An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells
© Wang et al; licensee BioMed Central Ltd. 2008
Received: 21 May 2008
Accepted: 24 November 2008
Published: 24 November 2008
Virus-binding activity is one of the important functions of fibronectin (FN). It has been reported that a high concentration of FN in blood improves the transmission frequency of hepatitis viruses. Therefore, to investigate a protease that hydrolyzes FN rapidly is useful to decrease the FN concentration in blood and HBV infection. So far, however, no specific protease digesting FN in serum has been reported.
We employed a purified earthworm protease to digest serum proteins. The rapidly cleaved protein (FN) was identified by MALDI-TOF MS and western blotting. The cleavage sites were determined by N-terminus amino acid residues sequencing. The protease was orally administrated to rats to investigate whether serum FN in vivo became decreased. The serum FN was determined by western blotting and ELISA. In cytological studies, the protease was added to the medium in the culture of HepG2.2.15 cells and then HBsAg and HBeAg were determined by ELISA.
The protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase). The cleavage sites on FN by the FNase were at R and K, exhibiting a trypsin alkaline serine-like function. The earthworm fibronectinase (EFNase) cleaved FN at four sites, R259, R1005, K1557 and R2039, among which the digested fragments at R259, K1557 and R2039 were related to the virus-binding activity as reported. The serum FN was significantly decreased when the earthworm fibronectinase was orally administrated to rats. The ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.
The earthworm fibronectinase (EFNase) cleaves FN much faster than the other proteins in serum, showing a potential to inhibit HBV infection through its suppressing the level of HBeAg. This suggests that EFNase is probably used as one of the candidates for the therapeutic agents to treat hepatitis virus infection.
Fibronectin (FN) is a glycoprotein composed of two nearly identical ~250 kDa subunits linked covalently near their C termini by a pair of disulfide bonds . Each subunit is a mosaic of a series of repeating modules: 12 Type I, 2 Type II, 15 to 17 (depending on splicing) Type III modules, and a variable (V) sequence that is not homologous to other parts of FN. The 2 Type III modules called extradomain A (EDA) and extradomain B (EDB) are subject to alternative splicing . On the basis of its solubility, FN is subdivided into two forms: soluble plasma FN and less-soluble cellular FN . Plasma FN that is synthesized in hepatocytes contains neither EDA nor EDB whereas cellular FN (synthesized locally in tissues) contains variable amounts of either or both EDA and EDB. In plasma FN dimers, only one of the subunits contains the V region, whereas almost all cellular FN subunits contain this region . Plasma FN is an abundant soluble constituent in plasma with a concentration about 300 μg/ml ; and cellular FN is an extracellular matrix that provides the architectural scaffolding in tissues .
FN is a multifunctional molecule involving many physiological and pathological processes such as infections with different viruses [4, 5], cellular adhesion, embryogenesis, tissue damage and repair [6, 7], atherosclerosis, myocardial infarction [2, 8] and tumor therapy . Studies have demonstrated that FN is involved in the virus infection, including rabies virus , human immunodeficiency virus (HIV) , cytomegalovirus , Rous sarcoma virus (RSV)  and infectious hematopoietic necrosis virus . Recently, the interaction of hepatitis B virus (HBV) with FN has been investigated by several laboratories and their results have clarified that FN is related to HBV infection [14–18].
HBV infection is a worldwide public health problem, especially in the eastern countries. Globally, more than 350 million people are infected by HBV and 10~25% of them die of either liver cirrhosis or hepatocellular carcinoma . Preventing the evolvement from transient HBV infection into chronic HBV, liver cirrhosis and hepatocellular carcinoma, human beings are encountering tremendous challenge. There are several therapeutic agents including interferon-α (IFN-α), peglyated IFN (PEG-IFN) and four oral nucleotide analogues (lamivudine, adefovir, entecavir and tonofovir) currently approved for the treatment of chronic HBV . But there are no proteases as drugs in the therapeutic agents.
