Molecular Cloning and Characterization of Cystatin, a Cysteine Protease Inhibitor, from Bufo Melanostictus

Biosci. Biotechnol. Biochem., 77 (10), 2077–2081, 2013

Molecular Cloning and Characterization of Cystatin, a Cysteine Protease Inhibitor, from Bufo melanostictus

y Wa LIU,1 Senlin JI,1 A-Mei ZHANG,2 Qinqin HAN,1 Yue FENG,2 and Yuzhu SONG1;

1Engineering Research Center for Molecular Diagnosis, Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, Yunnan 650500, China 2Laboratory of Molecular Virology, Faculty of Life Sciences and Technology, Kunming University of Science and Technology, Kunming, Yunnan 650500, China

Received May 31, 2013; Accepted July 17, 2013; Online Publication, October 7, 2013 [doi:10.1271/bbb.130424]

Cystatins are efficient inhibitors of papain-like cys- inhibit pathogens, such as CP1 from green kiwi fruit, teine proteinases, and they serve various important which exhibits antifungal activity against Alternaria physiological functions. In this study, a novel cystatin, radicina and Botrytis cinerea both in vitro and in vivo;2) Cystatin-X, was cloned from a cDNA library of the skin the cystatin gene in wheat, which provides resistance of Bufo melanostictus. The single nonglycosylated poly- against Karnal bunt, caused by Tilletia indica;3) and peptide chain of Cystatin-X consisted of 102 amino acid chicken cystatins, which inhibit the growth of Porphyr- residues, including seven cysteines. Evolutionary analy- omonas gingivalis.4) A small number of cystatins from sis indicated that Cystatin-X can be grouped with family amphibians have been identified by means of genome 1 cystatins. It contains cystatin-conserved motifs known and transcriptome sequencing, but their functions have to interact with the active site of cysteine proteinases. not been studied. Recombinant Cystatin-X expressed and purified from Cathepsin B has been co-expressed with cystatin A in Escherichia coli exhibited obvious inhibitory activity primary tumors and metastases, suggesting that cystatin against cathepsin B. rCystatin-X at a concentration of A suppresses metastasis via inhibition of cathepsin B.5) 8 M inhibited nearly 80% of cathepsin B activity within Cathepsin B is a lysosomal cysteine protease primarily 15 s, and about 90% of cathepsin B activity within involved in the degradation and processing of lysosomal 15 min. The Cystatin-X identified in this study can play proteins. Under certain conditions, it has been found to an important role in host immunity and in the medical be involved in additional cellular processes, including effect of B. melanostictus. cell invasion, vesicle trafficking, inflammasome forma- tion, and cell death.6) Key words: amphibian skin; cysteine proteinase inhib- The stratum corneum exuviated by the black-specta- itor; cDNA screening cled toad (Bufo melanostictus) is one of the most popular sources of chanyi, a traditional Chinese medi- Cystatins, inhibitors of papain-like cysteine proteases, cine that is used to treat hepatitis B, sores, and various are ubiquitous in plants and animals. They are catego- cancers. Certain bioactive ingredients have been isolated rized into three major families: (i) Family 1 (stefins) are from the skin of B. melanostictus, including bufadieno- unglycosylated inhibitors of about 11 kDa that lack lide sulfates and BMP1.7,8) The stratum corneum of the signal sequences and disulfide bonds. Stefin A and B skin is an important barrier of the host immune system (also known as cystatin A and B) belong to this family. against external injury. Amphibian skin contains abun- (ii) Family 2 (cystatins) have molecular masses ranging dant bioactive peptides, particularly antimicrobial pep- from 13 kDa to 14 kDa and contain signal peptide and tides and protease inhibitors, which provide efficient disulfide bonds at the carboxy terminus. Some members protection. Up to 2,000 antimicrobial peptides and 200 of this family are glycosylated, and the family is protease inhibitors have been identified in amphibian represented by cystatin C, D, S, SA, and SN. (iii) skin,9) but none have been reported for B. melanostictus. Family 3 (kininogens) have molecular masses ranging In our previous study, no antimicrobial activity was from 88 kDa to 114 kDa, are glycosylated, and have detected in the skin secretion of B. melanostictus three family 2 cystatin domains, two of which (domains (unpublished data). Considering the multiple functions 2 and 3) exhibit protease inhibitory activity.1) of cystatin, it is reasonabe to hypothesize that it serves Cystatins can efficiently inhibit endogenous and an important role in host immunity and officinal effect. extraneous cysteine proteinases, and have important Here we report the cloning, prokaryotic expression, physiological functions in the immune system, including and biological functions of cystatin obtained from steadying host-parasite interaction and defending against B. melanostictus (Cystatin-X). pathogens. They can function as the dominant biochem- ical skin barrier. Cystatin A, which is produced by Materials and Methods keratinocytes, can eliminate the enzymatic activity of parasite cysteine proteases and prevent them from cDNA library construction. Adult B. melanostictus was collected stimulating keratinocytes. Some cystatins can directly from Xishuangbanna, Yunnan Province, China. Total RNA was

y To whom correspondence should be addressed. Fax: +86-871-65939528; E-mail: [email protected] 2078 W. LIU et al. extracted from the dorsal skin with TRIzol reagent (Invitrogen, pH dependency assay was done by the procedure reported earlier by Carlsbad, USA). First-strand cDNA was synthesized with a Liu et al., but with minor modifications. In brief, purified rCystatin-X SMARTer polymerase chain reaction (PCR) cDNA Synthesis Kit (2 mM) was pre-incubated with assay buffers of different pH values, (Clontech, Mountain View, USA) with CDS III primer (50-ATTGAC- sodium acetate buffer (pH 3, 4, 5, and 6), sodium phosphate buffer TCGAGTCGACATCGATTTTTTTTTTTTTTTTTT-30) and SMART (pH 7 and 8), and Tris–HCl buffer (pH 9), for 5 min at 37 C. The II A oligonucleotide (50-AAGCAGTGGTATCAACGCAGAGTAC- mixture was added to the substrate, and this was incubated at 37 C for XXXXX-30). The second strand was amplified with ExTaq polymerase 30 min. Finally, the absorbance at 405 nm was measured.18) (Takara, Otsu, Japan) with 50 PCR primer II A (50-AAGCAGTGGTA- TCAACGCAGAGT-30) and 30 primer R1 (50-ATTGACTCGAGTCG- Results ACATCGA-30).10) Molecular cloning and sequence analysis Molecular cloning and sequence analysis. The synthesized cDNA Upon screening the skin cDNA library as indicated was used as template for PCR to screen the cDNAs encoding cystatin A. Two oligonucleotide primers, S1 (50-CCYGCCACYSCAGARRT- above, several clones containing inserts of about 400 ICA-30) and S2 (50-CAAGTNGTBGCHGGRDIVA-30), in the sense base pairs were identified and isolated. A cystatin direction, were designed according to the conserved sequences of homolog, Cystatin-X, was deduced from the nucleotide cystatin A from the vertebrata. Nested PCRs were performed together sequences (GenBank accession no. KF019630). The with R1 in the antisense direction. The PCR reaction was performed in open reading frames were composed of 102 amino acids a volume of 50 mL as follows: initial denaturation at 95 C for 3 min, 25 (Fig. 1). cycles of denaturation at 94 C for 30 s, annealing at 54.8 C for 30 s, Based on SignalP analysis, Cystatin-X may not extension at 72 C for 30 s, and final extension at 72 C for 5 min. The PCR products were cloned into pMD19-T Vector (Takara, Otsu, contain a signal peptide and is unlikely to be exposed Japan). DNA sequencing was performed on an Applied Biosystems to the glycosylation machinery. Glycosylation site DNA sequencer, model ABI PRISM 377. Another primer, R2 (50- prediction using NetOGlyc 3.1 and NetNGlyc 1.0 server CTAAAAATAACAGATGGGGTCT-30), was then designed according also indicated that Cystatin-X might not contain poten- to the sequenced nucleotide sequence, together with 50 PCR Primer II tial motifs to be glycosylated. 0 A, to amplify the 5 ends of the cDNA. Figure 2a compares the primary sequence for Cys- The nucleotide sequence were analyzed by BLAST and Translate Tool (http://web.expasy.org/translate/) to deduce the amino acid tatin-X with homologs from the vertebrate. Cystatin-X sequence.11) Then signal peptide and glycosylation prediction were contained the three conserved cysteine protease inhibitor performed using SignalP and NetOGlyc/NetNGlyc respectively.12,13) signature sequences critical to the biological function of Three-dimensional structure was predicted at Quark server (http: cystatins. Evolutionary analysis indicated that Cystatin- //zhanglab.ccmb.med.umich.edu/QUARK/).14) Evolutionary analyses X can be grouped among family 1 cystatins (Fig. 2b). 15) were conducted by MEGA5. In brief, multiple alignment of the The structure of Cystatin-X was predicted with the amino acid sequences were done using MUSCLE, and then the Quark Server.19) Three structural elements, G in the evolutionary history was inferred by the minimum evolution method together with the bootstrap test (10,000 replicates). N-terminal region, QVVAG at the first binding loop connecting the second and third -strands, and LP at the Expression and purification of recombinant Cystatin-X. The second binding loop between the fourth and fifth strands protein-encoding fragment of the Cystatin-X cDNA was subcloned of the -sheet, were similar to cystatin A (PDB 1G63) into pET32a (Novagen, Darmstadt, Germany), and expression of the (Fig. 3). recombinant Cystatin-X (rCystatin-X) as a His-tagged protein in transformant Escherichia coli BL21 (DE3) was induced by adding Expression and purification of rCystatin-X isopropyl -D-thiogalactopyranoside (IPTG, final concentration Cystatin-X was expressed abundantly as a soluble, 0.4 mM)at30C.16) The bacteria were lysed and treated with DNase and RNase. Soluble rCystatin-X was purified with Ni Sepharose 6 recombinant, HIS-tagged protein in E. coli BL21 (DE3) Fast Flow affinity media (GE, Freiburg, USA) and cleaved with after induction with IPTG and purification by HIS-tag enterokinase following the manufacturer’s instructions. Briefly, a affinity chromatography under non-denaturing condi- concentrated expression solution was loaded after dialysis on an tions. The N-terminal fused HIS-tag was enterokinase- affinity column equilibrated with binding buffer (20 mM sodium cleaved. The purified rCystatin-X showed a single band phosphate pH 7.4, 500 mM NaCl, 20 mM imidazole). The specifically on SDS–PAGE of about 11 kDa (Fig. 4). binding proteins were eluted using the same buffer with increasing concentrations of imidazole. The eluted fractions exhibiting inhibitory activity were pooled, and the resulting mixture was subjected to ultrafiltration by an Amicon Ultra-15 centrifugal filter unit with the Ultracel-3 membrane (Millipore, Feltham, UK). The purified compo- nents were electrophoresed in 16% Tris-tricine polyacrylamide gels to evaluate the purity and molecular masses of the components. Then MALDI-TOF analysis was done to determine the molecular masses accurately.

Cysteine protease inhibition assay. The ability of recombinant Cystatin-X to inhibit the activity of human cathepsin B (Sigma, St. Louis, USA) was examined in an assay that employed the chromogenic peptide substrate carbobenzoxyphenylalanine-arginine-7-amino-4- methyl coumarin (CBZ-Phe-Arg-pNA, Sigma, St. Louis, USA). Cathepsin B (0.2 ng per reaction) was incubated at room temperature for 5 min in an assay buffer (0.1 M sodium acetate pH 5.5, containing 1mM DTT, 2 mM EDTA, and 4 mM cysteine). Various amounts of rCystatin-X (final concentration 0.1 mM to 2 mM) were added. After 5 min of pre-incubation at room temperature, the substrate was added Fig. 1. Nucleotide and Deduced Amino Acid Sequences of to a final concentration of 400 mM. The formation of p-nitroaniline was Cystatin-X. then monitored continuously at 405 nm for 30 min.17) The stop codon TAG is indicated by asterisks. Molecular Cloning and Characterization of Cystatin 2079 a

b

Fig. 3. The Predicted Structure of Cystatin-X (a) Was Similar to Cystatin A (PDB 1G63) (b). The side chains of the amino acids of three conservative reactive sites are shown. The seven cysteine residues (showed in yellow) are near each other in the three-dimensional structure.

