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Cloning and characterization of a metalloprotease from Vibrioharveyi strain AP6
Gene 303 (2003) 147–156 www.elsevier.com/locate/gene
Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6 Jeanette W.P. Teoa, Lian-Hui Zhangb, Chit Laa Poha,* a
Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117 597, Singapore b Laboratory of Biosignals and Bioengineering, Institute of Molecular and Cell Biology, National University of Singapore, 5 Science Drive 2, Singapore 117 597, Singapore Received 12 August 2002; received in revised form 4 November 2002; accepted 12 November 2002 Received by A.M. Campbell
Abstract A metalloprotease gene pap6 was cloned from Vibrio harveyi strain AP6. Sequence analysis showed that pap6 was 2034 bp in length and predicted to encode a peptide of 677 amino acids with a molecular mass of 75 kDa. SDS-PAGE analysis of the purified Pap6 revealed that it was 35 kDa in size. N-terminal amino acid sequencing established that the mature protein began at Leu-191, suggesting that the preprotein of Pap6 was processed to generate a mature protease. Purified Pap6 was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors such as 1, 10-phenanthroline, EGTA and EDTA. The deduced amino acid sequence revealed the presence of a zincbinding motif HEXXH , 19aa , E. Substitution of these active site residues by site-directed mutagenesis caused significant losses in enzyme activity, thus demonstrating their involvement in catalysis. Pap6 was shown to digest a range of host proteins, including gelatin, fibronectin, and type IV collagen, indicating a potential role in pathogenesis. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Zinc metalloprotease; Zinc-binding motif; Pathogenesis
1. Introduction The farming of panaeid shrimp is a significant aquaculture activity in many Asian countries, like Thailand, Indonesia and India. The industry is frequently plagued by bacterial infections, particularly vibriosis caused by luminous vibrios such as Vibrio harveyi. Luminous vibriosis results in mass mortality of the affected shrimp and consequently leads to extensive economical losses (Karunasagar et al., 1994). Bacterial proteases particularly those produced by pathogens act as toxic factors to their host (Miyoshi and Shinoda, 2000) and have been implicated in virulence and pathogenicity. Many of these bacterial proteases are zinccontaining metalloproteases (Ha¨se and Finkelstein, 1993). Abbreviations: aa, amino acids; bp, base pairs; kb, kilo bases, kDa, kilodaltons; Ap, ampicillin; LB, Luria-Bertani medium; BSA, bovine serum albumin, IPTG, isopropyl-b-D -thiogalactopyranoside; SDS, sodium dodecyl sulfate. * Corresponding author. Tel.: þ 65-6874-3674; fax: þ 65-6776-6872. E-mail address: [email protected] (C.L. Poh).
Several Vibrio proteases have been cloned and well characterized. For example, marine bacterium Vibrio vulnificus, an opportunistic human pathogen causes haemorrhagic wound infections and septicemia. The pathogen secretes a 46 kDa zinc metalloprotease (VVP) which promotes the haemorraghic reaction and enhances vascular permeability through the generation of inflammatory mediators (Miyoshi and Shinoda, 2000). Vibrio cholerae 01 secretes a zinc-dependant metalloprotease hemagglutinin/protease (HA/protease) (Booth et al., 1983) which has been well studied. In vitro studies showed that HA/protease was able to cleave several physiologically important host substrates like mucin, fibronectin and lactoferrin (Finkelstein et al., 1983). HA/protease was also found to activate the cholera toxin by nicking the cholera toxin A subunit and it was considered to play a role in the pathogenesis of cholera (Miyoshi and Shinoda, 2000). Much less is known about the virulence factors that are involved in the pathogenesis of V. harveyi. Studies on the pathogenicity of various paneid isolates of V. harveyi reveal that its extracellular enzymes possessed stronger proteo-
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 7 8 - 1 1 1 9 ( 0 2 ) 0 1 1 5 1 - 4
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lytic, haemolytic and phospholipase activity than the nonpaneid ATCC reference strain. This suggested that pathogenicity of V. harveyi could be attributed to the presence of extracellular virulence factors like proteases, haemolysins, chitinases and phospholipases (Liu et al., 1996). A cysteine protease from a pathogenic Taiwanese strain of V. harveyi 820514 was purified and characterized (Liu et al., 1997). It was shown to be a novel protease of 38 kDa that exhibited lethal toxicity to Panaeus monodon by interfering with hemostasis, presumably through the degradation of coagulogen, a prawn plasma component (Liu and Lee, 1999). Alkaline proteases had also been extracted from the supernatants of V. harveyi, although no further characterization has been reported (Fukasawa et al., 1988). In this paper, we describe the cloning, expression and characterization of a novel metalloprotease (Pap6) from V. harveyi strain AP6. A recombinant 6xHis tagged enzyme was purified and biochemical characterization indicated that it was a zinc-dependant metalloprotease.
Sequence Analysis at http://www.cbs.dtu.dk/services/SignalP/. The phylogenetic tree was constructed using the Treecon for Windows version 1.3b software package available at http://bioinformatics.weizmann.ac.il/pub/ software/evolve/draw/treecon/. 2.4. Protease assay Proteolytic activity was determined using azocasein (Sigma Chemical Co., St. Louis, MO) as the substrate. Briefly, 10 ml of purified enzyme solution was added to 100 ml of azocasein (5 mg/ml) in 50 mM Tris –HCl buffer (pH 8.0). The mixture was incubated at 378C for 2 h. The reaction was terminated by adding 500 ml of 10% trichloroacetic acid and keeping on ice for 30 min. The mixture was centrifuged at 12,000 rpm for 10 min and 500 ml of the supernatant were added to 500 ml of 1 M NaOH. After mixing, the absorbance was measured at 442 nm. One unit of protease activity was defined as the amount of enzyme that caused an increase of 0.01 absorbance unit after 2 h of incubation at 378C.
2. Materials and methods 2.5. Electrophoresis, zymography and Western blot analysis 2.1. Bacterial strains, plasmids and media V. harveyi strain AP6 (Suwanto et al., 1998) was obtained from the coastal waters of east Java, Indonesia. V. harveyi was grown routinely in LB or LB agar containing 100 mg/ml of ampicillin. LB agar containing 2% of skim milk agar was used for the selection of proteolytic clones. Proteolytic activity was detected by formation of a clear halo around the colonies. 2.2. DNA cloning and sequencing Genomic DNA from V. harveyi AP6 was purified using the phenol-chloroform method extraction as described by Sambrook et al. (1989). The DNA was digested partially with Spe I and the cleaved fragments were ligated into the same site of pUC18 vector (Yanisch-Perron et al., 1985). The ligation mixture was transformed into Escherichia coli TOP10 cells (Invitrogen, Carlsbad, CA). Transformants were selected for proteolytic activity on skim milk agar plates. Subsequently, a subclone (pUC88) carrying the pap6 gene was isolated. 2.3. DNA sequencing and protein analysis The 2.7 kb Eco RI-HindIII fragment from plasmid pUC88 was sequenced on both strands using ABI prism Dye Terminator Cycle Sequencing Ready Reaction Kit and ABI cycle sequencer A373 (Applied Biosystems/Perkin Elmer, Foster City, CA). DNA sequencing was performed by using M13 forward and reverse primers. The identification of signal peptides was carried out using the programme SignalP V1.1 at the Center for Biological
SDS-PAGE was performed according to Sambrook et al. (1989) using 5% stacking gel and 10% resolving gel. Gelatin-gel zymography analysis was used to characterize protease activity in the protein samples. Ten mg/ml of gelatin was co-polymerized in 10% SDS-PAGE gels. Samples were run under non-reducing conditions and were not boiled prior to loading. After electrophoresis, gels were washed in 50 mM Tris – HCl (pH 8.0) and 2.5% Triton X-100 for 1 h and incubated overnight at 378C in 50 mM Tris– HCl (pH 8.0) buffer containing 2 mM CaCl2 and 2 mM MgCl2. Protein bands with gelatinase activity in the gels were visualized by Coomassie blue staining. For enzyme substrate assay, purified Pap6 was incubated with different protein substrates (15 mg), including bovine serum albumin (BSA), fibrinogen, acid-soluble native and heat denatured types I and IV collagens (Sigma) at 378C overnight. Denatured collagens (gelatins) were obtained by heating native collagens at 708C for 15 min. Digestion products were analyzed on a 10% SDS-PAGE gel. For western blotting analysis, protein samples were separated on a 10% SDS-PAGE gel and electrophoretically transferred onto an Immun-Blot PVDF membrane (BioRad, Hercules, CA). The western blots were probed with anti-His antibody (Qiagen) and goat anti-mouse IgG alkaline-phosphatase (AP) conjugated secondary antibody (Bio-Rad). The bound antibody was detected using the AP conjugate substrate kit (Bio-Rad). 2.6. Site-directed mutagenesis Mutagenesis was performed using QuikChangee SiteDirected Mutagenesis Kit according to the manufacturer’s
