TY - JOUR
T1 - Dissecting the Active Site of the Collagenolytic Cathepsin L3 Protease of the Invasive Stage of Fasciola hepatica
AU - Corvo, Ileana
AU - O'Donoghue, Anthony J.
AU - Pastro, Lucía
AU - Pi-Denis, Natalia
AU - Eroy-Reveles, Alegra
AU - Roche, Leda
AU - McKerrow, James H.
AU - Dalton, John P.
AU - Craik, Charles S.
AU - Caffrey, Conor R.
AU - Tort, José F.
PY - 2013/7
Y1 - 2013/7
N2 - Background: A 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 Findings: Using 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/Significance: These 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.
AB - Background: A 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 Findings: Using 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/Significance: These 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.
UR - http://www.scopus.com/inward/record.url?scp=84880821250&partnerID=8YFLogxK
U2 - 10.1371/journal.pntd.0002269
DO - 10.1371/journal.pntd.0002269
M3 - Article
AN - SCOPUS:84880821250
SN - 1935-2727
VL - 7
JO - PLoS Neglected Tropical Diseases
JF - PLoS Neglected Tropical Diseases
IS - 7
M1 - e2269
ER -