The efficient expression of human fibroblast collagenase in Escherichia coli and the discovery of flavonoid inhibitors

Abstract

Human skin fibroblast collagenase also known as Matrix Metalloproteinase-1 (MMP-1) is a key enzyme in remodeling and degradation of extracellular matrix, and the inhibitors of human MMP-1 are effective drug candidates for the treatment of cancer. In this study, we report an improved method for high-level expression of soluble human MMP-1 catalytic domain (cd-MMP-1) in E.coli. The enzymatic activity is found maximum at pH 7.5 and temperature 40°C with a K_m value of 13.02 µM. Effects of 17 structure-related flavonoids on MMP-1 activity are evaluated using a fluorescent assay, 6 inhibitors are identified with IC₅₀ < 10 µM. Fisetin is the most active agent with an IC₅₀ value of 1.35 µM and is identified as a mixed type inhibitor. Our improved soluble cd-MMP-1 expression method provides a basis for inhibitors identification and may be beneficial to discover novel anti-cancer agent targeting human MMP-1.

Keywords::

• MMP-1
• catalytic domain
• soluble expression
• flavonoids

Introduction

Matrix metalloproteinases (MMPs) are a large family of calcium-dependent, zinc-containing endopeptidases that are involved in the tissue remodeling and degradation of extracellular matrix (ECM) and play key roles in cell development, bone remodeling, wound healing, and angiogenesis^1–3. Matrix metalloproteinases inhibitors (MMPIs) have been identified as potential therapeutic candidates for cancer metastasis, arthritis, chronic inflammation and wrinkle formation^4,5. Therefore, developing of MMP inhibitors may provide a new treatment for these important diseases. Recently, a few inhibitors screened out from a large number of compounds for inhibition potency have been filed as investigational new drug candidates to treat disease. Rapid and cost-effective identification of such novel lead compound need the simple and easy-handling preparation of functional target protein. In general, heterologous expression in E.coli is chosen in priority to produce target recombinant MMPs, because the bacterial expression system is easy to engineer, practical and immediate to obtain target proteins for inhibitors screening⁶.

Human skin fibroblast collagenase also known as Matrix Metallo Proteinase-1 (MMP-1) is a member of the MMPs family. In humans, MMP-1 is considered as a prototype for all interstitial collagenases and plays an important role in the turnover of collagen fibrils in most tissues^7,8. Various forms of human MMP-1 have been cloned and expressed, such as in fibroblasts⁹, CHO cells^10,11, COS cell¹², Methylotrophic yeast¹³ and E.coli^14–16. However, the recombinant expression of MMP-1 in mammalian cells and yeast were low and the expression of MMP-1 in E.coli was always as inclusion bodies and should be refolded to obtain activity.

MMP-1 is an attractive therapeutic target for cancer invasion and metastasis. In order to fit for the demands for MMP-1 inhibitor screening and the further structural and biochemical characterization of enzyme-inhibitor complexes studies, we reported an improved method for high level expression of soluble human MMP-1 catalytic domain (cd-MMP-1) in E.coli. The purified cd-MMP-1 was highly active in digesting substrates and has similar activity as other MMP-1 reported previously¹³.

Flavonoids represent one of the most prevalent classes of natural products that are widely distributed in fruits, vegetables and beverages. Flavonoids have shown antitumor, anti-inflammation, and antioxidant effects¹⁷. Natural occurring flavonoids have been shown to suppress MMP-2 and MMP-9 expression and activity in cancer cells. However, there is little study focused on the human MMP-1. In the present study, 17 structure-related flavonoids are used to investigate the inhibitory activities on cd-MMP-1 activity in vitro. We find that six flavonols markedly inhibited cd-MMP-1 enzymatic activity with IC₅₀ less than 10 µM and we also investigate the inhibition mechanism of the most potent compound.

Material and methods

Reagent

All of flavonoids were obtained from Sigma-Aldrich, J&K or Aladdin reagent (Shanghai). The purity of the compounds was ≥95%. Restriction enzymes were from NEB (Beijing). pET-15b was purchased from novagen. Fluorogenic peptide substrate was from Anaspec (USA). MMP inhibitor II (Product number 444247) and MMP inhibitor III (product number 444264) were from Merck.

Cloning of the cd-MMP-1 into expression vectors

The cDNA encodes the proenzyme form of human fibroblast collagenase (proMMP-1) was kindly provided by Prof. Christopher M. Overall (Centre for Blood Research, University of British Columbia), the catalytic domain of human MMP-1 (Phe100-Gln268) (cd-MMP-1) was sub-cloned into vector pET-15b using NdeI/BamHI restrict enzyme sites. The primers used for PCR amplification were listed as follows: (sense) 5′-TCCGTTTCATATGTTTGTCCTCACTGAG-3′, (anti-sense) 5′-AATGGATCCT CACTGGACAGGATTTTGG-3′, and the nucleotide sequence of the insert was confirmed by DNA sequencing. For expression of the recombinant protein, pET15b-cd-MMP-1 was transformed into E.coli strain BL21(DE3).

Expression of cd-MMP-1 in E. coli

The 169-residue cd-MMP-1 was expressed in E.coliBL21(DE3) containing the plasmid described above. An overnight culture was started in Luria broth (LB) with 100 μM ampicillin from a signal colony. The culture was dilute to 200-fold using the same media and continually incubated at 37°C with shaking, until OD₆₀₀ was between 0.8 and 1.0. IPTG was added to a final concentration of 0.5 mM and the incubation was continued for 8 h. The cells were harvested by centrifugation at 4000 rpm for 20 min and frozen at −80°C overnight for next purification.

Purification of cd-MMP-1

The frozen cell pellet was suspended in 20 ml buffer A (50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl₂, 10 μM ZnCl₂, 1 mM DTT, 0.02% NaN3, pH 7.5), the cell suspension was lysed by sonication, and the disrupted cell lysate was centrifuged on 10,000 rpm for 30 min. The supernatant was load on a nickel-nitrilotriacetic acid (Ni-NTA) resin (Qiagen) column (1 mL) pre-equilibrated with buffer A. After 4 h, the column was washed with buffer A and buffer B (20 mM imidazole in Buffer A), the protein of interest was eluted with 10 mL elution buffer (50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl₂, 10 μM ZnCl₂, 0.02% NaN₃, 300 mM imidazole pH 7.5). The fractions containing the target protein were identified by SDS-PAGE. Imidazole was removed by dialysis against the corresponding buffer (50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl₂, 10 μM ZnCl₂, 0.02% NaN₃, pH 7.5). The proteins were concentrated with a 10-kDa cut-off membrane (Amicon) at 4°C and the concentration was measured by the standard Bradford method.

cd-MMP-1 activity assay

The activity of cd-MMP-1 was measured using a fluorescence-based assay. It was performed in white 96-well half area microplate (Greiner) in a final volume of 100 μL. The reaction buffer contained 7.6 nM cd-MMP-1 in assay buffer (50 mM Tris-HCl, 0.15 M NaCl, 10 mM CaCl₂, 10 µM ZnCl₂, pH 7.5). The reaction was initiated by the addition of 50 μl of quenched fluorogenic peptide substrate: Dnp-Pro-beta-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH2 (final concentration 5 μM). The cd-MMP-1 activity was determined in an Synergy^™ 2 Multi-Mode Microplate Reader(BioTek) at an excitation wavelength of 360 nm and an emission wavelength of 460 nm.

Effect of pH on cd-MMP-1 enzymatic activity

The pH optima of purified cd-MMP-1 were investigated using a pH range of 6.0–9.0. The buffer system used was a constant ionic strength containing 0.15 M NaCl, 10 mM CaCl₂ and 10 µM ZnCl₂, the protein concentration was 7.6 nM. The activities were determined as mentioned in cd-MMP-1 activity assay section.

Effect of temperature on cd-MMP-1 enzymatic activity

To investigate the effect of temperature on MMP-1 enzymatic activity, the assay contents were equilibrated for 20 min in Biotek Synergy^™ 2 at appropriate temperatures. The temperature range used was from 10 to 50°C. The reaction was started by addition of a substrate solution and fluorescence was read for 20 min.

Effect of zinc and calcium ions on cd-MMP-1 activity

Since zinc and calcium ions are important for cd-MMP-1 structure and activity, we also investigate the effects of above two ions on our cd-MMP-1 catalytic activity. The effects of zinc and calcium ion concentrations on cd-MMP-1 activity were evaluated using 4.7 nM cd-MMP-1 in buffer containing 50 mM HEPES buffer (pH 7.5). In the presence of 5 mM CaCl₂, the protein activity was determined with ZnCl₂ concentration ranging from 100 nM to 1 mM. In the presence of 1 µM ZnCl₂, the protein activity was determined with CaCl₂ concentration ranging from 50 µM to 25 mM.

Effect of flavonoids on cd-MMP-1 activity

Seventeen structure-related flavonoids were dissolved in DMSO as stock solution. The enzyme was pre-incubated with varied concentration of compounds for 20 min at room temperature and the activity was tested as described above. The MMP inhibitor II (Product number 444247) and MMP inhibitor III (product number 444264) from Merck were used as positive control to evaluate the inhibitors screening system.

Results

Protein expression and purification

The expression vector encoding human MMP-1 catalytic domain (residues Phe100-Gln268) was constructed using plasmid of pET-15b. The expression of 169-residue cd-MMP-1 construct was carried out using E.coli.BL21(DE3) as host system in LB media with 100 μM ampicillin and was induced by IPTG. After cell harvest, lysing and purification, the purified soluble protein was obtained and generated band of 21 kDa (shown as Figure 1). The yield from the purification was about 12–15 mg per half liter (protein concentration was determined using the bradford method with BSA as standard).

Figure 1.  SDS-PAGE analysis of cd-MMP-1 expression and purification procedures. Lane M, the standard protein mass markers; lane 1, the total cell lysate before induced; lane 2, the total cell lysate after induced; lane 3, pellet of cell lysate after induced; lane 4, supernatant of cell lysate after induced; lane 5, purified cd-MMP-1 protein.

For all recombinant MMPs reported so far, MMP-1 was always expressed in E.coli as insoluble inclusions body. The protein refolding is always complicated and time-consuming¹⁸. In present study, using soluble expression, we can get large amount of active cd-MMP-1 easily in a short time. The efficient preparation of active human cd-MMP-1 will facilitate the establishment of high-throughput screening assays for MMP-1 and further marking rapid and cost-effective identification of novel drug candidates for the treatment of diseases.

Characterization of cd-MMP-1

In order to verify the activity of our soluble cd-MMP-1, the data of the quenched peptide hydrolysis by cd-MMP-1 (3.8 nM) was measured at substrate concentration of 23.2, 11.6, 5.8, 2.32 µM at room temperature. The K_m value was calculated as 13.02 µM, V_max value was calculated as 28.72 ΔF/min/μg and V_max/K_m was 2.21 ΔF/min/μg/μM through the double-reciprocal form of the Michaelis–Menten equation, which plotting 1/v (reciprocal of rate) vs. 1/[s] (reciprocal of substrate concentration) (Figure 2).

Figure 2.  Measurement the K_m of human cd-MMP-1. Lineweaver-Burk plot analysis yielded the K_m of cd-MMP-1 for peptide substrate was 13.02 μM. The data points are means ± SE for triplicate experiments.

The pH optima of purified cd-MMP-1 were determined around 7.5 from the activities at various pH values (Figure 3A). Cd-MMP-1 revealed low activity at low pH values and the activity gradually increased with increasing pH. Enzyme activity reached a maximum value at pH 7.5 and thereafter a drastic decrease occurred at higher pH.