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instructions (Stratagene, La Jolla, CA). The plasmid pUC88, carrying pap6 was used as the template for mutagenesis. Mutants lacking proteolytic activity were identified by skim milk agar plate assay and later confirmed by nucleotide sequencing. 2.7. Protein expression and purification The Pap6 gene was expressed as a carboxy-terminal 6xHis-tagged recombinant protein. PCR amplification was carried out using plasmid pUC88 as the template together with primers protF (50 -CATGCCATGGATGCGAAACGTC ACTTTA-30 ) and protR (50 -CGGGATCCATAGTCGTTAC AGCTGCCTT-30 ) (Nco I and Bam HI sites are underlined respectively) (Fig. 1) The amplified gene was cloned into the Nco I and Bam HI sites of expression vector pQE60 to generate the plasmid pQEP10. E. coli BL21 (DE3) (Novagen, Madison, WI) was transformed with pQEP10 and grown in LB broth with 100 mg/ml of ampicillin. The cells were induced with 0.5 mM of IPTG and cultures were incubated with shaking at 308C overnight. After harvesting, the cells were resuspended in phosphate buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl) and sonicated. The cell lysate was centrifuged and the supernatant was applied onto a Ni-NTA spin column (Qiagen). The column was washed four times with washing buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM imidazole) to remove non-specific proteins. The 6xHis-tagged Pap6 was eluted with an elution buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole). 2.8. Determination of N-terminal sequence Purified Pap6 was run on a 10% SDS-PAGE gel and electrotransferred onto Sequi-Blot PVDF membrane (BioRad). After staining with Coomassie blue, the 35 kDa band was excised and the N-terminal sequence was determined by Edman degradation using the Applied Biosystems 494 Procise Protein Sequencing system. 2.9. Characterization of enzyme activity The effect of different ions (Mg2þ, Ca2þ and Zn2þ) on protein activity were studied by incubating the recombinant protein with 5 mg/ml of azocasein at 378C for 2 h. The effect of inhibitors on protease activity was examined using ethylenediaminetetraacetic acid (EDTA), ethylene glycolbis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), 1, 10-phenanthroline, phenylmethysulfonyl fluoride (PMSF), pepstatin, E-64 and diethylpyrocarbonate (DEPC) The protease was preincubated with the inhibitors for 30 min at 378C and the residual protease activity was measured using 5 mg/ml of azocasein. The effect of pH on caseinolytic assay was determined using the following buffers: 100 mM citric acid (pH 3.0 – 6.0), 100 mM Tris –HCl (pH 7.0, 8.0) and 100 mM glycine-NaOH (pH 9.0, 10.0 and 11.0). The effect of temperature on protease activity was tested by incubating the
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recombinant Pap6 protein at 4, 16, 22, 30, 37, 42 and 658C for 2 h using azocasein as a substrate. Elastinolytic activity was determined with elastin Congored (Sigma) as the substrate. Purified enzyme was added to 5 mg/ml of elastin-Congo red resuspended in 1 ml of 50 mM NaH2PO4 (pH 7.0) buffer. Reactions were incubated at 378C for 2 h and terminated by adding EGTA to a final concentration of 10 mM. The absorbance of the supernatants obtained by centrifugation was measured at 495 nm. 2.10. Nucleotide sequence accession number The nucleotide sequence data of Pap6 has been deposited in the GenBank database under the accession number AF508306.
3. Results 3.1. DNA cloning and sequence analysis of Pap6 gene Spe I digested genomic DNA library of V. harveyi strain AP6 was constructed using pUC18 and the recombinant plasmids were transformed into E. coli TOP10 cells. Screening of proteolytic clones was carried out on skim milk agar. One recombinant, pAS1, which possessed proteolytic activity, was selected for further study. Plasmid pAS1 had a 9 kb insert and the DNA fragment conferring proteolytic activity was subcloned in pUC18 to yield pUC88. Sequencing of the 2.7 kb insert present in pUC88 revealed one major open reading frame which was 2034 bp in length. It was predicted to encode a polypeptide (designated Pap6) of 677 amino acids with a molecular mass of about 75 kDa. A potential signal peptide of 15 amino acids was found at the N-terminus of the predicted protein (Fig. 1). Sequence homology analysis reveals that Pap6 is a novel enzyme having less than 50% identity with the metalloproteases of marine Vibrio and Aeromonas sp. The best homologs are HA/Protease of V. cholerae (41%), Aeromonas caviae (41%), V. vulnificus (40%), LasB of Pseudomonas aeruginosa (41%), and elastase of Aeromonas hydrophila (39%) (Fig. 2). The zinc-binding motif, HEXXH-E which is known to be highly conserved in zinc-metalloproteases, is present in the deduced amino acid sequence of Pap6 at positions 330– 354 (Ha¨se and Finkelstein, 1993) (Fig. 1). It is noted that three active site residues are required to co-ordinate a zinc cation (Vallee and Auld, 1990a). The histidine residues of the HEXXH motif function as the first and second zinc ligands whilst the location of the third zinc ligand, which is usually a glutamic residue (E), can vary (Vallee and Auld, 1990a). Based on the location of the third zinc ligand, bacterial zinc metalloproteases with the HEXXH sequence can be subdivided into three families (Miyoshi and Shinoda, 2000). By alignment with zinc metalloproteases of V.
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Fig. 1. Nucleotide and peptide sequences of Pap6. The vertical arrow pointing downwards indicates a possible signal peptide cleavage site. The conserved zinc motif HEXXH , 19aa , E shared by metalloproteases of the thermolysin family is underlined. Residues identified by N-terminal sequencing of mature Pap6 are double-underlined. Arrows indicate the location of PCR primers (protF and protR) used to construct the Pap6 expression vector.
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Fig. 2. Dendrogram of selected Vibrio and Aeromonas metalloproteases. Branch lengths are drawn to scale and proportional to the number of amino acid changes. The bootstrap values are indicated at each node. The metalloproteases used to comparison were VVP, EmpV from V. vulnificus (GenBank accession numbers JC5756 and AAC45343, respectively), vibrolysin from V. proteolyticus (GenBank accession number Q00971), EmpA from V. anguillarum (GenBank accession number P43147), HA/P from V. cholerae (GenBank accession number NP_233251), LasB from P. aeruginosa (GenBank accession number NP_252413), AphB from A. hydrophila (GenBank accession number AAF07184), and elastase from A. caviae (GenBank accession BAA95457).
cholerae, Vibrio anguillarum, and Vibrio proteolyticus, we found the potential third zinc ligand of Pap6 was located 19 bases downstream from the second histidine of the zincbinding motif (HEXXH , 19aa , E) (Table 1). Therefore, Pap6 likely belongs to the thermolysin family of zinc metalloproteases (Miyoshi and Shinoda, 2000). 3.2. Expression and purification of Pap6 The full length pap6 coding region was amplified by PCR (Fig. 1) and fused to the His-tag expression vector pQE60. The carboxy-terminal 6xHis tagged Pap6 was expressed from plasmid pQEP10 in E. coli BL21 and
purified with affinity chromatography using a Ni-NTA column. Pap6 was purified 295-fold from crude cell lysates and had a specific activity of 678.6 EU/mg. Full length Pap6 together with 6xHis-tag was predicted to be 75 kDa whereas SDS-PAGE analysis showed that the purified Pap6 had an estimated molecular mass of 35 kDa (Fig. 3A). Therefore, it is likely that Pap6 is processed into its mature protein by cleavage of the signal peptide and the N-terminal propeptide. To test the possibility, the 35 kDa protein band was prepared and its N-terminal sequence was determined. The first ten amino acids ‘Leu-Glu-Ala-Glu-Gly-Pro-Gly-GlyAsn-Gln’ of the matured protein corresponded to positions 191 – 200 in the predicted Pap6 full length peptide sequence
Table 1 Comparison of the conserved HEXXH , 19aa , E zinc-binding domain of Vibrio metalloproteases Metalloprotease
Amino acids of conserved domaina
Location
EmpA vibriolysin VVP HA/P Pap6
†A † A † AHEVSHGFTEQNSGLVYQNMSGGMNEAFSD AHEVSHGFTEQNSGLVYENMSGGMNEAFSD AHEVSHGFTEQNSGLIYSNMSGGMNEAFSD AHEVSHGFTEQNSGLVYRDMSGGINEAFSD AHEVSHGFTEQNSGLEYRGMSGGMYESFSD
345 –374 342 –371 342 –371 342 –371 329 –358
a
Zinc-binding residues and active site residues are indicated by black circles and open boxes, respectively.
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(Fig. 1). The purified 35 kDa Pap6 was proteolytically active as demonstrated by gelatin zymography (Fig. 3B). Sonicated cell lysates or supernatants made from E. coli BL21 showed no proteolytic activity (data not shown). Gelatin zymograms obtained using the culture supernatant of E. coli BL21 containing pQEP10 revealed that the proteolytic band was similar to that of the expressed recombinant Pap6 (Fig. 3B). Zymograms of the supernatant of wild type V. harveyi AP6 suggests that besides Pap6, several other proteases are also secreted as evidenced by the presence of proteolytic bands other than Pap6 (Fig. 3B). Western blot analysis was used to determine whether Pap6 had a C-terminal propeptide. Immunodetection using anti-His antibody did not detect the presence of a His-tag on the 35 kDa Pap6, suggesting that the His-tag was likely to have been part of a C-terminal propeptide which was cleaved during the maturation of Pap6 (Fig. 3C). His-tagged Sup35p protein from Saccharomyces cerevisiae (Parham et al., 2001) was used as positive control whilst E. coli BL21 sonicated cell lysates was the negative control (Fig. 3C). 3.3. Characterization of recombinant Pap6 Purified 6xHis tagged recombinant protein Pap6 was dialyzed overnight in 50 mM Tris – HCl (pH 8.0) before being used in the following enzyme assays. The effect of inhibitors on the recombinant protein Pap6 was examined. Zinc specific metal chelator 1, 10 phenanthroline (10 mM) and EGTA (10 mM) strongly inhibited enzyme activity and
almost caused a total loss of enzyme activity whilst EDTA (10 mM) reduced the enzyme activity by almost 50% (Table 2). Pap6 was uninhibited by serine protease inhibitor PMSF (10 mM), aspartate protease inhibitor pepstatin (10 mM) and cysteine protease inhibitor E-64 (10 mM) (Table 2). The addition of Mg2þ (0.1 – 10 mM) and Ca2þ (0.1 – 10 mM) did not significantly affect enzyme activity. However, Zn2þ in a concentration greater than 0.1 mM inhibited enzyme activity, with 10 mM Zn2þ resulting in an almost complete reduction of Pap6 activity (Table 2). These results suggest that Pap6 is a zinc dependant metalloprotease. The histidine modifying reagent DEPC almost completely inactivated the enzyme, thus supporting the role of histidine residues as part of the active site in Pap6. The effect of pH on Pap6 activity was tested using buffers with pH ranging from 3.0 to 11.0. Optimal caseinolytic activity was detected in an alkaline medium of pH 9.0 (Fig. 4A). A drastic drop in activity, about 50%, was observed when the pH was changed from 7.0 to 6.0 and from 9.0 to 10.0. The optimum temperature for the recombinant protein Pap6 was at 378C. However, enzyme activity was still retained over a wide temperature range of between 4 and 658C (Fig. 4B). 3.4. Site-directed mutagenesis of the HEXXH , 19aa , E zinc motif The HEXXH , 19aa , E zinc-binding motif (Vallee and Auld, 1990a) of Pap6 is located in positions 330– 354.
Fig. 3. SDS-PAGE and gelatin-gel zymography analyses of Pap6. (A) SDS-PAGE analysis of Pap6 purified through a Ni-NTA column. Electrophoresis was carried out on a 10% polyacrylamide gel. (B) Proteolytic activity was detected using a 10% SDS-PAGE gel co-polymerized with 10 mg/ml of gelatin. Lane 1, purified Pap6; lane 2, culture supernatant fraction of E. coli BL21 carrying pQEP10; lane 3, culture supernatant fraction of V. harveyi AP6. Horizontal arrow indicates the possible position of the 35 kDa Pap6 from the culture supernatant of V. harveyi AP6. (C) Western blot analysis using anti-His antibodies. Lane 1, His-tagged Sup35p from Saccharomyces cerevisiae was used as the positive control; lane 2, purified 35 kDa Pap6; lane 3, E. coli BL21 sonicated cell lysates was used as the negative control.
J.W.P. Teo et al. / Gene 303 (2003) 147–156 Table 2 Effect of metal ions and inhibitors on the activity of Pap6 protease Compound
Concentration (mM)a
None
% enzyme activityb 100
0.01 0.1 1 10
105.5 ^ 5.7 78.5 ^ 2.8 5 ^ 0.7 1.8 ^ 1.4
CaCl2
0.1 1 10
96.9 ^ 2.4 110.6 ^ 2.4 116.2 ^ 10.6
MgCl2
0.1 1 10
107.5 ^ 5.9 105.0 ^ 5.7 104.4 ^ 5.2
1, 10-phenanthroline DEPC EGTA EDTA E-64 PMSF Pepstatin
10 10 10 10 10 10 10
2.3 ^ 0.6 6.7 ^ 3.8 11.4 ^ 2.1 60.5 ^ 1.7 95.5 ^ 1.5 101.5 ^ 0.3 106.1 ^ 3.1
ZnCl2
a Enzyme samples were incubated with the metal ions in 5 mg/ml of azocasein solution for 2 h at 378C. Inhibitors were preincubated with the enzyme for 30 min prior to the addition of 5 mg/ml of azocasein solution. b Caseinolytic activity of recombinant Pap6 without the addition of metal ions or inhibitors was taken to be 100%. The effect of metal ions and inhibitors on Pap6 activity was based on averages of three independent experiments.
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showed low sequence homology to elastases of A. hydrophila and LasB of P. aeruginosa. However, no elastolytic activity was detected when the recombinant Pap6 protein was incubated with elastin congo red, indicating that it was not an elastase (data not shown).
4. Discussion In this paper, we describe the cloning, expression and characterization of Pap6, a novel metalloprotease from V. harveyi strain AP6. Sequence analysis of Pap6 predicts that it encodes a large 677 amino acid polypeptide with a predicted molecular mass of 75 kDa but SDS-PAGE analysis showed that biologically active Pap6 had a molecular mass of 35 kDa. Peptide sequence alignments and site-directed mutagenesis confirmed that Pap6 is a member of the thermolysin family of zinc metalloproteases. Members of the thermolysin family are first synthesized as inactive preproteins which are processed to the mature form (Ha¨se and Finkelstein, 1993; Miyoshi and Shinoda, 2000). Previous studies have shown that zinc metalloprotease VVP of V. vulnificus is first produced as a large precursor and is later cleaved to yield a 35
Residues His330, His334 and Glu354, thought to be involved in zinc binding were replaced with non-zinc-binding residues Ser, Pro and Val, respectively (Vallee and Auld, 1990b). Residues Glu331 and Tyr345 are probably involved in catalysis. Glu331 was replaced with Asp which had similar properties as Glu. Tyr345 was replaced with a non-polar Gly residue. These site-directed mutants were assayed for activity. Mutants H330S, E331D, H334P, Y345G and E354V had less than 10% of wild type activity (Fig. 5A). Mutant E331D had reduced enzymatic activity despite being replaced by a fairly similar residue. The results were consistent with the proposed roles of the residues in enzymatic catalysis. SDS-PAGE analysis of Pap6 and its mutants (Fig. 5B) showed that Pap6 and its mutants were expressed to similar levels; hence the loss of caseinolytic activity observed in the mutants was due to the replacement of critical amino acid residues and not the result of impaired protein expression. 3.5. Substrate specificity of Pap6 Digestion of various protein substrates by recombinant Pap6 was examined. It was able to degrade BSA, fibrinogen, native type IV collagen and type IV gelatin (Figs. 6A –D). It was unable to degrade native type I collagen (Fig. 6E) but was able to use type I gelatin as a substrate (Fig. 6F). Pap6
Fig. 4. (A) Effect of pH and temperature on Pap6 activity. Buffers of different pH containing 5 mg/ml of azocasein were used. Activity at pH 9.0 was defined as 100%. (B) Enzyme reaction was in 100 ml of 50 mM Tris – HCl (pH 8.0) containing 5 mg/ml of azocasein at varying temperatures. The enzyme activity at 378C was defined as 100%. Experimental details are described in Section 2. The relative activities are averages based on two independent experiments.
