Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Mokhtarian K

PROVIDER: S-EPMC7461709 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Mokhtarian Kobra K Falak Reza R Heidari Zahra Z

Iranian journal of biotechnology 20200101 1

<h4>Background</h4>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.<h4>Objectives</h4>In this study, we evaluated proteolytic activity of F. hepatica recombinant cathepsin L1 (rCL1) against gelatin and collagen as common substrates.<h4>Material and methods</h4>The coding sequences of F. hepatica CL1 were cloned and expressed in E. coli, in our previous study. The rCL1 was pu ...[more]

PMID: 32884958

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Project description:BackgroundA family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2.Methodology/principal findingsUsing a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS), we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL). Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3.Conclusions/significanceThese data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage parasite proteinases.

Dataset Information

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

Publications

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

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