Previous studies have shown that FN is involved in HBV infection, liver cirrhosis and hepatocellular carcinoma. Budkowska and colleagues  have found that FN of human liver sinusoids is able to bind to HBV via the pre-s2 domain. This in vivo interaction of HBV with liver FN may play a role in the initial steps of virus's entry into the target cells, subsequently mediating by specific hepatocyte receptors. FN expression increases markedly in sinusoidal endothelial cells, and the matrixes deposit by these cells stimulate the conversion of liver lipocytes to myofibroblasts in rat hepatic fibrosis model. Matsui et al.  reported that cellular FN participates in the hepatic fibrogenesis and the hepatocellular carcinoma. In 2006, Wang and colleagues  used an antisense oligonucleotide (ASODN), fibronectin antibody, and a small molecular compound to treat HepG2.2.15 cell line, targeting to FN. Their results showed that a decrease in the concentration of FN has an anti-HBV activity in HepG2.2.15 cells.
Earthworms have long been used as materials to make drug for antipyretic and diuretic purposes in Chinese traditional medicine. Furthermore, some of earthworm proteases have been prepared into enteric-coated capsules for clinical use in China for more than 10 years [21, 22]. Nakajima and co-workers  characterized six isozymes of serine proteases with fibrinolytic activity from the earthworm (E. fetida and L. rubellus). On the basis of their cleavage specificity against peptide substrates, some isozymes have both trypsin- and chymotrypsin-like activities, and some act in elastase-like function. Investigations of the enzymes are focused on cardiovascular system for a long time in this lab [24, 25]. We found that the proteases can activate not only prothrombin but also plasminogen. However, whether the earthworm E. fetida synthesizes a protease to degrade FN rapidly has been still unknown.
In this study, we show a novel protease from E. fetida, which cleaves FN much more quickly than the other serum proteins in vitro and in vivo. Sequentially, the cleavage sites on FN by EFNase and the effect on the production of HBV antigens are also exhibited.
Human serum was got from health volunteers. FN (from human plasma, the homogeneity is evaluated by immunoelectrophoresis) and fibronectin antibody (developed in rabbit affinity isolated) were from Sigma-Aldrich (USA). Peroxidase-conjugated IgG was from Beijing Zhongshan Golden Bridge Biotechnology Co., LTD (China). Lamivudine was the product from Beijing Nordhuns Chemical Technology Co., LTD (China) and its purity was higher than 99% measured by HPLC. Sprague-Dawley (SD) rats were obtained from Vitalriver (China). The animal experiments adhered to the principles of care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Peking University Health Science Center.
HepG2.2.15 cells were kindly provided by Dr Ding in the Beijing Institute of Radiation Medicine who got them from the Mount Sinai Medical Center in New York. Media for HepG2.2.15 cell culture consisted of Dulbecco's modified eagle medium (high glucose, Gibco, Invitrogen Corporation, USA) supplemented with 10% fetal bovine serum (PAA, Austria), antibiotic G-418 sulfate (final concentration 380 μg/ml, Amresco, USA) and penicillin-streptomycin solution (final concentrations 100 units/ml penicillin and 100 μg/ml streptomycin, HyClone, USA).
Purification of EFNase
EFNase was purified by affinity chromatography as described . Briefly, crude earthworm proteases were obtained from the ammonium sulfate precipitation of the soluble proteins of earthworm E. fetida. EFNase was purified on a 4-aminobenzamidine dihydrochloride-coupled Sepharose CL-6B affinity column (Pharmacia, USA) eluted with a gradient of denaturant from 0.1 to 1 M (AKTA prime plus, GE healthcare, Bio-science, USA). Eight amino acid residues of the N-terminus were sequenced (Applied Biosystem Automated Protein Sequencer, Applied Biosystem Inc., USA).