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Fig. 5. (A) Enzymatic activity of Pap6 and its derivatives. Proteolytic activity of mutant enzymes was expressed in relation to the activity of wild type Pap6 (100%). The results are the averages of triplicate independent experiments. (B) Analysis of the expression level of wild type Pap6 and its various site-directed mutants using a 10% SDS-PAGE gel.
kDa mature protein via removal of a 44.7 kDa amino acid Nterminal as well as a 10 kDa C-terminal propeptide (Miyoshi et al., 1997). The preprotein of Pap6 is predicted to be 75 kDa and even with the removal of the signal peptide (1.7 kDa) and the 19.2 kDa N-terminal propeptide, the size of the remaining peptide should be about 54.3 kDa. This implies that like VVP, Pap6 may be further processed, probably through the removal of a C-terminal propeptide, to produce a mature enzyme of 35 kDa. This was supported by western blot analysis which indicated that the 35 kDa Pap6 was not Histagged. Therefore, we suggest that Pap6 preprotein is likely to have four domains: a 15 amino acid signal peptide, a 19.2 kDa (175 amino acid) N-terminal propeptide, a 35 kDa mature protein and possibly a 19.1 kDa C-terminal propeptide. Under the conditions used in this study, we were unable to observe the 19.1 kDa band corresponding to the C-terminal propeptide. Perhaps stronger elution conditions such as a higher concentration of imidazole in the elution buffer may have been required to liberate the C-terminal propeptide from the affinity column. It is most probable that the propeptide of Pap6 was processed before being secreted as gelatin zymograms showed that the Pap6 protein purified from the sonicated cell lysates has a similar molecular weight as that derived from the supernatant of E. coli BL21 harboring pQEP10. Crystal structure determinations of several zinc containing enzymes have allowed the catalytic and structural features of the zinc binding sites to be defined (Ha¨se and
Finkelstein 1993; Vallee and Auld, 1990a). X-ray crystallographic analyses of members of the thermolysin zinc metalloprotease superfamily such as thermolysin from Bacillus thermoproteolyticus (Colman et al., 1972), elastase from P. aeruginosa (Thayer et al., 1991) and aureolysin from Staphylococcus aureus (Banbula et al., 1998) showed that a catalytic zinc atom is coordinated to two histidine residues and one glutamate residue of the enzyme, together with an active water molecule. The zinc ligands are the two histidines in the HEXXH motif and a glutamate residue separated by a 19 base long spacer (Vallee and Auld, 1990a). Peptide sequence alignment showed that Pap6 contains a zinc-binding motif 330HEXXH , 19aa , E354. Site-directed mutagenesis of the putative zinc binding residues of Pap6, i.e. histidines 330, 334 and glutamate 354, showed that they were indeed essential for activity. The enzyme inhibition by DEPC also lends support to the importance of histidine residues in catalysis. The zinccoordinating ligands of metalloproteases with the HEXXH , 19aa , E motif have been investigated by mutational analysis. His 415 and His 419 (equivalent to His 330 and His 334, respectively in Pap6) of Clostridium histolyticum collagenase were mutated and this caused catalytic activity and zinc-binding to be abolished (Jung et al., 1999). The replacement of Glu 408 in mouse aminopeptidase A (equivalent to Glu 354 in Pap6) generated mutants that had lost their catalytic and zinc-binding activity (Vazeux et al., 1996). Assaying the zinc content of these mutant enzyme should help confirm the importance of these residues in zinc-binding and coordination. The conserved zinc-binding motif is shared by these metalloproteases and mutations to these active site residues reveal a similar loss of enzyme activity. It is highly probable that the catalytic mechanism of Pap6 may be similar to other zinc metalloproteases. The replacement of two other active site residues, Glu 331 and Tyr 345 led to a loss of proteolytic activities thus demonstrating that these active site residues were also important for catalytic activity. The importance of these residues was similarly demonstrated in a thermolysinlike B. subtilis neutral protease which had a mutation occurring at Glu 143 (equivalent to Glu 331 of Pap6) (Toma et al., 1989). When Tyr 157 of B. thermoproteolyticus thermolysin (equivalent to Tyr 345 in Pap6) was substituted, a loss of catalytic activity was observed (Marie-Claire et al., 1998). Therefore, Tyr and Glu could be considered key active site residues. Enzyme inhibitor studies showed that Pap6 is most likely a zinc-dependant metalloprotease as it was strongly inhibited by the zinc-specific chelator 1, 10-phenanthroline and general metal chelators such as EGTA and EDTA. Pap6 was unaffected by protease inhibitors such as the serine protease inhibitor, PMSF; aspartate protease inhibitor, pepstatin and cysteine protease inhibitor, E-64. Our results showed that zinc concentrations greater than 1 mM completely inhibited the protease activity of Pap6. It has been noted that high zinc concentrations often inhibit
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Fig. 6. Analysis of protein digestion by Pap6. Each substrate (15 mg) was incubated in the absence (lanes 1) or in the presence (lanes 2) of Pap6 at 378C for overnight. Lane 3 is purified Pap6. (A) BSA; (B) fibrinogen; (C) type IV collagen; (D) type IV gelatin; (E) type I collagen; and (F) type I gelatin. Digested substrates were separated on a 10% SDS-PAGE gel. The horizontal arrow marks the position of recombinant Pap6.
metalloproteases. The inhibition is due to formation of zinc monohydroxide that bridges the catalytic zinc ion to the side chain of the active site of the enzyme (Larsen and Auld, 1991). The extracellular matrix (ECM) consists of collagens, non-collagenous glycoproteins, and proteoglycans, with collagens being the most abundant structural components (Aumailley and Gayraud, 1998). Protein substrate studies with Pap6 indicate that fibronectin, a non-collagenous glycoprotein component of the ECM was hydrolyzed by Pap6. Type IV collagen, the major component of the basement membrane was also digested by Pap6 whereas type I collagen was not readily digested. Gelatins (heat denatured types I and IV collagens) which are also components of the ECM were also digested by Pap6. VVP, the V. vulnificus protease, also possesses proteolytic activity against type IV collagen and it is speculated that the destruction of type IV collagen framework could be responsible for the breakdown of the basement membrane, thus leading to the hemorrhagic reaction typically seen in V. vulnificus infections (Miyoshi and Shinoda, 2000). In a similar manner, Pap6 may elicit tissue damage on prawn host by proteolyzing the ECM components. Further work could be carried out to determine the pathological actions of the Pap6 protein on a wider range of host substances such as plasma components and muscle proteins. It would also be interesting to investigate the role
of Pap6 in bacterial pathogenesis in vivo. This could be carried out by comparing the invasiveness of wild-type V. harveyi and a mutant deficient in Pap6 activity. A variety of exoenzymes like phospholipase, chitinase and haemolysin have been detected in paneid V. harveyi strains (Liu et al., 1996), and our study has also shown the presence of proteolytically active proteases other than Pap6 in the supernatants of V. harveyi strain AP6. It is highly likely that bacterial invasion could be the result of the synergistic actions of these pathogenic factors.
Acknowledgements We thank YiHu Dong for his suggestions during the course of this study and A. Suwanto for the gift of V. harveyi strain AP6, and Haiwei Song for the His-tagged Sup35p protein. This work was supported by a grant (No. 0451/2000) from the National Medical Research Council of Singapore.
References Aumailley, M., Gayraud, B., 1998. Structure and biological activity of the extracellular matrix. J. Mol. Med. 76, 253–265. Banbula, A., Potempa, J., Travis, J., Fernandez-Catalan, C., Mann, K., Huber, R., Bode, W., Medrano, F., 1998. Amino-acid sequence and
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three dimensional structure of the Staphylococcus aureus metalloproteinase at 1.72 A resolution. Structure 6, 1185–1193. Booth, B.A., Boesman-Finkelstein, M., Finkelstein, R.A., 1983. Vibrio cholerae soluble hemagglutinin/protease is a metalloenzyme. J. Bacteriol. 170, 4309– 4314. Colman, P.M., Jansonius, J.N., Matthews, B.W., 1972. The structure of thermolysin: an electron density map at 2.3 A resolution. J. Mol. Biol. 70, 701 –724. Finkelstein, R.A., Boseman-Finkelstein, M., Holt, P., 1983. Vibrio cholerae hemagglutinin/lectin/protease hydrolyzes fibronectin and ovomucin: F.M. Burnet revisited. Proc. Natl. Acad. Sci. USA 80, 1092–1095. Fukasawa, S., Nakamura, K., Miyahira, M., Kurata, M., 1988. Some properties of two proteinases from luminous bacteria, Vibrio harveyi strain FLN-108. Biol. Chem. 52, 3009–3014. Ha¨se, C.C., Finkelstein, R.A., 1993. Bacterial extracellular zinc-containing metalloproteases. Microbiol. Rev. 57, 823–837. Jung, C.M., Matsushita, O., Katayama, S., Minami, J., Sakurai, J., Okabe, A., 1999. Identification of metal ligands in the Clostridium histolyticum ColH collagenase. J. Bacteriol. 9, 2816– 2822. Karunasagar, I., Pai, R., Malathi, G.R., Karunasagar, I., 1994. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture 128, 203– 209. Larsen, K.S., Auld, D.S., 1991. Characterization of an inhibitory metal binding site in carboxypeptidase A. Biochemistry 30, 2610–2613. Liu, P.C., Lee, K.K., 1999. Cysteine protease is a major exotoxin of pathogenic luminous Vibrio harveyi in the tiger prawn, Penaeus monodon. Lett. Appl. Microbiol. 28, 428–430. Liu, P.C., Lee, K.K., Chen, S.N., 1996. Pathogenicity of different isolates of Vibrio harveyi in tiger prawn, Penaeus monodon. Lett. Appl. Microbiol. 22, 413 –416. Liu, P.C., Lee, K.K., Tu, C.C., Chen, S.N., 1997. Purification and characterization of a cysteine protease produced by pathogenic luminous Vibrio harveyi. Curr. Microbiol. 35, 32–39. Marie-Claire, C., Ruffet, E., Tiraboschi, G., Fournie-Zaluski, M.-C., 1998.
