Evaluation of Gelatinolytic and Collagenolytic Activity of Fasciola hepatica Recombinant Cathepsin-L1.

BACKGROUND

Cysteine proteases of the liver fluke, Fasciola hepatica, participate in catabolism of proteins, migration of the fluke through host tissues and combat host immune system.

OBJECTIVES

In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substrates.

MATERIAL AND METHODS

The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was purified by nickel affinity chromatography with a HisTrap Column. The protein concentrations of the purified fractions were determined by Bradford assay. Rat collagen type-1 was treated with distinct amounts of rCL1 at 37 °C, overnight, and the byproduct was analyzed by SDS-PAGE. Furthermore, we used bovine skin gelatin as zymography substrate to evaluate the gelatinolytic activity of the purified rCL1.

RESULTS

Recombinant CL1 was capable to digest intact type-1 collagen within 24 h and the gelatinlytic activity of rCL1 was visible at approximately 37 kDa region, with optimal activity at acidified conditions (pH 4).

CONCLUSION

Findings provide a possible mechanism by which a major secretory molecule of F. hepatica could be involved in parasite survival as well as its pathogenesis.