Cloning of a novel collagenase gene from Gram -negative

1

Cloning of a novel collagenase gene from Gram-negative 1

bacterium Grimontia (Vibrio) hollisae 1706B and its efficient 2

expression in Brevibacillus choshinensis 3

4

Naoko Teramura1, a

, Keisuke Tanaka1, a,

*, Katsumasa Iijima1, Osamu Hayashida

1, Koki 5

Suzuki2, Shunji Hattori

1, 2 and Shinkichi Irie

1, 2 6

1Nippi Research Institute of Biomatrix, Toride, Ibaraki 302-0017 Japan 7

2Japan Institute of Leather Research, Adachi, Tokyo 120-8601, Japan 8

a These two authors contributed equally to this work 9

10

Running title: Collagenase gene of G. hollisae 11

12

*Address correspondence to: Keisuke Tanaka, Nippi Research Institute of Biomatrix, 13

520-11, Kuwabara, Toride, Ibaraki 302-0017, Japan, Phone: +81-297-71-3045, Fax: 14

+81-297-71-3041, E-mail: [email protected] 15

Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.J. Bacteriol. doi:10.1128/JB.01528-10 JB Accepts, published online ahead of print on 22 April 2011

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Abstract. 1

The collagenase gene was cloned from Grimontia (Vibrio) hollisae 1706B, 2

and its complete nucleotide sequence was determined. Nucleotide sequencing 3

showed that the open reading frame was 2,301 bp in length and encoded an 84 kDa 4

protein of 767 amino acid residues. The deduced amino acid sequence contains a 5

putative signal sequence and a zinc metalloprotease consensus sequence, the HEXXH 6

motif. G. hollisae collagenase showed 60% and 59% amino acid sequence identity 7

to Vibrio parahaemolyticus and Vibrio alginolyticus collagenase, respectively. In 8

contrast, this enzyme showed less than 20% sequence identity with Clostridium 9

histolyticum collagenase. When the recombinant mature collagenase, which 10

consisted of 680 amino acids with a calculated molecular mass of 74 kDa, was 11

produced by the Brevibacillus expression system, a major gelatinolytic protein band 12

of approximately 60 kDa was determined by zymographic analysis. This result 13

suggested that cloned collagenase might undergo processing after secretion. 14

Moreover, the purified recombinant enzyme was shown to possess a specific activity 15

of 5,314 units/mg, an approximately 4-fold greater activity than that of C. 16

histolyticum collagenase. 17


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Introduction 1

Bacterial collagenases are metalloproteases containing a consensus motif for 2

zinc proteases, the HEXXH sequence, and are capable of digesting both native and 3

denatured collagen. They make multiple cleavages at the Y-Gly bond in repeating 4

X-Y-Gly sequences within triple helical regions, where proline and hydroxyproline 5

residues are most common in the X and Y positions, respectively (17). Because of 6

their characteristics, bacterial collagenases have been widely used in biological 7

experiment as tissue-dispersing enzymes, as well as in medical procedures such as the 8

isolation of pancreatic islet cells for transplantation (14) and the treatment for 9

Dupuytren’s disease (6). 10

Much of our knowledge of bacterial collagenases has come from studies of the 11

enzymes produced by Clostridium histolyticum (13, 15-17, 34). Analysis of the 12

primary structure of the gene product from C. histolyticum has revealed that 13

clostridial collagenases consist of three domains (catalytic domain, polycystic kidney 14

disease (PKD) domain and collagen-binding domain (CBD)) in their molecules. 15

Moreover, CBD has utilized for anchoring molecule that growth factors fused to CBD 16

can be functional to bind to collagen fibrils and maintain biological activities (21). 17

On the other hand, one of the other well-investigated bacterial collagenases is Vibrio 18

alginolyticus collagenase (7, 10, 11, 28). The collagenase activity of V. alginolyticus 19

collagenase is higher than that of any other bacterial collagenase, and it was found 20

highly efficient in debridement of necrotic burns, ulcers and decubitus. To date, 21

bacterial collagenases have been purified from various species, and their genes have 22

been cloned and sequenced (8, 12, 18, 24, 35). However, many collagenases have 23

not yet been both enzymatically and structurally characterized. 24

Vibrio hollisae is a Gram-negative bacterium first described in 1982 (4) and 25


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recently reclassified as the novel genus Grimontia (29). Grimontia (Vibrio) hollisae 1

has been reported as a toxic bacterium, whose toxin was clarified as thermostable 2

direct hemolysin (22), and is primarily known to cause moderate to severe cases of 3

gastroenteritis in healthy people (6). G. hollisae strain 1706B was isolated from 4

seashore sand collected from the Shin-Kiba coast in Tokyo (27). This organism 5

produces a collagenase with a very high specific activity in the presence of gelatin, 6

and this enzyme even degrades the tanned leather (26). The characteristics of this 7

organism and purified collagenase have been described in a series of papers (25-27). 8

