the GenBank database using the BLASTX algorithms, followed by BLASTP searches of the open reading frames [13]. ESTs having homology to previously identified sequences were given a tentative name based on the name of the sequence to which it was homologous. By this method, cystatin ESTs were identified. The cDNAs of cystatin were completely sequenced on each DNA strand and were found to be full-length. 2.3. Construction of cystatin J expression vector pETTrx/cystatin J The open reading frame of this cDNA sequence encoding the mature cystatin was amplified by PCR using a upstream primer (with tailing KpnI site and recognition site, DDDDK, for enterokinase underlined), 5'-GCT GAA TTC GGT ACC GAC GAC GAC GAC AAA CTA CTT CCT GGC GGA ATA AGA CG–3'; and a downstream primer (with NotI site), 5'-CGT GCG GCC GCT TAT CAA GCT CGG AGG CAT TTT GT–3'. The PCR product was digested with Kpn I and Not I, followed by ligation to pETTrx vector (a self-made expression vector in this laboratory, Fig. 1), which was previously linearized with Kpn I and Not I, resulting in the expression plasmid pETTrx/cystatin. The positive clones were sequenced with a BigDye™ DNA Sequencing Kit (Applied Biosystems) to ensure that it contained no mutations introduced by PCR. 2.4. Production and purification of recombinant Trx–cystatin J The original vector (pETTrx) as well as the recombinant plasmid (pETTrx/ cystatin) was transformed into E. coli
strain BL21 (DE3) bacteria and propagated in luria broth (LB) in the presence of 100 µg/ml ampicillin for selection of the cells transformed with the pETTrx/cystatin expression plasmid. The expression of recombinant protein was induced in exponentially growing bacteria (A600 = 0.6–0.8) with 0.1 mM isopropyl-1-thio–b-D-galactopyranoside for 8 h at 25 °C with vigorous agitation. The bacteria were harvested by centrifugation, washed with TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and resuspended in lysis buffer, 50 mM Tris-HCl, pH 8.0, containing 100 mM NaCl. Cells were lysed on ice by mild sonication, and the suspension was centrifuged at 5000 rpm for 30 min. All subsequent purification steps were carried out at 25 °C. The lysis supernatant was applied to a Ni2+ chelate column (Pharmacia Biotech), equilibrated with lysis buffer. The column was washed with the same buffer and bound protein was then eluted by 50 mM Tris-HCl, pH 7.0, containing 100 mM NaCl and a series of increasing imidazole concentrations. Analysis of the fractions was carried out on SDS-PAGE. Gels were stained with Coomassie Blue R-250. Fractions containing fusion proteins were pooled and applied to a Sephadex G-25 column (Pharmacia Biotech) equilibrated with 50 mM Tris-HCl, pH 7.5, containing 100 mM NaCl. Protein concentrations were determined by the Lowry Protein Quantitation Kit (Biocolor Co. Ltd.) using a standard curve generated with BSA. The Trx carrier was proteolytically cleaved from the fusion protein with enterokinase (produced by recombinant expression in this laboratory). The reaction was carried out at an enzyme to protein volume ratio of 1:500 for 4 h at 4 °C in 50 mM Tris-HCl, 100 mM NaCl, pH 7.5. Cleaved protein was applied to a SP-Sepharose column (Pharmacia Biotech), equilibrated with PB, pH 6.3. The column was washed with the same buffer and bound protein was then eluted by PB, pH 7.0, containing 150 mM NaCl. The purity of cystatin was assessed by SDS-PAGE.