Human serum fibronectin digested by EFNase
Human serum (25 μl) was incubated with different concentrations of EFNase (0, 0.4, 0.8, 1.6, 3.2, 6.4 and 10 μM) at 37°C. After 30 min incubation, aliquots were taken and diluted (10 times) immediately. The reaction was terminated by addition of 2 × sample buffer and boiling for 10 min. Samples (18 μl each) were electrophoresed on 8% SDS-PAGE. The protein bands in the gel were visualized with Coomassie Brilliant Blue R-250. Then, under the same conditions, the serum degradation in the presence of EFNase (final concentration 6.4 μM) was observed at different time intervals. Serum in the absence of EFNase and in the presence of trypsin (final concentration 6.4 μM) was used as negative and positive controls, respectively.
Identification of 'fibronectin' in the serum using mass spectrometry
The quick-diminished protein band 'fibronectin' was digested by trypsin and subjected to MALDI-TOF MS . The protein band of interest was excised from the gel and cut into small pieces. Another piece from a protein-free region of the gel was used as a parallel control for the digestion to identify trypsin autoproteolysis products. 25 mM NH4HCO3 in 50% acetonitrile was added to wash the gel particles and the gel particles dried in a vacuum centrifuge. 10 mM dithiothreitol in 25 mM NH4HCO3 was added, enough to cover the gel pieces and reduce at 56°C for 1 hr. Cooled to room temperature, the dithiothreitol solution was replaced roughly by the same volume 55 mM iodoacetamide in 25 mM NH4HCO3. Samples were incubated at room temperature in the dark with occasional vortexing for 45 min. The gel pieces were washed with 25 mM NH4HCO3 for 10 min while vortexing. They were dehydrated with 25 mM NH4HCO3 in 50% acetonitrile and rehydrated with 25 mM NH4HCO3 and dehydrated again. The liquid phase was removed and the gel pieces dried in a vacuum centrifuge. The gel particles were rehydrated in 25 mM NH4HCO3 (pH 8) containing 0.05–0.1 mg/ml trypsin by vortexing for 5 min. After incubated at 37°C for 12–16 h, the peptides were recovered from the gel particles and used for MALDI-TOF MS analysis (AXIMA-CFRTMPlus, Shimadzu, Japan). The peptide fingerprint was processed in the NCBI database using the MASCOT search program.
Proof of the band 'fibronectin' by western blotting
Western blotting was performed to prove the character of the protein 'fibronectin' . Different concentrations of EFNase were incubated with human serum at 37°C for 2 h or EFNase (final concentration 6.4 μM) was incubated with human serum at 37°C for different time intervals. Aliquots were taken for 8% SDS-PAGE. The proteins in the gel were electrotransferred onto nitrocellulose membrane (Millipore, USA). The membrane was blocked in phosphate buffer solution (PBS) containing 5.0% nonfat milk and then incubated with primary fibronectin antibody at 37°C for 2 h. After rinsing with PBS plus 0.2% Tween 20 (PBST), the membrane was subsequently exposed to the second antibody peroxidase-conjuated IgG in the same solution at 37°C for 2 h. Immunoreactive proteins were visualized by 3,3'-diaminobenzidine (DAB) staining, which generated a brown-colored product.
Fibronectin concentration change after oral administration of EFNase
Six male rats (SD, weighing 200–250 g) were fasted overnight. Before oral administration of EFNase, blood samples were drawn from the ocular vein into tubes. Then EFNase was given by gavage. Blood samples were drawn at different time intervals (0.5, 1, 1.5, 2, 3, 4, 6, 8, and 26 h). Sera were prepared and stored at -20°C until determination. Western blotting of the sera was conducted as mentioned above to test the change of FN. The concentrations of FN in sera were determined by ELISA as described by Özkan et al [29–31] with some modification. Sera samples were diluted 1:50 and 50 μl aliquots were deposited on 96 well microtitration plates (Costar, USA). The plates were incubated at 37°C for 2 h followed by five washing with PBST buffer. Then the plates were blocked with 5% horse serum in PBS at 37°C for 2 h. The primary fibronectin antibody (diluted with PBS plus 5% horse serum at a ratio of 1:10,000) was added 50μl per well and the plates were incubated at 37°C for 2 h. The plates were then washed five times with PBST. Peroxidase-conjuated Ig G (diluted with PBS at a ratio 1:5000) was added 100 μl per well and the plates were incubated at 37°C for 2 h. After the plates were washed five times with PBST, substrate solution (3,3',5,5'-tetramethylbenzidine, TMB) 100 μl were dispensed per well with a multichannel pipet. The reaction was stopped by adding 50 μl/well 2.0 M sulfuric acid. After sufficient color development, absorbance values were determined with an ELISA reader at a wavelength of 450 nm immediately.