The properties of this collagenase are as follows: (i) it has a molecular weight of 60 9

kDa, (ii) degrades insoluble collagen, soluble collagen, Z-GPLGP peptide and 10

Pz-PLGPR peptide, but not casein, (iii) has an optimum pH of 7.0-8.0 for insoluble 11

collagen hydrolysis, (iv) is stable in the range between pH 4.5 and 11 (25). In order 12

to clarify its enzymic characteristics and to utilize it for biological applications, the 13

primary structure of the collagenase needs to be elucidate. 14

In the present study, we cloned and sequenced a novel collagenase gene from 15

G. hollisae 1706B to elucidate its primary structure, and demonstrated the expression 16

and characterization of recombinant mature collagenase using the Brevibacillus 17

expression system. Moreover, we discussed the characteristics of the corresponding 18

amino acid sequence of this enzyme and its similarity to those of other bacterial 19

collagenases. 20


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Materials and Methods 1

Bacterial strains and plasmids 2

G. hollisae strain 1706B was obtained around the shore of Shin-Kiba, Tokyo, 3

Japan and used throughout this study (27). The plasmid pGEM-T Easy and the E. 4

coli competent cells JM109 (Promega, Madison, WI) were used as subcloning vector 5

and host, respectively. The expression plasmid vector pCC1BAC and the host 6

TransforMaxTM

EPI300TM

E. coli (EPICENTRE Biotechnologies, Madison, WI) were 7

used to make a BAC clone library of G. hollisae. Bacillus brevis expression vector 8

pNY326 and Brevibacillus choshinensis S5 (Takara Bio, Shiga, Japan) were used for 9

expression of recombinant proteins. 10

11

Construction of a genomic library from G. hollisae 1706B 12

The genomic DNA of G. hollisae 1706B was purified with a QIA genomic 13

DNA extraction kit (QIAGEN, Hilden, Germany). The purified DNA was digested 14

with the restriction enzyme EcoRI and separated on a 0.6%(w/v) agarose gel. Only 15

DNA fragments over 2kb were ligated into the E. coli expression vector, pCC1BAC 16

(EPICENTRE Biotechnologies). Then, pCC1BAC was transformed into 17

TransforMaxTM

EPI300TM

E. coli by electroporation, and the transformants were 18

plated onto LB agar plates containing 50 µg/ml ampicillin. 19

20

DNA probe preparation 21

Degenerate primers were designed based on the internal peptide sequence of 22

original collagenase (Fig. 2, No. 6) for F1 and the consensus sequence of catalytic site 23

from V. alginolyticus and V. parahaemolyticus collagenase (Fig.2, underlined) for R1. 24

The primer sets F1 (5’-GAGGCNATCTTTAGCTCCAATCATATGTAYAAY-3’) 25


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and R1 (5’-ATCTAAGTAATGCACGTATTCATGYTCNAGRTT-3’) were used for 1

amplification of probe sequence. Y, R and N represent C/T, A/G and A/C/T/G, 2

respectively. Amplification was performed in 50 cycles of 0.5 min at 95°C, 0.5 min 3

at 45°C, and 1 min at 72°C. The amplified 1,080bp PCR product was electro-eluted 4

from 1% agarose gel, ligated into pGEM-T Easy vector (Promega), and then 5

transformed into E.coli strain JM109. The purified plasmid was used as a template 6

to create DIG-labeled DNA probes using DIG-high prime labeling reagent (Roche, 7

Basel, Switzerland) according to the manufacturer’s instructions. 8

9

Screening of the genomic library with DNA probe 10

The genomic library was screened by colony hybridization according to the 11

manufacturer’s instructions (Roche). Briefly, the ampicillin-resistant transformants 12

were blotted onto a nylon membrane (Roche), and lysed with 0.5 M NaOH. The 13

denatured DNA was then immobilized, followed by a protease K treatment. The 14

DIG-labeled DNA probe was used for hybridization. Positive clones were picked 15

from cultured LB agar plates, and subcultured in LB liquid medium with ampicillin at 16

37°C. BAC DNA of collagenase-positive colonies was prepared using the QIA 17

genomic DNA extraction kit (QIAGEN). 18

19

DNA sequencing and alignment of deduced amino acid sequence 20

Purified BAC DNA was amplified with cycle sequencing using a thermal 21

cycler (Takara Bio), and sequenced with a DNA auto sequencer (ABI PRISM 310, 22

ABI). Appropriate oligonucleotide primers (Sigma-Aldrich, St. Louis, MO) and 23

Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carlsbad, CA) were used 24

to walk along the sequence. The sequencing strategy for the pCC1BAC-2 insert was 25


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outlined in Fig. 1. Computer analysis of the DNA sequence data was performed 1

using GenBank database and BLAST search programs. The deduced amino acid 2

sequence alignment and homology data were generated using the CLUSTAL W2 3

program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). 4

5

Recombinant collagenase preparation 6

pCC1BAC-2 was used as the DNA template. To add NcoI site to the 5’ 7

region and HindIII site to the 3’ region of the mature collagenase gene, the primers 8

were designed as follows: Forward: 9

5’-AAACCATGGCTTTCGCTGCGGTTGAACAGTGTGATCT-3’; Reverse: 10

5’-AAAAAGCTTTTACTGACGACACTGGTTAC-3’. Restriction sites are 11

represented by bold letters and underlined. The mature domain of 2.1kb collagenase 12

gene was amplified with Expand High Fidelity PCR system (Roche). After the 13

treatment with NcoI and HindIII, the double digested fragment was ligated into the 14

multi cloning site of the Brevibacillus expression vector pNY326, which was located 15

downstream of Brevibacillus signal sequence (pNY326-Col2). Plasmid 16

pNY326-Col2, harboring the complete mature collagenase gene, was transformed into 17

Brevibacillus choshinensis S5 to express the recombinant enzyme. The 18

Brevibacillus transformant was aerobically cultured in 2SLN medium containing 19

neomycin (50 µg/ml). After centrifugation, the supernatant was purified with a 20

DEAE-Sepharose column (26 × 100 mm) with an FPLC system under gradation of 21

sodium concentration (0.2-1.1 M NaCl concentration). The column was eluted 22

isocratically with 0.2 M NaCl, 50 mM Tris-HCl (pH 7.5) for 10 min at 5 ml/min 23

followed by a linear gradient to 1.1 M NaCl for 50 min. The purified recombinant 24

protein was concentrated by ultrafiltration with a 30 kDa cutoff (PALL, Port 25


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Washington, NY), and dialyzed against 50 mM Tris-HCl buffer (pH 7.5) at 4°C. 1

2

SDS-PAGE 3

SDS-PAGE was carried out on a 7.5% or 10% polyacrylamide gel 4

according to Laemmli (9) unless otherwise stated. After electrophoresis, the gel was 5

stained with 0.25% Coomassie brilliant blue R-250 in 50% methanol and 10% acetic 6

acid, and destained with 5% methanol and 7.5% acetic acid. 7

8

Determination of collagenolytic activity 9

The collagenolytic activity of recombinant collagenase was measured using 10

FITC-labeled type I collagen as previously described (20). Briefly, the enzyme 11

solution was mixed with 50 mM Tris-HCl (pH 7.5) containing 0.05% FITC-labeled 12

type I collagen, 5 mM CaCl2 and 200 mM NaCl, and incubated at 30°C for 30 min. 13

After adding EDTA to stop the enzymatic reaction, the degraded FITC-labeled 14

collagen fragment was extracted with 50 mM Tris-HCl (pH 9.5) containing 70% 15

ethanol. The fluorescence intensity of the supernatant was measured by 16

fluorescence spectrophotometry (530 nm Em/ 485 nm Ex). One unit of 17

collagenolytic activity was defined as the amount degrading 1 µg of FITC-labeled 18

collagen at 30°C per min. Protein concentrations were determined using the 19

Coomassie Plus – The Better BradfordTM

Assay Reagent (Thermo Scientific, 20

Rockford, IL). Collagenase from Clostridium histolyticum (Amano Enzyme, 21

Nagoya, Japan) was used as a reaction standard. All assays were carried out in 22

triplicate. 23

For measurement of enzyme kinetics, 0.5 µg of enzyme was incubated with 24

various amounts of FITC-labeled type I collagen (10-50 µg) at 30°C for 5 min and the 25