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
1035
2.5. Active site titration Papain (3 nM final concentration) was incubated with increasing amounts of cystatin (0–9 nM final concentration) [14] in 500 µl of 20 mM PB, pH 6.2, containing 0.5 mM EDTA, 0.06 mM b-ME and 2 mM cysteine. After 10 min of incubation, added to 7.5 ml BAEE (Sigma) solution, pH 6.2, containing 1 M NaCl, 0.4 mM EDTA and 2 mM cysteine. The residual activity of papain was calculated by the volume of acid hydrolyzed by BAEE, as described by Stellmach [15]. 2.6. Inhibition kinetics of papain by cystatin J The kinetics of the reaction between cystatin and papain was analyzed under various concentrations of BAEE (i.e. 1.5, 3, 6 mM). The residual activity of papain was monitored as described above. The inhibition constant, Ki value, of cystatin J was obtained from a Dixon plot [16,17]. All the experiments were repeated at least three times. 2.7. Stability of cystatin J The thermal stability of cystatin J inhibitory activity in 20 mM PB, pH 6.2 was investigated by two methods: one was by incubating aliquots of inhibitor for 10 min at selected temperatures between 20 and 90 °C, then cooled on ice prior to determination of residual activity towards papain [9]; The other was by incubating at 4, 30, 60 and 90 °C for various times from 5 min to 72 h, the remaining inhibitor activity was determined by papain assay [16,17]. The pH stability of cystatin J inhibitory activity was investigated from pH 3–13 at 4 °C for 24 h. Two micromolar sodium phosphate buffer was used for pH 3–9 adjusting pH with 1 N HCl or NaOH, 2 mM Tris–HCl buffer was used for pH 10–13. The remaining inhibitory activity of cystatin J was analyzed with BAEE as a substrate after adjusting the pH to 6.2 with 200 mM PB [16,17]. 3. Results and discussion 3.1. Discovery of a cDNA encoding a novel cystatin On analysis of EST sequences obtained from cDNA library of C. capillata tentacle, several clones with low but significant homology to the Family 2 cystatin sequence were identified. The full-length cDNA clone contained an open reading frame encoding a 131-residue preprotein (Fig. 2), whose first 18 amino acids probably constituted the signal peptide were analyzed by SignalP V2.0 program. The cDNA started translation at the first ATG codon according to an alignment with Cyprinus carpio and Oncorhynchus keta cystatin sequence and had a purine (A) at -3 position [18]. The stop codon was followed by a 3'-untranslated sequence of 86 nucleotides with a polyadenylation signal, AATAAA [19], and a poly (A) tail (Fig. 2). The deduced mature protein sequence was just 26% identical to that of O. keta Family 2 cystatin, showed similar
Fig. 2. Nucleotide and deduced amino acid sequence of cDNA encoding cystatin J. Complete amino acid sequence of cystatin J inferred from the cDNA is given in one-letter code below the first nucleotide of each codon. The polyadenylation signal is underlined. The cleavage site of the signal peptide is shown by an arrow. Residues involved in enzyme-binding for cystatins are boxed.
resemblance (23–26% identity) to the sequences of the other known human Family 2 cystatins C, D, E/M, S, SN (Fig. 3) and cystatins from different species including carp, rat, chicken, salmon, and puff adder. The similarity to cystatin domain 1 of human kininogen was relatively high (34% identity), whereas it showed no significant similarity to the domain 2 and 3, as well as to Family 1 cystatins, A and B. The mature cystatin J contained a Gly4 residue at the active site near the N terminals, liked the Gly11 of cystatin C and Gly9 of chicken cystatin [20,21]. The novel cystatin had exactly the same central Gln–Xaa–Val–Xaa–Gly motif as the other cystatin sequences, and also a Trp98 residue toward the C-terminal end of the translation product, conserved in Family 2 or 3 cystatins. The sequence also contained four Cys residues towards the C-terminal end, corresponding to form two disulfide bridges in cystatin C and chicken cystatin, thereby stabilizing the cystatin structure [21,22]. Since the novel protein showed significantly similarity in overall sequence to known cystatins, and especially in the portion essential for structure and function, it should belong to the cystatin superfamily. But according to the significantly low sequence similarities, this novel protein should be considered as a new member in the superfamily. Therefore, this novel cystatin was designated as cystatin J, just for the first letter of jellyfish. The GenBank accession number of cystatin J gene is AY167572. The novel cystatin contained some notable characteristics, including some insertion and deletion of residues among the sequence, which did not locate in the enzyme-binding site [11] and seemed not to affect the inhibitory function. But, it was remarkable that the Pro-Trp pair at the C-terminal, which
1036
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
Fig. 3. Relationship between jellyfish cystatin and other cystatins. (A) Alignment of cystatin J sequence with other Family 2 cystatins. Numbering of amino acid sequence begins at the first residue of the mature protein. Dashes indicate gaps inserted for an optimal alignment. Vertical lines indicate residues identical in cystatin J and the other Family 2 cystatins. Boxes indicate residues that are involved in the cystatin inhibitory activity of the Family 2 cystatins and cystatin J. The four Cys residues shown to form two disulfide bridges in Cystatin C are marked with solid brackets. (B) The phylogenetic relationship between cystatin J and other cystatins. The phylogenetic tree was constructed using program on the web site (clustalw. genome. ad. jp). The tree indicated that cystatin J may belong to a new family.
