Characterization of cysteine proteases from the carcinogenic liver fluke, Opisthorchis viverrini.
Protease activities in extracts of Opisthorchis viverrini were investigated using gelatin zymography and fluorogenic peptide substrates. Using gelatin-impregnated X-ray film, 2 microg of O. viverrini excretory-secretory products (Ov-ES) and adult somatic extract (Ov-SE) showed proteolytic activity. Zymography of both O. viverrini extracts revealed bands at approximately 30 kDa. Using fluorogenic peptide substrates, the majority of O. viverrini activity was determined to be cathepsin L-like cysteine protease (cleaved Z-Phe-Arg-aminomethylcoumarin (AMC)) whereas little or no activity was ascribable to other classes of proteases. The O. viverrini cysteine protease activity was greatest at pH 6.0 and the activity was inhibited by the class-specific inhibitors, E-64 and Z-Ala-CHN2. Chromatographic purification of O. viverrini cysteine proteases on thiol-sepharose enriched for protein(s) of approximately 30 kDa from Ov-ES and Ov-SE. The activity profile of the purified enzyme was similar to that of the cathepsin L-like activity characterized in Ov-SE and Ov-ES. Furthermore, determination of cysteine protease activity in several developmental stages of the parasite revealed the highest protease activity in metacercariae soluble extract, followed by Ov-ES, egg soluble extract, and Ov-SE. These findings demonstrated that O. viverrini has a cathepsin L-like cysteine protease(s) and suggested that abundant cysteine protease activity was present in metacercariae where the hydrolase might be involved in cyst excystation during mammalian infection.
Kaewpitoon
Natthawut
N
Graduate School, Khon Kaen University, Khon Kaen, Thailand.
Laha
Thewarach
T
Kaewkes
Sasithorn
S
Yongvanit
Puangrat
P
Brindley
Paul J
PJ
Loukas
Alex
A
Sripa
Banchob
B
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D005780:Gelatin / Q000378:metabolism; D015801:Helminth Proteins / Q000378:metabolism; D009891:Opisthorchis / Q000201:enzymology* / Q000254:growth & development
D015801:Helminth Proteins; D005780:Gelatin; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L