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fluorescence intensity of the supernatant was measured. Specific collagenase 1

substrate FALGPA (N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala) (Bachem AG, 2

Bubendorf, Switzerland) was also chosen to determine enzyme activity. Assay with 3

FALGPA was performed according to the modified method of a previous report (31). 4

Briefly, the enzyme and FALGPA were mixed in 50 mM Tricine buffer (pH 7.5) 5

containing 0.4 M NaCl and 40 mM CaCl2, and incubated at 30°C for 5 min. After 6

incubation, the absorbance change at 345 nm was measured using a CORONA 7

SH-9000 microplate reader (Corona Electric, Ibaraki, Japan). The FALGPA 8

concentrations were varied from 0.5 mM to 3.0 mM. In the FALGPA assay, one 9

unit of activity was defined as the amount degrading 1 µmol of FALGPA peptide at 10

30°C per min. Vmax and Km values for hydrolysis of native collagen and FALGPA 11

were estimated from the Lineweaver-Burk plot using the reaction rates at different 12

substrate concentrations. 13

14

Real-time zymography 15

Real-time zymography was performed as previously described (3). Briefly, 16

recombinant collagenase was subjected to non-reducing SDS-PAGE using a 10% gel 17

containing 0.05% FITC-labeled gelatin. After electrophoresis at 4°C, the gel was 18

washed in 50 mM Tris-HCl (pH 7.5) containing 2.5% TritonX-100 for 30 min, and 19

then was incubated in 50 mM Tris-HCl (pH 7.5) containing 5 mM CaCl2 and 200 mM 20

NaCl at 37°C for 5 h. The collagenase-digested FITC-labeled gelatin was visualized 21

using a transilluminator. 22

The effect of protease inhibitors on collagenase activity was determined by 23

adding the inhibitors to the incubation buffer. In inhibition studies, protease 24

inhibitors such as EDTA, o-phenanthroline, NEM or PMSF were used at final 25


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concentrations of 20, 2.0, 5.0 and 1.0 mM, respectively. 1

2

Amino acid sequence 3

Amino acid sequence analysis of N-terminal or internal peptide fragments 4

of original collagenase was performed as described previously (19). Briefly, internal 5

sequences were determined by lysyl endoprotease, trypsine and V8 protease digestion. 6

The enzyme-cleaved fragments and purified collagenase were separated by 10% 7

SDS-PAGE, and electrophoretically transferred to Immobilon-P (Millipore, Billerica, 8

MA). The membrane was stained with Coomassie brilliant blue R-250, and the 9

protein band was excised from the membrane, and then washed extensively with 10

deionized distilled water. The N-terminal sequence was analyzed using a Procise 11

491 protein sequencer (Applied Biosystems, Carlsbad, CA). Detected fragments 12

were shown in Fig. 2. 13

14

Nucleotide sequence accession number 15

The determined nucleotide sequence was deposited in the DDBJ database 16

under accession number AB600550. 17

18


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Results. 1

Cloning of collagenase gene from G. hollisae 1706B 2

In order to amplify a fragment of the G. hollisae collagenase coding gene, 3

degenerate primers were designed. A primer set yielded a single amplification 4

product, and nucleotide sequencing revealed that the amino acid sequence deduced 5

from this PCR product contained four partial peptide sequences (Fig. 2, No. 2, 3, 4 6

and 7). Therefore, the plasmid containing this PCR product was used as a template 7

to create DIG-labeled DNA probes as a hybridization probe for genomic library 8

screening. 9

A partial genomic library was constructed with EcoRI-digested genomic DNA 10

fragments from G. hollisae using a pCC1BAC vector system. The plasmids were 11

transformed into TransforMaxTM

EPI300TM

E. coli using electroporation, yielding 12

about 5,000 colonies on LB-ampicillin plates. By using colony hybridization 13

technique, 30 positive clones were picked up from the library and all the purified 14

BAC contained 2.3 kb inserts encoding the collagenase gene. One of the former 15

clones, designated as pCC1BAC-2, was chosen for further study. Restriction 16

analysis revealed that pCC1BAC-2 contained a >50 kb EcoRI insert (data not shown). 17

18

Nucleotide sequence of G. hollisae collagenase gene 19

The nucleotide sequence of the pCC1BAC-2 insert was sequenced using the 20

strategy outlined in Fig. 1. The obtained sequences were aligned by their overlaps to 21

form a single contiguous sequence, and the 2.3 kb sequence of G. hollisae collagenase 22

was determined (Fig. 2). The entire open reading frame (ORF) of G. hollisae 23

collagenase was sequenced on both strands. Analysis of the sequence revealed a 24

complete ORF extending from an ATG codon at nucleotide +1 to a TAA stop codon 25


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at position 2,301, which encodes a protein of 767 amino acids. A Shine-Dalgarno 1

sequence (AGAAGAA) is observed at 5-11 bp upstream from the ATG codon. A 2

stem-loop sequence (nucleotide position 2,328 to 2,347) with a short run of T’s is 3

present downstream of the termination codon. 4

The N-terminal amino acid sequence 5

(Ala-Val-Glu-Gln-Cys-Asp-Lys-Ser-Glu) of the purified original enzyme 6

corresponded perfectly to the deduced amino acid sequences of our determined 7

sequence in position 262 to 288 downstream of the ATG codon. Moreover, the 8

deduced amino acid sequence of ORF includes a zinc metalloprotease HEXXH 9

consensus motif, which was detected as HEYVH in position 1474 to 1488. It is 10

known that the amino acid sequence HEXXH is important in facilitating the electron 11

transfer with zinc in enzyme catalysis (30). Moreover, the internal peptide 12

sequences were found to agree completely with the protein sequence deduced from 13

DNA sequencing. 14

We next compared the deduced primary sequence of G. hollisae collagenase 15

gene with other known protein sequences using the BLASTP programs of the NCBI. 16

The predicted amino acid sequence of G. hollisae collagenase showed 59% and 60% 17

identity with collagenase from V. alginolyticus (28) and V. parahaemolyticus (8), 18

respectively (Fig. 3A). Moreover, the alignment predicted that G. hollisae 19

collagenase consists of a pre-pro region (aa 1-87), catalytic domain (aa 88-615) and 20

bacterial pre-peptidase C-terminal (PPC) domain (aa 688-749) (33) (Fig. 3B). 21

Furthermore, a database search revealed that G. hollisae collagenase showed less than 22

35% similarity with any other reported Vibrio metalloproteases (data not shown). 23

On the other hand, G. hollisae collagenase showed 12% and 11% identity to ColG 24

and ColH, respectively, from Clostridium histolyticum (data not shown). 25


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1

Expression and characterization of recombinant collagenase in Brevibacillus 2

To examine whether the product of the G. hollisae collagenase gene possesses 3

similar proteolytic activity as the original enzyme, we first made a construct for 4

expressing the recombinant mature collagenase (aa 88-767) and produced the 5

recombinant enzyme using the Brevibacillus expression system. When the 6

collagenolytic activity of the culture supernatant of transformants (carrying 7

pNY326-Col2) was measured using FITC-labeled collagen, the activity was found to 8

be higher than that of mock transformants (data not shown). This result indicated 9

that recombinant collagenase was successively produced. 10

To characterize certain biochemical properties, recombinant G. hollisae 11

collagenase was purified from Brevibacillus culture medium using a 12

DEAE-Sepharose column with FPLC system. The expression typically yielded 0.2 g 13

of pure collagenase from 1 L of culture. When the Brevibacillus culture medium 14

and purified recombinant enzyme were confirmed on SDS-PAGE, three bands (74, 60 15

and 40 kDa) derived from this enzyme were detected (Fig. 4A, lane 3). The deduced 16

molecular mass of mature protein, without the possible pre-pro peptide of 87 amino 17

acids, was 74 kDa, and matched with the molecular mass determined by SDS-PAGE. 18

The major 60 kDa form was the same molecular mass as the purified form from the 19

original bacterial collagenase. Zyomography showed that all three forms of 20

collagenase possessed gelatinase activity; however, the activity of the 40 kDa form 21

was weaker than that of the other forms (Fig. 4B). Moreover, the gelatinolytic band 22

disappeared in the presence of the metal ion chelators, EDTA and o-phenanthroline, 23

but not cysteine and serine protease inhibitors, such as NEM and PMSF (Fig. 4C). 24

We also found that the three forms of collagenase have the same N-terminal amino 25


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acid sequence (data not shown), suggesting that the C-terminal region of the mature 1

enzyme would be autodegraded and then become 60 kDa and 40 kDa enzymes. 2

Furthermore, the 60 kDa enzyme seems to be the most stable form. The purified 3

recombinant enzyme could digest insoluble and soluble type I collagen, and 4

collagenolytic activity assay showed a specific activity of 5,841 units/mg using 5

FITC-collagen, which was elevated by approximately 5.8-fold compared to the 6

culture medium (data not shown). In contrast, this recombinant enzyme could not 7

degrade casein (data not shown). 8

9

Kinetic parameters for the hydrolysis of type I collagen by collagenase 10

The kinetic parameters of collagenase were determined using native type I 11

collagen and the synthetic peptide substrate, FALGPA (Table 1). G. hollisae 12

collagenase showed a 4.2 times lower Km value and a slightly higher Vmax value 13

against FITC-collagen than C. histolyticum collagenase, resulting in a higher specific 14

constant (approximately 6.7 times higher). On the other hand, the two enzymes have 15

comparable substrate affinity to FALGPA, whereas the Vmax values increased 17-fold 16

in G. hollisae when compared to C. histolyticum collagenase. As a result, G. 17

hollisae collagenase showed a high specific constant also against FALGPA 18

(approximately 27 times higher). These results help to explain the specific activity 19

of the two enzymes. Previously, V. parahaemolyticus collagenase has been reported 20

to have a Km value of 1.06 mM toward FALGPA, at pH 8.0 and 25°C (35), and this 21

Km value was comparable to that of the present collagenase. Based on the specific 22

constant, G. hollisae collagenase had a higher specificity for type I collagen and 23

FALGPA than C. histolyticum collagenase.24


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Discussion 1

In the present study, we isolated the G. hollisae collagenase gene by a 2

cloning and sequencing method. The isolated gene consisted of 2,301 nucleotides, 3

and the 767 amino acid protein deduced from the ORF revealed high homology to 4

several Vibrio collagenases. Moreover, we succeeded in the effective expression of 5

its recombinant enzyme in Brevibacillus. It hydrolyzed type I collagen, gelatin and 6

FALGPA peptide more efficiently than C. histolyticum collagenase, and the inhibition 7

study showed that the enzyme was inhibited by metal ion chelators, such as EDTA, 8

indicating that the cloned collagenase was a metalloprotease. 9

BLASTP search indicated that the deduced amino acid sequences of G. 10

hollisae collagenase show extensive homology with V. alginolyticus and V. 11

parahaemolyticus metalloproteases. On the other hand, G. hollisae collagenase 12

shared less than 20% identity with ColG and ColH from C. histolyticum, and ColA 13

from C. perfringens (data not shown). This result indicates that G. hollisae 14

collagenase can be classified into the M9A subfamily in the MEROPS database (23). 15

In the previous study, Vibrio metalloproteases were classified into three subgroups 16

(class I, II and III) based on the HEXXH sequence and substrate specificity (8). 17

According to this classification, G. hollisae collagenase belongs to the class III group 18

that includes V. alginolyticus and V. parahaemolyticus. However, G. hollisae 19

collagenase has little caseinase activity (data not shown), while V. alginolyticus and V. 20

parahaemolyticus collagenase reportedly possess apparent caseinase activity. One 21

of the possible reasons for this difference in substrate specificity may be the 22

difference in domain structures among these enzymes. Given that the PKD domain 23

was absent from G. hollisae collagenase (Fig. 3A and 3B), this domain might 24

participate in caseinase activity when bound to substrate. These results indicate that 25


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G. hollisae collagenase should be classified into a new group among the Vibrio 1

collagenases, and suggest that the three enzymes, which differ in function and origin, 2

are evolutionary related. 3

Surprisingly, we found that G. hollisae collagenase possessed markedly 4

greater activity compared to C. histolyticum collagenase (Table 1). Since G. hollisae 5

collagenase showed high affinity to native collagen and high catalytic activity to 6

FALGPA as compared to C. histolyticum collagenase, these results suggested that the 7

degradation mechanism of the two collagenases appear to be different against 8

collagen or gelatin. However, C. histolyticum collagenase used in this study should 9

be considered as a commercially available enzyme, which is a purified native protein. 10