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
Fig. 4. SDS-PAGE electrophoresis of cystatin J. Purification and enterokinase cleavage of recombinant Trx–cystatin fusion protein were analyzed by 15% SDS-PAGE at reducing conditions and stained with Coomassie Brilliant Blue R-250. Lane 1, recombinant Trx-cystatin fusion protein purified by Ni2+ chelate column; Lane 2, enterokinase cleavage of recombinant Trx-cystatin fusion protein. These step caused some unspecific degradation; Lane 3, SP-Sepharose column washed with the equilibrious buffer. The fraction contained fusion protein and Trx; Lane 4, purified cystatin J; Lane M, molecular weight markers with sizes of relevant bands indicated to the left.
was rather conserved in cystatin superfamily [11,23], was replaced by Ser97-Trp98 pair that was not found previously in anyone of cystatin superfamily. The Pro-Trp pair had been shown to be involved in a hydrophobic patch important for the integrity of the enzyme-binding surface of the inhibitor [23]. Since Ser residue had polarity and hydrophilicity, the substitution of Ser for Pro in cystatin J probably destroyed the hydrophobic contact between cystatin and enzyme, and then influenced the enzyme-binding property of the protein. The phylogenetic relationship between cystatin J and other cystatins (Fig. 3B) together with the low sequence similarities and unusual enzyme-binding domain indicated that the novel cystatin probably belongs to a new cystatin family, which differed from classic cystatin, named Family 4.
1037
Fig. 5. Active site titration of cystatin J with papain. Inhibition of 3 nM papain with increasing concentration of cystatin J. When the ratio of cystatin to papain reached 1:1, the activity of the enzyme was completely inhibited. This indicated that the cystatin bound with 1:1 stoichiometry to papain.
papain, and the residual enzyme activity was measured at various ratios of cystatin to papain. When papain incubated with increasing concentrations of cystatin, there was an obvious decrease in the activity of the enzyme (Fig. 5), which indicated that the novel cystatin is able to completely inhibit papain activity against BAEE by forming a 1:1 complex with the enzyme. The Ki value of cystatin J against papain was studied under various concentration of BAEE (Fig. 6). The Dixon plot indicated the cystatin was a competitive inhibitor against papain, with a Ki below 0.5 nM [16]. The Ki value of the novel cystatin was in similar level with cystatins extracted from chum salmon and sea anemone [14,24], which suggested that the substitution of hydrophilic Ser for hydrophobic Pro was not affecting the reaction between cystatin and
3.2. Production and characterization of recombinant cystatin J The cDNA encoding the mature cystatin J was amplified using a pair of specific primers. The PCR product, which did not contain the signal peptide, was cloned into the pETTrx expression vector and expressed in E. coli as a Trx-cystatin fusion protein. The fusion protein was purified by Ni2+ chelate column, resulting in a >95% pure protein band according to SDS-PAGE. This band indicated that the protein’s Mr was about 25,700 (Fig. 4, lane 1) and did not present in control extracts (not shown). The Trx carrier was cleaved from the purified fusion protein by proteolytic digestion at the enterokinase site (Fig. 4, lane 2). Cleaved protein was further purified by SP-Sepharose column and eluted as a single band with a relative molecular mass of 12,800. Since unspecific degradation occurred in the proteolytic reaction, the cleaved cystatin J showed a relatively low yield (Fig. 4, lane 4). 3.3. Cysteine proteinase inhibitory activity of cystatin J To investigate a potential protease inhibitory function of cystatin J, the recombinant protein was allowed to react with
Fig. 6. Dixon plots of papain inhibition with cystatin J using various concentration of BAEE. The concentration of BAEE was 1.5 mM ( ), 3 mM ("), 6 mM (m), respectively. The plot indicated the cystatin was a competitive inhibitor against papain, with a Ki below 0.5 nM.
1038
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
Fig. 9. pH stability of cystatin J inhibitory activity. The cystatin was incubated in pH 3–13 for 24 h at 4 °C. The residual inhibitory activity was tested. Fig. 7. Effect of temperature on the stability of cystatin J. Cystatin was incubated at various temperatures for 10 min and was immediately put on ice. The inhibitory activity towards papain was tested.
papain. To identify whether the mutation of cystatin J would bring higher inhibitory activity, the recombinant protein with Ser-97 was mutated to Pro-97 and expressed in the prokaryotic system. However, the mutant protein was degraded by a proteolytic reaction. The probable reason is that the mutation might affect the stable folding of the protein. Hence, it became difficult to investigate the protease inhibitory function of the mutant. The inhibitory activity of recombinant Trx–cystatin fusion protein, of which the N-terminal of cystatin is not dissociative, was also studied. The Ki value of the fusion protein was >20 nM and the protein–papain affinity was about 50–fold lower than cystatin, which indicated that the correct folding of the N-terminal of cystatin was important for papain inhibition. 3.4. Stability of cystatin J Heat stability of cystatin J was investigated by two methods. The cystatin was incubated between 20 and 90 °C for 10 min, its activity was stable at 20–40 °C and had an obvious decrease to 82% at 50 °C, then reduced to 27% when the temperature reached 90 °C (Fig. 7). The investigation was also performed by incubating the cystatin at 4, 30, 60 and
90 °C for various times from 5 min to 72 h. The activity was very stable at 4 °C, and still retained 82% after incubation for 72 h at 30 °C. Whereas, the activity decreased significantly when incubating at 60 and 90 °C, and remained about 9% after incubation for 12 h at 60 °C and for 1 h at 90 °C (Fig. 8A). The inactive constants (Kinact) of cystatin J were determined to be 0 for 4 °C, 4.17 × 10–5/min for 30 °C, 1.26 × 10–3/min for 60 °C, 1.52 × 10–2/min for 90 °C. The relationship between temperature (Kelvin) and Kinact was shown by Arrhenius plots (Fig. 8B) [16]. Activation energy, Ea, is a property of the given reaction. The larger the Ea, the greater is the effect the temperature has on the reaction. Ea of heat denaturation for the novel cystatin was determined from the slope of the plot as 10.28 kcal/mol (slope = –Ea/R) [25]. The heat sensibility of cystatin J showed a little higher than human cystatin C [26] and cystatin extract from chum salmon [9], which had remarkable stability at 70 and 90 °C, respectively. The inhibitory activity of cystatin J against papain was very stable in pH 5–8 for 24 h at 4 °C and was still maintained above 80% in pH 4 and pH 9–11. But, in pH 3 and pH 13 the activity was obviously reduced to 45% and 55%, respectively (Fig. 9). Cystatin J seemed to have no significant difference from the cystatins extract from fishes and plants, which showed high stability at pH 4–10 [16].