Differences in transition state stabilization between thermolysin (EC 3.4.24.27) and neprilysin (EC 3.4.24.11). FEBS Lett. 438, 215–219. Miyoshi, S., Shinoda, S., 2000. Microbial metalloproteases and pathogenesis. Microbes Infect. 2, 91– 98. Miyoshi, S., Wakae, H., Tomochika, K., Shinoda, S., 1997. Functional domains of a zinc metalloprotease from Vibrio vulnificus. J. Bacteriol. 179, 7606–7609. Parham, S.N., Resende, C.G., Tuite, M.F., 2001. Oligopeptide repeats in the yeast protein Sup35p stabilize intermolecular prion interactions. EMBO J. 20, 2111– 2119. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Suwanto, A., Yuhana, M., Herawaty, E., Angka, S.L., 1998. Genetic diversity of luminous Vibrio isolated from shrimp larvae. In: Flegel, T.W., (Ed.), Advances in Shrimp Biotechnology, National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand, pp. 217 –224. Thayer, M.M., Flaherty, K.M., McKay, D.B., 1991. Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-A resolution. J. Biol. Chem. 5, 2864– 2871. Toma, S., Campagnoli, S., De Gregoriis, E., Gianna, R., Margarit, I., Zan, M., Grandi, G., 1989. Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease. Protein Eng. 2, 359 –364. Vallee, B.L., Auld, D.S., 1990a. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry 29, 5647–5659. Vallee, B.L., Auld, D.S., 1990b. Active-site zinc ligands and activated H2O of zinc enzymes. Proc. Natl. Acad. Sci. USA 87, 220–224. Vazeux, G., Wang, J.Y., Corvol, P., Llorens-Corte`s, C., 1996. Identification of glutamate residues essential for catalytic activity and zinc coordination in aminopeptidase A. J. Biol. Chem. 271, 9069–9074. Yanisch-Perron, C., Vieira, J., Messing, J., 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103 –119.
Cloning and characterization of a metalloprotease from Vibrio harveyi strain AP6 Jeanette W.P. Teoa, Lian-Hui Zhangb, Chit Laa Poha,* a
Programme in Environmental Microbiology, Department of Microbiology, Faculty of Medicine, National University of Singapore, 5 Science Drive 2, Singapore 117 597, Singapore b Laboratory of Biosignals and Bioengineering, Institute of Molecular and Cell Biology, National University of Singapore, 5 Science Drive 2, Singapore 117 597, Singapore Received 12 August 2002; received in revised form 4 November 2002; accepted 12 November 2002 Received by A.M. Campbell
Abstract A metalloprotease gene pap6 was cloned from Vibrio harveyi strain AP6. Sequence analysis showed that pap6 was 2034 bp in length and predicted to encode a peptide of 677 amino acids with a molecular mass of 75 kDa. SDS-PAGE analysis of the purified Pap6 revealed that it was 35 kDa in size. N-terminal amino acid sequencing established that the mature protein began at Leu-191, suggesting that the preprotein of Pap6 was processed to generate a mature protease. Purified Pap6 was characterized as a zinc metalloprotease as it was inhibited by zinc- and metal-specific inhibitors such as 1, 10-phenanthroline, EGTA and EDTA. The deduced amino acid sequence revealed the presence of a zincbinding motif HEXXH , 19aa , E. Substitution of these active site residues by site-directed mutagenesis caused significant losses in enzyme activity, thus demonstrating their involvement in catalysis. Pap6 was shown to digest a range of host proteins, including gelatin, fibronectin, and type IV collagen, indicating a potential role in pathogenesis. q 2002 Elsevier Science B.V. All rights reserved. Keywords: Zinc metalloprotease; Zinc-binding motif; Pathogenesis
1. Introduction The farming of panaeid shrimp is a significant aquaculture activity in many Asian countries, like Thailand, Indonesia and India. The industry is frequently plagued by bacterial infections, particularly vibriosis caused by luminous vibrios such as Vibrio harveyi. Luminous vibriosis results in mass mortality of the affected shrimp and consequently leads to extensive economical losses (Karunasagar et al., 1994). Bacterial proteases particularly those produced by pathogens act as toxic factors to their host (Miyoshi and Shinoda, 2000) and have been implicated in virulence and pathogenicity. Many of these bacterial proteases are zinccontaining metalloproteases (Ha¨se and Finkelstein, 1993). Abbreviations: aa, amino acids; bp, base pairs; kb, kilo bases, kDa, kilodaltons; Ap, ampicillin; LB, Luria-Bertani medium; BSA, bovine serum albumin, IPTG, isopropyl-b-D -thiogalactopyranoside; SDS, sodium dodecyl sulfate. * Corresponding author. Tel.: þ 65-6874-3674; fax: þ 65-6776-6872. E-mail address: [email protected] (C.L. Poh).
Several Vibrio proteases have been cloned and well characterized. For example, marine bacterium Vibrio vulnificus, an opportunistic human pathogen causes haemorrhagic wound infections and septicemia. The pathogen secretes a 46 kDa zinc metalloprotease (VVP) which promotes the haemorraghic reaction and enhances vascular permeability through the generation of inflammatory mediators (Miyoshi and Shinoda, 2000). Vibrio cholerae 01 secretes a zinc-dependant metalloprotease hemagglutinin/protease (HA/protease) (Booth et al., 1983) which has been well studied. In vitro studies showed that HA/protease was able to cleave several physiologically important host substrates like mucin, fibronectin and lactoferrin (Finkelstein et al., 1983). HA/protease was also found to activate the cholera toxin by nicking the cholera toxin A subunit and it was considered to play a role in the pathogenesis of cholera (Miyoshi and Shinoda, 2000). Much less is known about the virulence factors that are involved in the pathogenesis of V. harveyi. Studies on the pathogenicity of various paneid isolates of V. harveyi reveal that its extracellular enzymes possessed stronger proteo-
0378-1119/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. doi: 1 0 . 1 0 1 6 / S 0 3 7 8 - 1 1 1 9 ( 0 2 ) 0 1 1 5 1 - 4
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lytic, haemolytic and phospholipase activity than the nonpaneid ATCC reference strain. This suggested that pathogenicity of V. harveyi could be attributed to the presence of extracellular virulence factors like proteases, haemolysins, chitinases and phospholipases (Liu et al., 1996). A cysteine protease from a pathogenic Taiwanese strain of V. harveyi 820514 was purified and characterized (Liu et al., 1997). It was shown to be a novel protease of 38 kDa that exhibited lethal toxicity to Panaeus monodon by interfering with hemostasis, presumably through the degradation of coagulogen, a prawn plasma component (Liu and Lee, 1999). Alkaline proteases had also been extracted from the supernatants of V. harveyi, although no further characterization has been reported (Fukasawa et al., 1988). In this paper, we describe the cloning, expression and characterization of a novel metalloprotease (Pap6) from V. harveyi strain AP6. A recombinant 6xHis tagged enzyme was purified and biochemical characterization indicated that it was a zinc-dependant metalloprotease.
Sequence Analysis at http://www.cbs.dtu.dk/services/SignalP/. The phylogenetic tree was constructed using the Treecon for Windows version 1.3b software package available at http://bioinformatics.weizmann.ac.il/pub/ software/evolve/draw/treecon/. 2.4. Protease assay Proteolytic activity was determined using azocasein (Sigma Chemical Co., St. Louis, MO) as the substrate. Briefly, 10 ml of purified enzyme solution was added to 100 ml of azocasein (5 mg/ml) in 50 mM Tris –HCl buffer (pH 8.0). The mixture was incubated at 378C for 2 h. The reaction was terminated by adding 500 ml of 10% trichloroacetic acid and keeping on ice for 30 min. The mixture was centrifuged at 12,000 rpm for 10 min and 500 ml of the supernatant were added to 500 ml of 1 M NaOH. After mixing, the absorbance was measured at 442 nm. One unit of protease activity was defined as the amount of enzyme that caused an increase of 0.01 absorbance unit after 2 h of incubation at 378C.