Since we found that CBD and PKD domains were absent from this purchased enzyme 11

by SDS-PAGE (data not shown), this result raises the possibility that the loss of CBD 12

and PKD domains may lead to a decrease in activity of this purchased enzyme as 13

compared to the intact form. 14

Analysis of kinetic parameters led to our considerable interest in why and how 15

G. hollisae collagenase degrades collagen effectively. Because collagen is highly 16

resistant to most proteases, collagenase seems to possess an effective degradation 17

mechanism against collagen. For example, a recent study indicated that mammalian 18

collagenases locally unwind the triple-helical structure through the co-ordinated 19

action of the catalytic domain and collagen-binding domain (called the hemopexin 20

domain), and then hydrolyze the peptide bonds (1). Moreover, the PKD domain of 21

deseasin MCP-01, which is a bacterial collagenolytic serine protease, is reported to 22

bind collagen and to swell collagen fascicles, suggesting that the PKD domain may 23

improve the collagenolytic efficiency of the catalytic domain (32, 36). However, G. 24

hollisae collagenase possesses neither a CBD nor PKD domain. Since CBD has 25


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been reported to be necessary for the collagenolytic activity of mammalian and 1

bacterial collagenase (2, 16), G. hollisae collagenase may contain an unidentified 2

CBD in the 60 kDa form and/or possess a novel degradation mechanism against 3

collagen. The domain structure/function relationship remains to be clarified in order 4

to elucidate the mechanism of action of this enzyme. 5

It is noteworthy that the recombinant collagenase of G. hollisae was produced 6

with stable activity using the Brevibacillus expression system. Since Brevibacillus 7

is a Gram-positive bacterium, this system leads to the expression of recombinant 8

proteins with low endotoxin contamination, which has been known to enhance the 9

immunological response of higher animals. In addition, this recombinant enzyme 10

can be used for dispersion of human fibroblasts in collagen gel, and appears to have 11

no obvious cytotoxicity (data not shown). Therefore, it can be utilized for biological 12

applications, specifically for medical applications. 13

In conclusion, we cloned a novel collagenase gene from G. hollisae 1706B 14

and produced a high yield of recombinant enzyme using the Brevibacillus expression 15

system. Moreover, we provided evidence that this enzyme showed higher 16

collagenolytic activity than C. histolyticum collagenase, indicating that G. hollisae 17

collagenase is suitable for both basic and applied research. 18


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Acknowledgements 1

We thank Dr. Kiyoko Ogawa-Goto and Tomonori Ueno for their technical advice and 2

support. We also thank Dr. Osamu Matsushita and Dr. Takehiko Mima (Department 3

of Microbiology, Kitasato University School of Medicine) for their stimulating 4

discussions.5

6


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Abbreviations 1

PKD, polycystic kidney disease; CBD, collagen-binding domain; PCR, polymerase chain 2

reaction; DIG, digoxigenin; FPLC, fast protein liquid chromatography; SDS-PAGE, sodium 3

dodecyl sulfate-polyacrylamide gel electrophoresis; FITC, fluorescein isothiocyanate; NEM, 4

N-ethylmaleimide; PMSF, phenylmethylsulfonyl fluoride; ORF, open reading frame; PPC, 5

pre-peptide C-terminal 6


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Figure Legends 1

Fig. 1 Sequencing strategy for the collagenase gene inserted into pCC1BAC-2. 2

The thick line represents the collagenase gene inserted into pCC1BAC-2 plasmid. The 3

thin line indicates probe region used for cloning. The lower arrows indicate the 4

direction of sequence determinations, starting from specific primers. 5

6

Fig. 2 DNA sequence and deduced amino acid sequence of Grimontia hollisae 7

collagenase gene. 8

The N-terminal amino acid sequence of 74 kDa and 60 kDa collagenase are indicated by 9

box 1. Numbered boxes indicate biochemically identified peptides. No. 2, 3 and 4, 10

lysyl endoprotease-digested fragments; No.5, trypsin-digested fragment; No. 6 and 7, 11

V8 protease-digested fragments. The zinc metalloprotease HEXXH consensus motif is 12

underlined. The SD site is indicated by dotted line. The putative transcriptional 13

terminator sequence is indicated by arrows. 14

15

Fig. 3 Amino acid sequence comparison of Grimontia hollisae collagenase with 16

homologous collagenase. 17

(A) The amino acid sequences from G. hollisae (this study), Vibrio parahaemolyticus 18

