Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica

Abstract

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.

Excretion/secretion products from Schistosoma mansoni adults, eggs and schistosomula have unique peptidase specificity profiles

“…Cleavage sites that were sensitive to AEBSF had a trypsin-like substrate specificity profile using the MSP-MS assay. Interestingly, the cleavage sites that were sensitive to E-64 also had a strong P1 preference for Arg and Gln and generally accepted hydrophobic residues at P2, consistent with that known for cysteine cathepsins [75–77]. …”

SecretedCandida parapsilosislipase modulates the immune response of primary human macrophages

“…Secretion of hydrolytic enzymes has been associated with virulence in pathogenic bacteria, parasites, 3 and fungi. 4 Secreted aspartyl proteinases have been extensively studied and investigations have demonstrated that they play a role in the virulence of both C. albicans 5 and C. parapsilosis.…”

Complementary Proteomic and Biochemical Analysis of Peptidases in Lobster Gastric Juice Uncovers the Functional Role of Individual Enzymes in Food Digestion

“…Fasciola cathepsin L3 is capable of degrading the proline-rich triple helical regions of collagen while cathepsin L1 cannot (Corvo et al 2013). We can therefore deduce that H. americanus cathepsin L2 is likely to be the enzyme responsible for the collagen-degrading activity in gastric juice (Chen 1991).…”