Enzymolysis sites on fibronectin by EFNase
To investigate enzymolysis characteristics of EFNase, FN from human plasma was used. First, FN (1 mg/ml, 10 μl) incubated with EFNase at different final concentrations (0, 0.01, 0.03, 0.1, 0.3, and 3 μM, respectively) at 37°C for 15 min to determine the suitable concentration of EFNase to cleave FN. Then the proper concentration of EFNase (final concentration 0.3 μM) was incubated with FN for 1, 5, 10, 30 and 60 min, respectively. The cleaved products were electrophoresed on 8% reduced and non-reduced SDS-PAGE and electrotransferred onto polyvinylidene difluoride membrane (PVDF, Millipore, USA). After staining with Commassie Brilliant Blue R-250, the main digested fragments were cut out for amino acid sequencing to identify the main cleavage sites.
Treatment of HepG2.2.15 cells by EFNase and lamivudine
HepG2.2.15 cells were seeded 1 × 104 cells per well in 96-well cell culture plates . EFNase and lamivudine (an anti-HBV drug which is widely used in the therapy of chronic hepatitis B) were added to the media 3 days after seeding. Cells were grown in the presence of EFNase or lamivudine for 6 days with changes of medium every 3 days. Six days after treatment by EFNase or lamivudine. The culture supernatants were collected and subjected to HBsAg and HBeAg analysis.
Assays of hepatitis B surface antigen and hepatitis B e antigen
Hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) in culture media were determined using diagnostic kits for HBsAg and for HBeAg as described in the manufacturer's manual (Sino-American Biotechnology Co., China). The inhibition rates were calculated according to the following formula: inhibition rate (%) = (Acontrol - Atest)/Acontrol × 100% .
Cell viability assay
A colorimetric assay using CCK-8 was to assess cell viability . After the cells in 96-well plates were treated with EFNase or lamivudine, the media were aspirated, and 10 μl of CCK-8 (Dojindo Labs, Japan) was added to each well and incubated at 37°C for 1 h. The absorbance at 450 nm that stands for cell growth was measured with the reference wavelength at 595 nm.
Degradation of fibronectin in the presence of earthworm fibronectinase
Cleavage sites on fibronectin
Cleavage sites on fibronectin by EFNase
No. cleavage site
from Q1 to R259
The main cleavage sites were at R and K, indicating a trypsin alkaline serine-like protease of EFNase. BSA was used as a control for the degradation, but the EFNase (0.3 μM) depicted a little hydrolysis on BSA (Additional file 4B). This suggests again that EFNase has a relatively high specificity to FN.
HBsAg and HBeAg assays after the cells treated by EFNase and lamivudine
Because fibronectinolysis may disturb the virus binding activity to FN, EFNase was used to treat HepG2.2.15 cells to test its potential function in HBV infection. HepG2.2.15 cells were the clonal cells derived from HepG2 cells, transfected with a plasmid containing HBV DNA. The cells secreted not only all HBV proteins but also hepatitis B virions, whilst they were remarkably down-regulated by lamivudine . As shown in Figure 8A and 8B, EFNase and lamivudine were observed to inhibit the secretion of HBsAg and HBeAg in HepG2.2.15 cell culture medium. The ELISA results indicated that the secretion of HBeAg was decreased by EFNase much more obvious than by lamivudine. However, the inhibition of HBsAg secretion in the presence of EFNase was less than lamivudine under the experimental conditions.