Fig. 4. SDS–PAGE Analyses of Expressed and Purified rCystatin-X. Lane M, protein molecular weight marker; lane 1, total cell extract of E. coli without induction; lane 2, total cell extract after induction of rCystatin by IPTG; lane 3, soluble cell lysates after IPTG induction; lane 4, rCystatin-X with fusion protein purified from lysates; lane 5, rCystatin-X released from the fusion protein by enzymatic cleavage.

activity) at pH 8, and retained only 23% of its activity Fig. 2. Multiple Sequence Alignment and Evolutionary Analysis. at pH 3. (a) Multiple sequence alignment of cystatin-X and its homologs from Danio rerio (Dre Cystatin-A, Genbank accession no. Discussion NP 001070742), Bos taurus (Bta Cystatin-A, Genbank accession no. NP 001161296), and Homo sapiens (Hsa Cystatin-A, Genbank accession no. NP 005204; Hsa Cystatin-B, Genbank accession no. Cysteine proteases, a family of proteases found in AAF44059). The three conservative reactive sites, Gly at the N nearly all living organisms, are involved in various terminus, Gln-X-Val-X-Gly in the middle, and Pro-Trp pair at the C biological and pathological processes.20) Cystatins, terminus, are boxed. (b) Evolutionary analysis of Cystatins. All 45 inhibitors of cysteine proteases, protect against the sequences can be clustered into three clades representing different Cystatin families. Cystatin-X is underlined. potentially harmful effects of free cysteine proteases by rapidly trapping the enzymes in tight complexes.21) Our results indicate that screening a toad skin cDNA library Cysteine protease inhibition assay allowed us successfully to clone and sequence toad To investigate the potential protease inhibitory Cystatin-X. The open reading frame consisted of 309 bp, activity of Cystatin-X, rCystatin-X was reacted with encoding 102 amino acid residues. Cystatin-X, similarly human cathepsin B in vitro. While incubating cathepsin to typical family 1 cystatins, might consist of non- B with increasing concentrations of the inhibitor, glycosylated polypeptide chains. But unlike family 1 residual enzyme activity was measured continuously cystatins, Cystatin-X possesses seven cysteines. Align- in the presence of the chromogenic substrate Z-Arg- ment with other cystatins indicated that it contains three Arg-pNA. As shown in Fig. 5, the inhibition percent- conserved cysteine protease inhibitor signature se- age increased with increasing rCystatin-X concentra- quences critical to the biological function of cystatins.22) tions. At 2 mM, rCystatin-X inhibited about 60% of rCystatin-X displayed strong inhibitory activity cathepsin B activity within 15 s, and at 15 min, it against cathepsin B. In the present study, cystatin inhibited about 75%. At a concentration of 8 mM, showed maximally 85% inhibitory activity at pH 8.18) rCystatin-X inhibited nearly 80% of cathepsin B Under the same conditions, rCystatin-X reached 93% activity within 15 s, and about 90% within 15 min. inhibitory activity. This indicates that Cystatin-X had rCystatin-X showed maximum activity (93% inhibitory potency similar to known cystatins. Many studies have 2080 W. LIU et al. a

b

Fig. 5. Cysteine Protease Inhibition Assay of rCystatin-X. Cathepsin B was inhibited by rCystatin-X in a dose-dependent and time-dependent manner (a). rCystatin-X was maximally active (93% inhibitory activity) at pH 8 and retained 23% of its activity at pH 3 (b). found that overexpression of cathepsin B is correlated cDNA into human EC9706 esophageal squamous cell with invasive and metastatic phenotypes in cancers.23) carcinoma cells inhibited tumor growth, angiogenesis, Suppressing the proteolytic activity of cathepsin B invasion, and metastasis, mainly by inhibiting cathepsin inhibits the infiltrative behavior of tumor cells.24) The B activity.29) It is reasonable to speculate that the skin of the black-spectacled toad is the main source of Cystatin-X identified in the present study plays an chanyi, which is used to treat various cancers, although important role in host immunity and the medical effect its active compounds remain unclear. Some cystatins of B. melanostictus, but the exact functions of Cystatin- inhibit pathogens and treat tumors.25) It has been X, such as anti-tumor activity, and structure-function reported that both chicken cystatin and salivary cystatin relationship, require further study. S inhibit the growth of the oral pathogen Porphyromo- In summary, a novel cystatin, Cystatin-X, was nas gingivalis.4) Wesierska et al. found that Escherichia isolated from the skin cDNA library of B. melanostictus. coli, Pseudomonas aeruginosa, and Staphylococcus Further study of the function of Cystatin-X is required to aureus are highly sensitive to low concentrations of interpret its role in host immunity and its medical effect. chicken egg white cystatin.26) Cystatin as a tumor suppressor has been confirmed by observations of breast, Acknowledgments prostate, and esophageal tumors. 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