Secreted cysteine proteases of the carcinogenic liver fluke, Opisthorchis viverrini: regulation of cathepsin F activation by autocatalysis and trans-processing by cathepsin B

Abstract

SummaryOpisthorchis viverrini is an important helminth pathogen of humans that is endemic in Thailand and Laos. Adult flukes reside within host bile ducts and feed on epithelial tissue and blood cells. Chronic opisthorchiasis is associated with severe hepatobiliary diseases such as cholangiocarcinoma. Here we report that adult O. viverrini secrete two major cysteine proteases: cathepsin F (Ov-CF-1) and cathepsin B1 (Ov-CB-1). Ov-CF-1 is secreted as an inactive zymogen that autocatalytically processes and activates to a mature enzyme at pH 4.5 via an intermolecular cleavage at the prosegment-mature domain junction. Ov-CB-1 is also secreted as a zymogen but, in contrast to Ov-CF-1, is fully active against peptide and macromolecular substrates despite retaining the N-terminal prosegment. The active Ov-CB-1 zymogen was capable of trans-activating Ov-CF-1 by proteolytic removal of its prosegment at pH 5.5, a pH at which the Ov-CF-1 zymogen cannot autocatalytically activate. Both cathepsins hydrolyse human haemoglobin but their combined action more efficiently degrades haemoglobin to smaller peptides than each enzyme alone. Ov-CF-1 degraded extracellular matrix proteins more effectively than Ov-CB-1 at physiological pH. We propose that Ov-CB-1 regulates Ov-CF-1 activity and that both enzymes work together to degrade host tissue contributing to the development of liver fluke-associated cholangiocarcinoma.

Evaluation of liver fluke recombinant cathepsin B-1 protease as a serodiagnostic antigen for human opisthorchiasis

“…Affinity purified r Ov -CB-1 migrated in SDS-PAGE as a single species at ~44 kDa, which is the active form of the enzyme [14] (Figure 1, lane 3). Immunoblot analysis showed that r Ov -CB-1 was recognized by O. viverrini positive human sera, revealing a major band of recognition at 44 kDa, whereas control, non- O. viverrini infected sera showed no reactivity (Figure 1).…”

“…Recombinant O. viverrini cathepsin B1 (r Ov -CB-1) (GenBank accession number ACT99885) was produced in Pichia pastoris as described [14]. Recombinant proteins were purified using Ni-NTA affinity columns (Novagen) and dialyzed against phosphate buffered saline (PBS) at pH 7.2–7.4 through dialysis membrane (SnakeSkin TM Pleated Dialysis Tubing, Pierce) for 4 h at 4°C and stored at −20°C until used.…”

“…Protein concentration was determined by the method of Bradford [15]. The recombinant enzyme was catalytically active as described by us previously [14]. …”

Papain-like peptidases: structure, function, and evolution

“…There have been several reports relating cathepsin F to the molecular mechanisms of host invasion by parasitic worms such as Clonorchis sinensis (156) , Teladorsagia circumcincta (157) , Opisthorchis viverrini (158) , and Manduca secta (159) . Indeed, in Asian flukes from the genera Clonorchis , Paragonimus , and Opsthorchis , cathepsin F-like peptidases are the predominantly expressed cysteine peptidases (140) .…”

Structural Basis for Inhibition of Cathepsin B Drug Target from the Human Blood Fluke, Schistosoma mansoni

“…Thus, oligo/polypeptide fragments do not accumulate to the extent observed for hemoglobin digestion by helminth cathepsin L-type endopeptidases (65)(66)(67). With regard to hemoglobinolytic capability, SmCB1 resembles cathepsins B from the Southeast Asian liver fluke Opisthorchis viverrini and the hookworm Ancylostoma caninum (66,68) but differs from cathepsins B of the avian fluke Trichobilharzia regenti and the hookworm Necator americanus that cannot initiate hemoglobinolysis (69,70). We conclude that SmCB1 operates as an effective proteolytic machine to degrade the major protein in the parasite's blood meal.…”