(NP_797719) and Vibrio alginolyticus (CAA44501) were aligned using the CLUSTAL 19

W2 program. Identical residues among the three sequences are indicated by asterisks. 20

(B) Schematic representation of the domain architecture of G. hollisae (this study), V. 21

parahaemolyticus (NP_797719) and V. alginolyticus (CAA44501). Pre, signal 22


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peptide; pro, putative prodomain; PKD, polycystic kidney disease-like domain; PPC, 1

pre-peptidase C-terminal domain. 2

3

Fig. 4 Analysis of recombinant collagenase purified from Brevibacillus culture 4

media. 5

(A) Purified recombinant collagenase was analyzed by SDS-PAGE using a reducing 6

7.5% gel. Lane 1: molecular weight marker, lane 2: culture medium from 7

Brevibacillus (carrying pNY326-Col2), lane 3: purified collagenase. (B) Real-time 8

gelatin zymography using a non-reducing 10% gel. Lane 1: molecular weight marker, 9

Lane 2: SDS-PAGE of purified collagenase, lanes 3 and 4: gelatin zymogram of 10

purified collagenase after 2 h (lane 3) or 19 h (lanes 4) of incubation. (C) Inhibition 11

assay using real-time gelatin zymography. Lane 1: SDS-PAGE of purified collagenase, 12

lanes 2-6: gelatin zymogram of purified collagenase in the absence (lane 2) or presence 13

of inhibitors (lanes 3-6), lane 3: EDTA, lane 4: o-phenanthroline, lane 5: NEM, lane 6: 14

PMSF. 15


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Table 1. Kinetic constants of Grimontia hollisae collagenase

Table 1 Teramura et al.

Km

(mM)

8,889 ±±±± 924

5,556 ±±±± 962

Vmax

(mM/min)

3.18 ±±±± 0.27 ×××× 106

0.48 ±±±± 0.04 ×××× 106

Specific

constant

(Vmax

/Km

)

G. hollisae

Enzyme

C. hystolyticum

Specific

Activity

(U/mg)

5,314

1,289

33.2 ±±±± 1.11

1.35 ±±±± 0.12

13.8 ±±±± 0.81

0.57 ±±±± 0.07

G. hollisae

C. hystolyticum

11.8 ±±±± 1.95 ×××× 10-3

2.83 ±±±± 0.76 ×××× 10-3

2.40 ±±±± 0.52

2.41 ±±±± 0.19

FITC-

collagen

Substrate

FALGPA 7.40

0.39

* ** *

* *

The activities of G. hollisae and C. hystolyticum collagenase were determined using FITC labeled-

collagen or synthetic peptide substrate, FALGPA. Assays were carried out in 50 mM Tris-HCl,

0.3 M NaCl, 10 mM CaCl2, pH7.5 at 30°C for FITC labeled-collagen, or 50 mM Tricine, 0.4 M NaCl,

40 mM CaCl2, pH7.5 at 30°C for FALGPA. Each collagenase was used at the amount of 0.5 µg for

FITC labeled-collagen. When used for FALGPA, the amount of G. hollisae and C. hystolyticum

collagenase were 1.0 and 20 µg, respectively. The data represent the means ± SD of 3 separate

experiments. *P<0.01. **P<0.05.


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Fig.1 Teramura et al.

(bp)

F1

Sm

aI

1 23011497385

R1

Kp

nI

Ec

oR

V

Sp

hI

Sc

aI

Xh

oI

Sp

hI

Ec

oR

V


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7.

4.

3.

6.

2.

5.

1.

-12

89

189

289

389

489

589

689

789

889

989

1089

1189

1289

1389

1489

1589

1689

1789

1889

1989

2089

2189

2289

88

188

288

388

488

588

688

788

888

988

1088

1188

1288

1388

1488

1588

1688

1788

1888

1988

2088

2188

2288

2388


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Zn2+NH2 COOH

Pre Pro Catalytic domain PKD PPC

Zn2+NH2

COOH

Pre Pro Catalytic domain PPC

V. parahemolyticus

V. alginolyticus

G. hollisae

A

B


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11797

55

37

29

(kDa)

2 31 5 64

19

C

A

21 3

250

150

100

75

50

(kDa)

37

B

21 3

25

20

100

75

50

(kDa)

37

4


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Cloning of a novel collagenase gene from Gram -negative

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