Figure 3.  Effect of pH, metal ion and temperature on cd-MMP-1 activity. (A) pH effect on the catalytic activity of cd-MMP-1. Activity of cd-MMP-1 was monitored in 50 mM Bis-Tris buffer (pH 6.0–7.0, squares), 50 mM HEPES buffer (pH 7.0–8.0, circles) or 50 mM Tris buffer (pH 7.5–9.0, triangles). The data points are means ± SE for triplicate experiments. (B) Temperature optimum profiles of purified cd-MMP-1. The data points are means ± SE for triplicate experiments. (C) Effect of zinc and calcium concentration on catalytic activities of cd-MMP-1. Hydrolysis of the fluorogenic peptide substrate was monitored in HEPES buffer (pH 7.5). Activities at varied ZnCl₂ concentrations in the presence of 5 mM CaCl₂ are shown as open circles and activities at varied CaCl₂ concentrations in the presence of 1 µM ZnCl₂ are shown as closed circles. The data points are means ± SE for triplicate experiments.

The temperature optimum profiles for purified cd-MMP-1 were shown in Figure 3B. Protein showed an gradual increase in activity from 10 to 20°C, then followed by a steep increase in activity from 20 to 40°C, and thereafter do not show any significant change in activity until 50°C. From the result, we can find that the optimum temperature of cd-MMP-1 is around 37°C, which is the body temperature of human being.

Since zinc and calcium ions are important in the structure and activity of cd-MMP-1 protein, we also investigated the effects of zinc and calcium ions on cd-MMP-1 activity (Figure 3C). In the present of 5 mM CaCl₂, the optimal activity of cd-MMP-1 was achieved around 1 µM ZnCl₂, while the activity of cd-MMP-1 decreased significantly if the concentration of ZnCl₂ went higher. In the present of 1 µM ZnCl₂, cd-MMP-1 shown the best activity at the concentration of CaCl₂ around 5 mM. Our data shown that cd-MMP-1 required low concentration of zinc ions and higher concentration of calcium ions to maintain active, zinc concentration higher than 1µM could inhibit the cd-MMP-1 activity.

Inhibition of cd-MMP-1 by flavonoids

We evaluate the screening system by using the positive compounds. The inhibition effect of the positive compounds MMP inhibitor II and MMP inhibitor III from Merck were determined with an IC₅₀ value of 13.05 nM and 10.20 nM, respectively which are consistent with the references^19,20. For optimal signal to noise ratio, we chose 10 nM cd-MMP-1 and 10 µM of the compound concentration for the initial screening assay. 17 structure-related flavonoids including flavones, flavonols, flavanones, isoflavones, and flavanonol were used to investigate the inhibitory effect on cd-MMP-1 enzyme activity (Table 1). These screening procedures leading to the identification of six compounds (luteolin, fisetin, kaempferol, morin, myricetin and quercetin) with an IC₅₀ value less than 10 µM. Among these six active compounds, fisetin, kaempferol, morin, myricetin and quercetin belong to flavonols and luteolin belong to flavones. Flavonoids are well-known antitumor, antioxidant, and anti-inflammation agents and previous structure-activity analysis has indicated that number and location of OH substitutions are important for the biological effects of flavonoids²¹. Obviously, the active flavonoids in this study contain four or more hydroxyl groups and hydroxylation at the 3′- and or the 4′-position of the B ring seemed of special importance. Among the five active flavonlos, the most potent two compounds fisetin and quercetin both have the 3′-position hydroxyl group. This substitution may beneficial for cd-MMP-1 inhibition activity of flavonoids. Some flavones tested in the present study showed low free inhibition activities against cd-MMP-1, such as apigenin and chrysin, however luteolin exhibited intense activity because of the 3′-position hydroxyl group. Flavanones, isoflavones and flavanonol selected in this study have no inhibition activities for cd-MMP-1. In this study, flavonoids glycoside (Quercitrin, Rutin, Naringin) did not inhibit the enzymatic activity of cd-MMP-1 because the glycoside may serve as negative moieties on MMP inhibition²².

Table 1.  Inhibitions activity flavonoids against human MMP-1 catalytic domain. The inhibition rate was determined at the concentration of 10 μM.

	Compounds	Inhibition rate (%)	IC50 (μM)
Flavones	Apigenin	6.55
	Chrysin	5.69
	Luteolin	65.34	3.03
Flavonols	Fisetin	99.41	1.35
	Kaempferol	70.46	3.92
	Morin	71.55	4.85
	Myricetin	78.37	2.92
	Quercetin	87.78	1.49
	Quercitrin	15.92
	Rutin,	22.18
Flavanones	Hesperetin	17.50
	(±)-Naringenin	9.77
	Naringin	6.69
Isoflavones	Daidzein	1.02
	Genistein	9.75
	Puerarin	8.39
Flavanonol	Taxifolin	0.15
	MMP inhibitor II	99.52	0.013
	MMP inhibitor III	98.36	0.010

As presented in Table 1, fisetin was the most potent inhibitor with an IC₅₀ = 1.35 µM. To further investigate the inhibition kinetics, the most potent inhibitor of cd-MMP-1, fisetin was using the Lineweaver-Burk method (Figure 4B). The concentration of quenched fluorogenic peptide substrate: Dnp-Pro-beta-cyclohexyl-Ala-Gly-Cys(Me)-His-Ala-Lys(Nma)-NH2 was from 2.5 to 20 μM. The velocities of reactions were measured without or with inhibitor (2 or 5 μM). The inhibition kinetics analyzed by Lineweaver-Burk plots indicates that fisetin is a mixed type inhibitor of cd-MMP-1. In mixed inhibition, the inhibitor binds to a site different from the active site where the substrate binds. Luteolin was reported as a non-competitive or mixed type inhibitor of MMP-9 and supposed binding MMP-9 outside the active site²³. Fisetin may also employ the similar interaction mode with cd-MMP-1 as luteolin. Further structural information (like crystallography) is needed to determine the accurate interaction mode for fisetin towards cd-MMP-1. In summery, our result could help to provide a basis for the design of novel anti-cancer agents targeting human MMP-1.

Figure 4.  Inhibition activity of fisetin against cd-MMP-1. (A) Effect of fisetin against cd-MMP-1, the data points are means ± SE for triplicate experiments. (B) Lineweaver-Burk plots for inhibitory activity of Fisetin (0, 2 and 5 μM) on cd-MMP-1 activity with the concentration of the substrate range from 2.5 μM to 20 μM. Each value was the mean of three independent experiments.

Conclusion

The efficient production of purified protein is important for biomedical applications such as small molecular drug discovery and enzyme-inhibitor complex structure determination. Large scale soluble expression is usually a bottleneck for the recombinant preparation of MMPs. Since the importance of MMP-1, human MMP-1 has been expressed use many methods^9–15, however soluble and active MMP-1 are still difficult to achieve. Herein, we develop an efficient method for the high level production of soluble and active MMP-1 in E.coli. Our experiment results indicated that cd-MMP-1 prepared following our procedure has similar activity as other MMP-1 reported previously¹³, and the yield is around 12–15 mg per half liter. The cd-MMP-1 protein can serve as a screening enzyme, because catalytic domain and full-length MMP-1 show similar activity. In general, the inhibitors of cd-MMP-1 may also inhibit the activity of full-length MMP-1 with the same potency and selectivity²⁴. 17 structure-related flavonoids were tested for their cd-MMP-1 inhibition activities and six compounds (luteolin, fisetin, kaempferol, morin, myricetin and quercetin) were active with an IC₅₀ value less than 10 µM. Fisetin, the most potent inhibitor, was identified as a mixed type inhibitor of cd-MMP-1. High level expression of soluble cd-MMP-1 has allowed for kinetic study and inhibitors identification and could help to discover novel anti-cancer agent targeting human MMP-1.

Acknowledgments

This work was supported by the 111 Project (Grant B07023), the National Natural Science Foundation of China (Grants 10979072, 81102420), the Innovation Program of Shanghai Municipal Education Commission (Grant 10ZZ41), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant 20090074120012).

Declaration of interest

The authors report no conflicts of interest.Reference