To assess the potential toxicity of EFNase and lamivudine to HepG2.2.15 cells, the viability of treated cells was examined. The results showed that cell viability had little change when the concentration of EFNase was lower than 34 nM under the experimental conditions (Figure 8C). Lamivudine had little impact on the cell growth when its concentration reached 100 μM (Figure 8D).
In this study, the earthworm FNase has been found to recognize the ~250 kDa protein from human serum and digest it rapidly. We think that this serum protein is FN because of the following results. (1) The molecular mass of the protein is ~250 kDa, approximately equal to FN (from 210 to 250 kDa). (2) The N-terminal amino acid sequence is identical to that of FN (QAQQMVQPQ). (3) The mass-spectrum shows a fingerprint of FN for the trypsin digested serum protein. Finally, (4) EFNase degrades the purified FN at the specific cleavage sites as mentioned above.
It is necessary to explain why we call the earthworm protease as EFNase. First, we have known that R and K are the main cleavage sites for the protease, exhibiting the trypsin alkaline serine-like activity. 0.01 μM of the protease digests FN within 15 min, showing a high hydrolytic activity to FN. Definitely, the protease is able to recognize FN from a great number of proteins contained in human serum. Under the same conditions, trypsin as a control did not rapidly degrade the serum FN. The earthworm protease did not quickly digest BSA and the other serum proteins. By in vivo experiments we have demonstrated that the relative concentration of the serum FN will decrease when the earthworm protease are orally administrated to rats. These results have given us an enlightenment to consider the protease as an FNase.
As shown in the previous studies, the earthworm E. fetida contains 8 trypsin-like proteases and their molecular masses are around 30 kDa . The apparent molecular mass of the earthworm FNase is ~34 kDa (Figure 1), similar to these proteases. The N-terminal sequence (8 amino acid residues) of EFNase showed identical to that of one isozymes of the earthworm protease (Ef P-III-2, a trypsin-like protease). However, the complete gene and the intact amino acid sequence of EFNase have not been clarified. Whether EFNase is Ef P-III-2 remains further investigated.
To study the cleavage site of EFNase on FN, we have employed plasma FN as the substrate. Although plasma FN is distinct from cellular FN in some functions [36, 37], most of the amino acid sequence is similar between plasma FN and cellular FN except EDA, EDB and V regions. Plasma FN is much more soluble than the tissue FN. Thus, it is convenient for us to analyze the degraded fragments of plasma FN in the presence of EFNase. From the results of plasma FN, we could predict some changes of cellular FN in the presence of the earthworm proteases. In fact, our unpublished data show that the concentration of cellular FN in the HBV transgenic mice livers is indeed decreased.
One of the important functions of FN is virus-binding activity. A high concentration of FN in blood improves the transmission frequency of hepatitis viruses . Several domains have been reported to participate in the viruses binding to FN (see Figure 7). Liu and co-workers  have shown that FN2 (with a sequence from N-terminus to Type III3) mediates IHNV attachment and cell entry. HIV-1IIIB specifically interacts with the III1-C region within FN and the interaction may play a role in facilitating HIV infection in vivo . The C-terminal 120–140 kDa fragment that carries the cell-binding activity bound influenza A (H3N2, Bangkok strain) virus most efficiently . A binding site for HBsAg has been found in heparin binding region II, and between the variable sequence (V) and C-terminus . This virus-binding ability may explain the high rate of transmission of virus infections by plasma derivates enriched in FN . In this study, we hypothesized that the cleavage of FN by EFNase may have the possibility to influence the binding activity of viruses to FN and virus infections. However, the FN fragments may elicit quite different function from the intact molecule . The cleavage products increase or decrease the binding effect needs further investigation.