Fig. 8. Heat stability of cystatin J inhibitory activity. (A) Cystatin incubate at 4 (m), 30 (×), 60 ("), 90 ( ) °C for various times. The residual inhibitory activity was tested. (B) Arrhenius plots of the inactivation rate constants of cystatin J. Ea of heat denaturation for the novel cystatin was determined from the slope of the plot as 10.28 kcal/mol.
Y. Yang et al. / Biochimie 85 (2003) 1033–1039
4. Conclusion A novel jellyfish cystatin, cystatin J, had been characterized and included a nature mutation in the critical position of enzyme-binding site, but shared a similar inhibitory activity and stability to other cystatins. Due to its unique feature, this novel cystatin should belong to a new family, the Family 4.
Acknowledgements We thank Xiaofeng Zhong for providing us help in protein expression and purification. This work was supported by the National High-Tech Development Program (863 Program) of China (2001AA626010), (819-06-06), and the key project from the National Natural Science Foundation of China (69935020).
References [1]
G. Rottini, L. Gusmani, E. Parovel, M. Avian, P. Patriarca, Purification and properties of a cytolytic toxin in venom of the jellyfish Carybdea marsupialis, Toxicon 33 (1995) 315–326.
[2]
C.J. Cao, M.E. Eldefrawi, A.T. Eldefrawi, J.W. Burnett, R.J. Mioduszewski, D.E. Menking, J.J. Valdes, Toxicity of sea nettle toxin to human hepatocytes and the protective effects of phosphorylating and alkylating agents, Toxicon 36 (1998) 269–281.
[3]
L.K. Sun, Y. Yoshii, A. Hyodo, H. Tsurushima, A. Saito, T. Harakuni, Y.P. Li, M. Nozaki, N. Morine, Apoptosis induced by box jellyfish (Chiropsalmus quadrigatus) toxin in glioma and vascular endothelial cell lines, Toxicon 40 (2002) 441–446.
[4]
J.J. Chung, L.A. Ratnapala, I.M. Cooke, A.A. Yanagihara, Partial purification and characterization of a hemolysin (CAH1) from Hawaiian box jellyfish (Carybdea alata) venom, Toxicon 39 (2001) 981–990.
[5]
M. Torres, M.B. Aguilar, A. Falcon, L. Sanchez, F.F. Radwan, J.W. Burnett, E.P. Heimer-de la Cotera, R.O. Arellano, Electrophysiological and hemolytic activity elicited by the venom of the jellyfish Cassiopea xamachana, Toxicon 39 (2001) 1297–1307.
[6]
C.J. Grimmelikhuijzen, M. Hahn, K.L. Rinehart, A.N. Spencer, Isolation of pyroGlu–Leu–Leu–Gly–Gly–Arg–Phe–NH2 (Pol-RFamide), a novel neuropeptide from hydromedusae, Brain Res. 475 (1988) 198–203.
[7]
A. Moosler, K.L. Rinehart, C.J. Grimmelikhuijzen, Isolation of three novel neuropeptides, the Cyanea-RFamides I–III, from Scyphomedusae, Biochem. Biophys. Res. Commun. 236 (1997) 743–749.
[8]
S.L. Olsson, B. Ek, I. Bjork, The affinity and kinetics of inhibition of cysteine proteinases by intact recombinant bovine cystatin C, Biochim. Biophys. Acta 1432 (1999) 73–81.
[9]
M. Synnes, Purification and characterization of two cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.), Comp. Biochem. Physiol. B Biochem. Mol. Biol. 121 (1998) 257–264.
1039
[10] J. Ni, M. Abrahamson, M. Zhang, M.A. Fernandez, A. Grubb, J. Su, G.L. Yu, Y. Li, D. Parmelee, L. Xing, T.A. Coleman, S. Gentz, R. Thotakura, N. Nguyen, M. Hesselberg, R. Gentz, Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins, J. Biol. Chem. 272 (1997) 10853–10858. [11] M.T. Stubbs, B. Laber, W. Bode, R. Huber, R. Jerala, B. Lenarcic, V. Turk, The refined 2.4 A X–ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction, EMBO J. 9 (1990) 1939–1947. [12] M. Alvarez-Fernandez, A.J. Barrett, B. Gerhartz, P.M. Dando, J. Ni, M. Abrahamson, Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site, J. Biol. Chem. 274 (1999) 19195–19203. [13] S. Halfon, J. Ford, J. Foster, L. Dowling, L. Lucian, M. Sterling,Y. Xu, M. Weiss, M. Ikeda, D. Liggett, A. Helms, C. Caux, S. Lebecque, C. Hannum, S. Menon, T. McClanahan, D. Gorman, G. Zurawski, Leukocystatin, a new Class II cystatin expressed selectively by hematopoietic cells, J. Biol. Chem. 273 (1998) 16400–16408. [14] B. Lenarcic, A. Ritonja, B. Strukelj, B. Turk, V. Turk, Equistatin, a new inhibitor of cysteine proteinases from Actinia equina, is structurally related to thyroglobulin type-1 domain, J. Biol. Chem. 272 (1997) 13899–13903. [15] B. Stellmach (Ed.), The Mensuration of Enzyme, The People Publishers of China, Beijing, 1988, pp. 243–255. [16] J. Wu, N.F. Haard, Purification and characterization of a cystatin from the leaves of methyl jasmonate treated tomato plants, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 127 (2000) 209–220. [17] G. Chen, R.Q. Chou (Eds.), Enzymology, The Science and Technology Publishers, Hunan, 1987, pp. 311–350. [18] M. Kozak, Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs, Nucleic Acids Res. 12 (1984) 857–872. [19] N.J. Proudfoot, G.G. Brownlee, 3' non-coding region sequences in eukaryotic messenger RNA, Nature 263 (1976) 211–214. [20] L. Johansson, A. Grubb, M. Abrahamson, F. Kasprzykowski, R. Kasprzykowska, Z. Grzonka, U.H. Lerner, A peptidyl derivative structurally based on the inhibitory center of cystatin C inhibits bone resorption in vitro, Bone 26 (2000) 451–459. [21] G. Sotiropoulou, A. Anisowicz, R. Sager, Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer, J. Biol. Chem. 272 (1997) 903–910. [22] J. Ni, M.A. Fernandez, L. Danielsson, R.A. Chillakuru, J. Zhang, A. Grubb, J. Su, R. Gentz, M. Abrahamson, Cystatin F is a glycosylated human low molecular weight cysteine proteinase inhibitor, J. Biol. Chem. 273 (1998) 24797–24804. [23] W. Bode, R. Engh, D. Musil, U. Thiele, R. Huber, A. Karshikov, J. Brzin, J. Kos, V. Turk, The 2.0 A X–ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases, EMBO J. 7 (1988) 2593–2599. [24] M. Yamashita, S. Konagaya, A novel cysteine protease inhibitor of the egg of chum salmon, containing a cysteine-rich thyroglobulin-like motif, J. Biol. Chem. 271 (1996) 1282–1284. [25] N.N. Ulusu, M.S. Kus, N.L. Acan, E.F. Tezcan, A rapid method for the purification of glucose-6-phosphate dehydrogenase from bovine lens, Int. J. Biochem. Cell Biol. 31 (1999) 787–796. [26] A. Hall, K. Hakansson, R.W. Mason, A. Grubb, M. Abrahamson, Structural basis for the biological specificity of cystatin C. Identification of leucine 9 in the N–terminal binding region as a selectivityconferring residue in the inhibition of mammalian cysteine peptidases, J. Biol. Chem. 270 (1995) 5115–5121.