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An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells.

Wang XQ, Chen L, Pan R, Zhao J, Liu Y, He RQ - BMC Biochem. (2008)

Bottom Line: The protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase).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 ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Lab of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, 15 Da Tun Road, Chao Yang District, Beijing 100101, PR China. wangxq@bjmu.edu.cn

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

Digestion of human serum in the presence of EFNase. EFNase (final concentrations as indicated) was incubated with human serum (25 μl) at different time intervals during the degradation and then aliquots were taken for SDS-PAGE (panel A). Under the same conditions, serum in the absence of EFNase was used as negative control (panel B). Panel C was the comparison of the hydrolytic activity of EFNase and trypsin by densitomitric scanning analysis of Figure 2A and Additional file 1B.
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Figure 2: Digestion of human serum in the presence of EFNase. EFNase (final concentrations as indicated) was incubated with human serum (25 μl) at different time intervals during the degradation and then aliquots were taken for SDS-PAGE (panel A). Under the same conditions, serum in the absence of EFNase was used as negative control (panel B). Panel C was the comparison of the hydrolytic activity of EFNase and trypsin by densitomitric scanning analysis of Figure 2A and Additional file 1B.

Mentions: SDS-PAGE of the human serum treated by different protease concentration was showed in Additional file 1A. Observably, a band of ~250 kDa protein in serum was rapidly degraded (at 37°C in 15 min) in the presence of the protease (final concentration 6.4 μM) (Figure 2A). Serum in the absence of the protease was used as a negative control, showing little changes in the protein bands in serum (Figure 2B). Trypsin (final concentration 6.4 μM) used as a positive control (Additional file 1B) depicted a relatively low hydrolytic activity on the ~250 kDa protein by the densitomitric scanning analysis (Figure 2C). This suggests that the earthworm protease has a significant high enzymic activity on the ~250 kDa protein.


An earthworm protease cleaving serum fibronectin and decreasing HBeAg in HepG2.2.15 cells.

Wang XQ, Chen L, Pan R, Zhao J, Liu Y, He RQ - BMC Biochem. (2008)

Digestion of human serum in the presence of EFNase. EFNase (final concentrations as indicated) was incubated with human serum (25 μl) at different time intervals during the degradation and then aliquots were taken for SDS-PAGE (panel A). Under the same conditions, serum in the absence of EFNase was used as negative control (panel B). Panel C was the comparison of the hydrolytic activity of EFNase and trypsin by densitomitric scanning analysis of Figure 2A and Additional file 1B.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2611985&req=5

Figure 2: Digestion of human serum in the presence of EFNase. EFNase (final concentrations as indicated) was incubated with human serum (25 μl) at different time intervals during the degradation and then aliquots were taken for SDS-PAGE (panel A). Under the same conditions, serum in the absence of EFNase was used as negative control (panel B). Panel C was the comparison of the hydrolytic activity of EFNase and trypsin by densitomitric scanning analysis of Figure 2A and Additional file 1B.
Mentions: SDS-PAGE of the human serum treated by different protease concentration was showed in Additional file 1A. Observably, a band of ~250 kDa protein in serum was rapidly degraded (at 37°C in 15 min) in the presence of the protease (final concentration 6.4 μM) (Figure 2A). Serum in the absence of the protease was used as a negative control, showing little changes in the protein bands in serum (Figure 2B). Trypsin (final concentration 6.4 μM) used as a positive control (Additional file 1B) depicted a relatively low hydrolytic activity on the ~250 kDa protein by the densitomitric scanning analysis (Figure 2C). This suggests that the earthworm protease has a significant high enzymic activity on the ~250 kDa protein.

Bottom Line: The protease purified from earthworm Eisenia fetida was found to function as a fibronectinase (FNase).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 ELISA results showed that the secretion of HBeAg from HepG2.2.15 cells was significantly inhibited in the presence of the FNase.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Lab of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, 15 Da Tun Road, Chao Yang District, Beijing 100101, PR China. wangxq@bjmu.edu.cn

ABSTRACT

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus