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


CONCLUSIONS
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.
Wang
Xue-Qing
XQ
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
Chen
Lan
L
Pan
Rong
R
Zhao
Jing
J
Liu
Ying
Y
He
Rong-Qiao
RQ
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D006514:Hepatitis B Surface Antigens / Q000276:immunology; D006513:Hepatitis B e Antigens / Q000276:immunology*; D006515:Hepatitis B virus / Q000276:immunology; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology*; D051381:Rats; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology*; D044967:Serum / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005353:Fibronectins; D006514:Hepatitis B Surface Antigens; D006513:Hepatitis B e Antigens; D019259:Lamivudine; D012697:Serine Endopeptidases; C071222:fibronectinase