For the cytological experiments, HBsAg and HBeAg secreted by HepG2.2.15 cells were both inhibited when the cells were treated with EFNase, especially the HBeAg decreases more obviously. HBeAg has the best correlation to the presence of infectious HBV. It is an important marker of the infection. Once HBeAg seroconversion takes place, there may be transition to the nonreplicative phase, characterized by normal serum liver enzymes and low HBV DNA levels and reduced hepatic necroinflammation . The significant clearance of HBeAg by EFNase may endow EFNase a potential product to treat HBV infection diseases.
The mechanism for EFNase to decrease HBeAg level in the HepG2.2.15 cell culture is due to two possibilities. (1) The cleavage of FN by EFNase may disturb the virus-binding activity and may inhibit the initial steps of the virus entry into target cells. (2) EFNase degrades HBeAg directly to decrease the level of the antigen in the medium. But, this possibility should not exist because HBsAg would be digested if the EFNase substrate specificity was so broad that all the proteins in the medium should be destroyed. In general, the mechanism for EFNase to decreases HBeAg remains further to be studied.
We have tried to find a drug with the same mechanism as a positive control to the inhibition of the antigens preduced by HepG2.2.15 cells. But no drug with such a characteristic was found. Lamivudine is selected because it is the first-line drug in treating chronic HBV infections on clinic . In research on HBV infections, lamivudine is commonly used as a positive control. Meanwhile, lamivudine, an oral antiviral agent, has the same administration route with EFNase.
We adopted an oral administration route of the protease to develop an oral administration dosage form. The FN concentration in serum decreased when EFNase was orally administrated to rats. As we know, earthworm proteases have been formulated into the oral enteric-soluble capsules which protect the enzymes from degradation in stomach. This formulation whose efficiency is certified has been successfully used on clinic for more than ten years [21, 22]. Furthermore, previous [42–44] and our present (Additional file 5) investigations on the absorption of the proteases indicate that the proteases could be partially absorbed from intestines to blood.
As mentioned above, the enzyme has a high activity to hydrolyze FN in serum. However, the protease could hydrolyze some other proteins under the experimental conditions (Figure 2). Furthermore, this protease is supposed to digest some other proteins on the HepG 2.2.15 cells or in the serum of rats although we didn't detect the degradation of the proteins.
An important cleavage site should be emphasized here is R1005/G1006, which is involved in the binding of collagen (Type I6–I9 and II1–II2). Cutting at this site may destroy the interaction between FN and collagen. In fact, some HBV infection evolves into liver cirrhosis and hepatocellular carcinoma. FN is important in the fibrogenesis. During the early phase of active fibroplasia, FN production increases dramatically, and this augmentation is associated with the fibroblast proliferation thereafter responsible for excessive synthesis and deposition of the collagen protein. FN associated with collagen through the specific binding sites, which promotes the formation of fibre. The hydrolysis of FN by EFNase not only decreases FN concentration directly, but also prevents the association of collagen to FN, which probably delay the formation of fibre.
The current therapeutic agents (including interferon and nucleoside analogues) used for the treatment of chronic HBV are mainly prevent the replicate of the viruses. But once the cirrhosis has occurred, it is difficult to be reversed . EFNase is supposed to be able to prevent both the processes, which has some advantage over interferon and nucleoside analogues. Thus we think that EFNase may have the potential to be developed into a new medicine to treat chronic HBV, liver cirrhosis and hepatocellular carcinoma patients.
In conclusion, a novel EFNase that cleaved FN much faster than the other proteins in mammal serum was purified from E. fetida. The results suggest that EFNase may have the potential to inhibit HBV infection through its suppressing the level of FN and HBeAg.
The authors want to gratefully acknowledge Ms ZS Xie and Mr P. Xue who did MALDI-TOF MS measurements and analyzed the results. This study was supported by 973 projects of China (2006CB500703, 2006CB911003) and the Knowledge Innovation Program of the Chinese Academy of Sciences (Grant NO. KSCX2-YW-R-119-1).
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