2. Materials and methods 2.5. Electrophoresis, zymography and Western blot analysis 2.1. Bacterial strains, plasmids and media V. harveyi strain AP6 (Suwanto et al., 1998) was obtained from the coastal waters of east Java, Indonesia. V. harveyi was grown routinely in LB or LB agar containing 100 mg/ml of ampicillin. LB agar containing 2% of skim milk agar was used for the selection of proteolytic clones. Proteolytic activity was detected by formation of a clear halo around the colonies. 2.2. DNA cloning and sequencing Genomic DNA from V. harveyi AP6 was purified using the phenol-chloroform method extraction as described by Sambrook et al. (1989). The DNA was digested partially with Spe I and the cleaved fragments were ligated into the same site of pUC18 vector (Yanisch-Perron et al., 1985). The ligation mixture was transformed into Escherichia coli TOP10 cells (Invitrogen, Carlsbad, CA). Transformants were selected for proteolytic activity on skim milk agar plates. Subsequently, a subclone (pUC88) carrying the pap6 gene was isolated. 2.3. DNA sequencing and protein analysis The 2.7 kb Eco RI-HindIII fragment from plasmid pUC88 was sequenced on both strands using ABI prism Dye Terminator Cycle Sequencing Ready Reaction Kit and ABI cycle sequencer A373 (Applied Biosystems/Perkin Elmer, Foster City, CA). DNA sequencing was performed by using M13 forward and reverse primers. The identification of signal peptides was carried out using the programme SignalP V1.1 at the Center for Biological
SDS-PAGE was performed according to Sambrook et al. (1989) using 5% stacking gel and 10% resolving gel. Gelatin-gel zymography analysis was used to characterize protease activity in the protein samples. Ten mg/ml of gelatin was co-polymerized in 10% SDS-PAGE gels. Samples were run under non-reducing conditions and were not boiled prior to loading. After electrophoresis, gels were washed in 50 mM Tris – HCl (pH 8.0) and 2.5% Triton X-100 for 1 h and incubated overnight at 378C in 50 mM Tris– HCl (pH 8.0) buffer containing 2 mM CaCl2 and 2 mM MgCl2. Protein bands with gelatinase activity in the gels were visualized by Coomassie blue staining. For enzyme substrate assay, purified Pap6 was incubated with different protein substrates (15 mg), including bovine serum albumin (BSA), fibrinogen, acid-soluble native and heat denatured types I and IV collagens (Sigma) at 378C overnight. Denatured collagens (gelatins) were obtained by heating native collagens at 708C for 15 min. Digestion products were analyzed on a 10% SDS-PAGE gel. For western blotting analysis, protein samples were separated on a 10% SDS-PAGE gel and electrophoretically transferred onto an Immun-Blot PVDF membrane (BioRad, Hercules, CA). The western blots were probed with anti-His antibody (Qiagen) and goat anti-mouse IgG alkaline-phosphatase (AP) conjugated secondary antibody (Bio-Rad). The bound antibody was detected using the AP conjugate substrate kit (Bio-Rad). 2.6. Site-directed mutagenesis Mutagenesis was performed using QuikChangee SiteDirected Mutagenesis Kit according to the manufacturer’s
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instructions (Stratagene, La Jolla, CA). The plasmid pUC88, carrying pap6 was used as the template for mutagenesis. Mutants lacking proteolytic activity were identified by skim milk agar plate assay and later confirmed by nucleotide sequencing. 2.7. Protein expression and purification The Pap6 gene was expressed as a carboxy-terminal 6xHis-tagged recombinant protein. PCR amplification was carried out using plasmid pUC88 as the template together with primers protF (50 -CATGCCATGGATGCGAAACGTC ACTTTA-30 ) and protR (50 -CGGGATCCATAGTCGTTAC AGCTGCCTT-30 ) (Nco I and Bam HI sites are underlined respectively) (Fig. 1) The amplified gene was cloned into the Nco I and Bam HI sites of expression vector pQE60 to generate the plasmid pQEP10. E. coli BL21 (DE3) (Novagen, Madison, WI) was transformed with pQEP10 and grown in LB broth with 100 mg/ml of ampicillin. The cells were induced with 0.5 mM of IPTG and cultures were incubated with shaking at 308C overnight. After harvesting, the cells were resuspended in phosphate buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl) and sonicated. The cell lysate was centrifuged and the supernatant was applied onto a Ni-NTA spin column (Qiagen). The column was washed four times with washing buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 20 mM imidazole) to remove non-specific proteins. The 6xHis-tagged Pap6 was eluted with an elution buffer (50 mM NaH2PO4 pH 8.0, 300 mM NaCl, 250 mM imidazole). 2.8. Determination of N-terminal sequence Purified Pap6 was run on a 10% SDS-PAGE gel and electrotransferred onto Sequi-Blot PVDF membrane (BioRad). After staining with Coomassie blue, the 35 kDa band was excised and the N-terminal sequence was determined by Edman degradation using the Applied Biosystems 494 Procise Protein Sequencing system. 2.9. Characterization of enzyme activity The effect of different ions (Mg2þ, Ca2þ and Zn2þ) on protein activity were studied by incubating the recombinant protein with 5 mg/ml of azocasein at 378C for 2 h. The effect of inhibitors on protease activity was examined using ethylenediaminetetraacetic acid (EDTA), ethylene glycolbis(b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA), 1, 10-phenanthroline, phenylmethysulfonyl fluoride (PMSF), pepstatin, E-64 and diethylpyrocarbonate (DEPC) The protease was preincubated with the inhibitors for 30 min at 378C and the residual protease activity was measured using 5 mg/ml of azocasein. The effect of pH on caseinolytic assay was determined using the following buffers: 100 mM citric acid (pH 3.0 – 6.0), 100 mM Tris –HCl (pH 7.0, 8.0) and 100 mM glycine-NaOH (pH 9.0, 10.0 and 11.0). The effect of temperature on protease activity was tested by incubating the
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recombinant Pap6 protein at 4, 16, 22, 30, 37, 42 and 658C for 2 h using azocasein as a substrate. Elastinolytic activity was determined with elastin Congored (Sigma) as the substrate. Purified enzyme was added to 5 mg/ml of elastin-Congo red resuspended in 1 ml of 50 mM NaH2PO4 (pH 7.0) buffer. Reactions were incubated at 378C for 2 h and terminated by adding EGTA to a final concentration of 10 mM. The absorbance of the supernatants obtained by centrifugation was measured at 495 nm. 2.10. Nucleotide sequence accession number The nucleotide sequence data of Pap6 has been deposited in the GenBank database under the accession number AF508306.
3. Results 3.1. DNA cloning and sequence analysis of Pap6 gene Spe I digested genomic DNA library of V. harveyi strain AP6 was constructed using pUC18 and the recombinant plasmids were transformed into E. coli TOP10 cells. Screening of proteolytic clones was carried out on skim milk agar. One recombinant, pAS1, which possessed proteolytic activity, was selected for further study. Plasmid pAS1 had a 9 kb insert and the DNA fragment conferring proteolytic activity was subcloned in pUC18 to yield pUC88. Sequencing of the 2.7 kb insert present in pUC88 revealed one major open reading frame which was 2034 bp in length. It was predicted to encode a polypeptide (designated Pap6) of 677 amino acids with a molecular mass of about 75 kDa. A potential signal peptide of 15 amino acids was found at the N-terminus of the predicted protein (Fig. 1). Sequence homology analysis reveals that Pap6 is a novel enzyme having less than 50% identity with the metalloproteases of marine Vibrio and Aeromonas sp. The best homologs are HA/Protease of V. cholerae (41%), Aeromonas caviae (41%), V. vulnificus (40%), LasB of Pseudomonas aeruginosa (41%), and elastase of Aeromonas hydrophila (39%) (Fig. 2). The zinc-binding motif, HEXXH-E which is known to be highly conserved in zinc-metalloproteases, is present in the deduced amino acid sequence of Pap6 at positions 330– 354 (Ha¨se and Finkelstein, 1993) (Fig. 1). It is noted that three active site residues are required to co-ordinate a zinc cation (Vallee and Auld, 1990a). The histidine residues of the HEXXH motif function as the first and second zinc ligands whilst the location of the third zinc ligand, which is usually a glutamic residue (E), can vary (Vallee and Auld, 1990a). Based on the location of the third zinc ligand, bacterial zinc metalloproteases with the HEXXH sequence can be subdivided into three families (Miyoshi and Shinoda, 2000). By alignment with zinc metalloproteases of V.
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Fig. 1. Nucleotide and peptide sequences of Pap6. The vertical arrow pointing downwards indicates a possible signal peptide cleavage site. The conserved zinc motif HEXXH , 19aa , E shared by metalloproteases of the thermolysin family is underlined. Residues identified by N-terminal sequencing of mature Pap6 are double-underlined. Arrows indicate the location of PCR primers (protF and protR) used to construct the Pap6 expression vector.
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Fig. 2. Dendrogram of selected Vibrio and Aeromonas metalloproteases. Branch lengths are drawn to scale and proportional to the number of amino acid changes. The bootstrap values are indicated at each node. The metalloproteases used to comparison were VVP, EmpV from V. vulnificus (GenBank accession numbers JC5756 and AAC45343, respectively), vibrolysin from V. proteolyticus (GenBank accession number Q00971), EmpA from V. anguillarum (GenBank accession number P43147), HA/P from V. cholerae (GenBank accession number NP_233251), LasB from P. aeruginosa (GenBank accession number NP_252413), AphB from A. hydrophila (GenBank accession number AAF07184), and elastase from A. caviae (GenBank accession BAA95457).
cholerae, Vibrio anguillarum, and Vibrio proteolyticus, we found the potential third zinc ligand of Pap6 was located 19 bases downstream from the second histidine of the zincbinding motif (HEXXH , 19aa , E) (Table 1). Therefore, Pap6 likely belongs to the thermolysin family of zinc metalloproteases (Miyoshi and Shinoda, 2000). 3.2. Expression and purification of Pap6 The full length pap6 coding region was amplified by PCR (Fig. 1) and fused to the His-tag expression vector pQE60. The carboxy-terminal 6xHis tagged Pap6 was expressed from plasmid pQEP10 in E. coli BL21 and
purified with affinity chromatography using a Ni-NTA column. Pap6 was purified 295-fold from crude cell lysates and had a specific activity of 678.6 EU/mg. Full length Pap6 together with 6xHis-tag was predicted to be 75 kDa whereas SDS-PAGE analysis showed that the purified Pap6 had an estimated molecular mass of 35 kDa (Fig. 3A). Therefore, it is likely that Pap6 is processed into its mature protein by cleavage of the signal peptide and the N-terminal propeptide. To test the possibility, the 35 kDa protein band was prepared and its N-terminal sequence was determined. The first ten amino acids ‘Leu-Glu-Ala-Glu-Gly-Pro-Gly-GlyAsn-Gln’ of the matured protein corresponded to positions 191 – 200 in the predicted Pap6 full length peptide sequence
Table 1 Comparison of the conserved HEXXH , 19aa , E zinc-binding domain of Vibrio metalloproteases Metalloprotease
Amino acids of conserved domaina
Location
EmpA vibriolysin VVP HA/P Pap6
†A † A † AHEVSHGFTEQNSGLVYQNMSGGMNEAFSD AHEVSHGFTEQNSGLVYENMSGGMNEAFSD AHEVSHGFTEQNSGLIYSNMSGGMNEAFSD AHEVSHGFTEQNSGLVYRDMSGGINEAFSD AHEVSHGFTEQNSGLEYRGMSGGMYESFSD
345 –374 342 –371 342 –371 342 –371 329 –358
a
Zinc-binding residues and active site residues are indicated by black circles and open boxes, respectively.
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(Fig. 1). The purified 35 kDa Pap6 was proteolytically active as demonstrated by gelatin zymography (Fig. 3B). Sonicated cell lysates or supernatants made from E. coli BL21 showed no proteolytic activity (data not shown). Gelatin zymograms obtained using the culture supernatant of E. coli BL21 containing pQEP10 revealed that the proteolytic band was similar to that of the expressed recombinant Pap6 (Fig. 3B). Zymograms of the supernatant of wild type V. harveyi AP6 suggests that besides Pap6, several other proteases are also secreted as evidenced by the presence of proteolytic bands other than Pap6 (Fig. 3B). Western blot analysis was used to determine whether Pap6 had a C-terminal propeptide. Immunodetection using anti-His antibody did not detect the presence of a His-tag on the 35 kDa Pap6, suggesting that the His-tag was likely to have been part of a C-terminal propeptide which was cleaved during the maturation of Pap6 (Fig. 3C). His-tagged Sup35p protein from Saccharomyces cerevisiae (Parham et al., 2001) was used as positive control whilst E. coli BL21 sonicated cell lysates was the negative control (Fig. 3C). 3.3. Characterization of recombinant Pap6 Purified 6xHis tagged recombinant protein Pap6 was dialyzed overnight in 50 mM Tris – HCl (pH 8.0) before being used in the following enzyme assays. The effect of inhibitors on the recombinant protein Pap6 was examined. Zinc specific metal chelator 1, 10 phenanthroline (10 mM) and EGTA (10 mM) strongly inhibited enzyme activity and
almost caused a total loss of enzyme activity whilst EDTA (10 mM) reduced the enzyme activity by almost 50% (Table 2). Pap6 was uninhibited by serine protease inhibitor PMSF (10 mM), aspartate protease inhibitor pepstatin (10 mM) and cysteine protease inhibitor E-64 (10 mM) (Table 2). The addition of Mg2þ (0.1 – 10 mM) and Ca2þ (0.1 – 10 mM) did not significantly affect enzyme activity. However, Zn2þ in a concentration greater than 0.1 mM inhibited enzyme activity, with 10 mM Zn2þ resulting in an almost complete reduction of Pap6 activity (Table 2). These results suggest that Pap6 is a zinc dependant metalloprotease. The histidine modifying reagent DEPC almost completely inactivated the enzyme, thus supporting the role of histidine residues as part of the active site in Pap6. The effect of pH on Pap6 activity was tested using buffers with pH ranging from 3.0 to 11.0. Optimal caseinolytic activity was detected in an alkaline medium of pH 9.0 (Fig. 4A). A drastic drop in activity, about 50%, was observed when the pH was changed from 7.0 to 6.0 and from 9.0 to 10.0. The optimum temperature for the recombinant protein Pap6 was at 378C. However, enzyme activity was still retained over a wide temperature range of between 4 and 658C (Fig. 4B). 3.4. Site-directed mutagenesis of the HEXXH , 19aa , E zinc motif The HEXXH , 19aa , E zinc-binding motif (Vallee and Auld, 1990a) of Pap6 is located in positions 330– 354.
Fig. 3. SDS-PAGE and gelatin-gel zymography analyses of Pap6. (A) SDS-PAGE analysis of Pap6 purified through a Ni-NTA column. Electrophoresis was carried out on a 10% polyacrylamide gel. (B) Proteolytic activity was detected using a 10% SDS-PAGE gel co-polymerized with 10 mg/ml of gelatin. Lane 1, purified Pap6; lane 2, culture supernatant fraction of E. coli BL21 carrying pQEP10; lane 3, culture supernatant fraction of V. harveyi AP6. Horizontal arrow indicates the possible position of the 35 kDa Pap6 from the culture supernatant of V. harveyi AP6. (C) Western blot analysis using anti-His antibodies. Lane 1, His-tagged Sup35p from Saccharomyces cerevisiae was used as the positive control; lane 2, purified 35 kDa Pap6; lane 3, E. coli BL21 sonicated cell lysates was used as the negative control.
J.W.P. Teo et al. / Gene 303 (2003) 147–156 Table 2 Effect of metal ions and inhibitors on the activity of Pap6 protease Compound
Concentration (mM)a
None
% enzyme activityb 100
0.01 0.1 1 10
105.5 ^ 5.7 78.5 ^ 2.8 5 ^ 0.7 1.8 ^ 1.4
CaCl2
0.1 1 10
96.9 ^ 2.4 110.6 ^ 2.4 116.2 ^ 10.6
MgCl2
0.1 1 10
107.5 ^ 5.9 105.0 ^ 5.7 104.4 ^ 5.2
1, 10-phenanthroline DEPC EGTA EDTA E-64 PMSF Pepstatin
10 10 10 10 10 10 10
2.3 ^ 0.6 6.7 ^ 3.8 11.4 ^ 2.1 60.5 ^ 1.7 95.5 ^ 1.5 101.5 ^ 0.3 106.1 ^ 3.1
ZnCl2
a Enzyme samples were incubated with the metal ions in 5 mg/ml of azocasein solution for 2 h at 378C. Inhibitors were preincubated with the enzyme for 30 min prior to the addition of 5 mg/ml of azocasein solution. b Caseinolytic activity of recombinant Pap6 without the addition of metal ions or inhibitors was taken to be 100%. The effect of metal ions and inhibitors on Pap6 activity was based on averages of three independent experiments.
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showed low sequence homology to elastases of A. hydrophila and LasB of P. aeruginosa. However, no elastolytic activity was detected when the recombinant Pap6 protein was incubated with elastin congo red, indicating that it was not an elastase (data not shown).
