Research article

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

Xue-Qing Wang^1,2,3 , Lan Chen¹ , Rong Pan¹ , Jing Zhao¹ , Ying Liu¹ and Rong-Qiao He^1,3

¹State Key Lab of Brain and Cognitive Sciences, Institute of Biophysics, the Chinese Academy of Sciences, 15 Da Tun Road, Chao Yang District, Beijing 100101, PR China

²School of Pharmaceutical Sciences, Peking University, 38 Xue Yuan Road, Hai Dian District, Beijing 100191, PR China

³Graduate University of Chinese Academy of Science, 19 A Yuquan Road, Shijingshan District, Beijing 100049, PR China

author email corresponding author email

BMC Biochemistry 2008, 9:30doi:10.1186/1471-2091-9-30

Published:	24 November 2008

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.