4. Discussion In this paper, we describe the cloning, expression and characterization of Pap6, a novel metalloprotease from V. harveyi strain AP6. Sequence analysis of Pap6 predicts that it encodes a large 677 amino acid polypeptide with a predicted molecular mass of 75 kDa but SDS-PAGE analysis showed that biologically active Pap6 had a molecular mass of 35 kDa. Peptide sequence alignments and site-directed mutagenesis confirmed that Pap6 is a member of the thermolysin family of zinc metalloproteases. Members of the thermolysin family are first synthesized as inactive preproteins which are processed to the mature form (Ha¨se and Finkelstein, 1993; Miyoshi and Shinoda, 2000). Previous studies have shown that zinc metalloprotease VVP of V. vulnificus is first produced as a large precursor and is later cleaved to yield a 35
Residues His330, His334 and Glu354, thought to be involved in zinc binding were replaced with non-zinc-binding residues Ser, Pro and Val, respectively (Vallee and Auld, 1990b). Residues Glu331 and Tyr345 are probably involved in catalysis. Glu331 was replaced with Asp which had similar properties as Glu. Tyr345 was replaced with a non-polar Gly residue. These site-directed mutants were assayed for activity. Mutants H330S, E331D, H334P, Y345G and E354V had less than 10% of wild type activity (Fig. 5A). Mutant E331D had reduced enzymatic activity despite being replaced by a fairly similar residue. The results were consistent with the proposed roles of the residues in enzymatic catalysis. SDS-PAGE analysis of Pap6 and its mutants (Fig. 5B) showed that Pap6 and its mutants were expressed to similar levels; hence the loss of caseinolytic activity observed in the mutants was due to the replacement of critical amino acid residues and not the result of impaired protein expression. 3.5. Substrate specificity of Pap6 Digestion of various protein substrates by recombinant Pap6 was examined. It was able to degrade BSA, fibrinogen, native type IV collagen and type IV gelatin (Figs. 6A –D). It was unable to degrade native type I collagen (Fig. 6E) but was able to use type I gelatin as a substrate (Fig. 6F). Pap6
Fig. 4. (A) Effect of pH and temperature on Pap6 activity. Buffers of different pH containing 5 mg/ml of azocasein were used. Activity at pH 9.0 was defined as 100%. (B) Enzyme reaction was in 100 ml of 50 mM Tris – HCl (pH 8.0) containing 5 mg/ml of azocasein at varying temperatures. The enzyme activity at 378C was defined as 100%. Experimental details are described in Section 2. The relative activities are averages based on two independent experiments.
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Fig. 5. (A) Enzymatic activity of Pap6 and its derivatives. Proteolytic activity of mutant enzymes was expressed in relation to the activity of wild type Pap6 (100%). The results are the averages of triplicate independent experiments. (B) Analysis of the expression level of wild type Pap6 and its various site-directed mutants using a 10% SDS-PAGE gel.
kDa mature protein via removal of a 44.7 kDa amino acid Nterminal as well as a 10 kDa C-terminal propeptide (Miyoshi et al., 1997). The preprotein of Pap6 is predicted to be 75 kDa and even with the removal of the signal peptide (1.7 kDa) and the 19.2 kDa N-terminal propeptide, the size of the remaining peptide should be about 54.3 kDa. This implies that like VVP, Pap6 may be further processed, probably through the removal of a C-terminal propeptide, to produce a mature enzyme of 35 kDa. This was supported by western blot analysis which indicated that the 35 kDa Pap6 was not Histagged. Therefore, we suggest that Pap6 preprotein is likely to have four domains: a 15 amino acid signal peptide, a 19.2 kDa (175 amino acid) N-terminal propeptide, a 35 kDa mature protein and possibly a 19.1 kDa C-terminal propeptide. Under the conditions used in this study, we were unable to observe the 19.1 kDa band corresponding to the C-terminal propeptide. Perhaps stronger elution conditions such as a higher concentration of imidazole in the elution buffer may have been required to liberate the C-terminal propeptide from the affinity column. It is most probable that the propeptide of Pap6 was processed before being secreted as gelatin zymograms showed that the Pap6 protein purified from the sonicated cell lysates has a similar molecular weight as that derived from the supernatant of E. coli BL21 harboring pQEP10. Crystal structure determinations of several zinc containing enzymes have allowed the catalytic and structural features of the zinc binding sites to be defined (Ha¨se and
Finkelstein 1993; Vallee and Auld, 1990a). X-ray crystallographic analyses of members of the thermolysin zinc metalloprotease superfamily such as thermolysin from Bacillus thermoproteolyticus (Colman et al., 1972), elastase from P. aeruginosa (Thayer et al., 1991) and aureolysin from Staphylococcus aureus (Banbula et al., 1998) showed that a catalytic zinc atom is coordinated to two histidine residues and one glutamate residue of the enzyme, together with an active water molecule. The zinc ligands are the two histidines in the HEXXH motif and a glutamate residue separated by a 19 base long spacer (Vallee and Auld, 1990a). Peptide sequence alignment showed that Pap6 contains a zinc-binding motif 330HEXXH , 19aa , E354. Site-directed mutagenesis of the putative zinc binding residues of Pap6, i.e. histidines 330, 334 and glutamate 354, showed that they were indeed essential for activity. The enzyme inhibition by DEPC also lends support to the importance of histidine residues in catalysis. The zinccoordinating ligands of metalloproteases with the HEXXH , 19aa , E motif have been investigated by mutational analysis. His 415 and His 419 (equivalent to His 330 and His 334, respectively in Pap6) of Clostridium histolyticum collagenase were mutated and this caused catalytic activity and zinc-binding to be abolished (Jung et al., 1999). The replacement of Glu 408 in mouse aminopeptidase A (equivalent to Glu 354 in Pap6) generated mutants that had lost their catalytic and zinc-binding activity (Vazeux et al., 1996). Assaying the zinc content of these mutant enzyme should help confirm the importance of these residues in zinc-binding and coordination. The conserved zinc-binding motif is shared by these metalloproteases and mutations to these active site residues reveal a similar loss of enzyme activity. It is highly probable that the catalytic mechanism of Pap6 may be similar to other zinc metalloproteases. The replacement of two other active site residues, Glu 331 and Tyr 345 led to a loss of proteolytic activities thus demonstrating that these active site residues were also important for catalytic activity. The importance of these residues was similarly demonstrated in a thermolysinlike B. subtilis neutral protease which had a mutation occurring at Glu 143 (equivalent to Glu 331 of Pap6) (Toma et al., 1989). When Tyr 157 of B. thermoproteolyticus thermolysin (equivalent to Tyr 345 in Pap6) was substituted, a loss of catalytic activity was observed (Marie-Claire et al., 1998). Therefore, Tyr and Glu could be considered key active site residues. Enzyme inhibitor studies showed that Pap6 is most likely a zinc-dependant metalloprotease as it was strongly inhibited by the zinc-specific chelator 1, 10-phenanthroline and general metal chelators such as EGTA and EDTA. Pap6 was unaffected by protease inhibitors such as the serine protease inhibitor, PMSF; aspartate protease inhibitor, pepstatin and cysteine protease inhibitor, E-64. Our results showed that zinc concentrations greater than 1 mM completely inhibited the protease activity of Pap6. It has been noted that high zinc concentrations often inhibit
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Fig. 6. Analysis of protein digestion by Pap6. Each substrate (15 mg) was incubated in the absence (lanes 1) or in the presence (lanes 2) of Pap6 at 378C for overnight. Lane 3 is purified Pap6. (A) BSA; (B) fibrinogen; (C) type IV collagen; (D) type IV gelatin; (E) type I collagen; and (F) type I gelatin. Digested substrates were separated on a 10% SDS-PAGE gel. The horizontal arrow marks the position of recombinant Pap6.
metalloproteases. The inhibition is due to formation of zinc monohydroxide that bridges the catalytic zinc ion to the side chain of the active site of the enzyme (Larsen and Auld, 1991). The extracellular matrix (ECM) consists of collagens, non-collagenous glycoproteins, and proteoglycans, with collagens being the most abundant structural components (Aumailley and Gayraud, 1998). Protein substrate studies with Pap6 indicate that fibronectin, a non-collagenous glycoprotein component of the ECM was hydrolyzed by Pap6. Type IV collagen, the major component of the basement membrane was also digested by Pap6 whereas type I collagen was not readily digested. Gelatins (heat denatured types I and IV collagens) which are also components of the ECM were also digested by Pap6. VVP, the V. vulnificus protease, also possesses proteolytic activity against type IV collagen and it is speculated that the destruction of type IV collagen framework could be responsible for the breakdown of the basement membrane, thus leading to the hemorrhagic reaction typically seen in V. vulnificus infections (Miyoshi and Shinoda, 2000). In a similar manner, Pap6 may elicit tissue damage on prawn host by proteolyzing the ECM components. Further work could be carried out to determine the pathological actions of the Pap6 protein on a wider range of host substances such as plasma components and muscle proteins. It would also be interesting to investigate the role
of Pap6 in bacterial pathogenesis in vivo. This could be carried out by comparing the invasiveness of wild-type V. harveyi and a mutant deficient in Pap6 activity. A variety of exoenzymes like phospholipase, chitinase and haemolysin have been detected in paneid V. harveyi strains (Liu et al., 1996), and our study has also shown the presence of proteolytically active proteases other than Pap6 in the supernatants of V. harveyi strain AP6. It is highly likely that bacterial invasion could be the result of the synergistic actions of these pathogenic factors.
Acknowledgements We thank YiHu Dong for his suggestions during the course of this study and A. Suwanto for the gift of V. harveyi strain AP6, and Haiwei Song for the His-tagged Sup35p protein. This work was supported by a grant (No. 0451/2000) from the National Medical Research Council of Singapore.
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