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.

Mutations in ARX gene should be considered in patients with mental disability or/and epilepsy. It is an X-linked gene that has pleiotropic effects. Here, we report the case of a boy diagnosed with Ohtahara syndrome. We performed the molecular analysis of the gene and identified a new missense mutation.

Secreted cysteine proteases are major players in host-parasite interactions; in Fasciola hepatica, a distinct group of cathepsins L was found to be predominantly expressed in the juvenile stages, but their enzymatic properties were unknown. Cathepsin L3 (FhCL3) is a main component of the juvenile secretory products and may participate in invasion. To characterize the biochemical properties, the proenzyme was expressed in the methylotrophic yeast Hansenula polymorpha and the mature enzyme was obtained from the culture medium. FhCL3 exhibited optimal activity and stability at neutral pH and a noticeable restricted substrate specificity with 70-fold preference for Tos-Gly-Pro-Arg-AMC over typical cathepsin substrates with hydrophobic or aliphatic residues in the S2 position. Accordingly, FhCL3 efficiently cleaved type I collagen over different pH and temperature conditions, but it did not cleave immunoglobulin. While most cathepsin cysteine proteinases are unable to digest collagen, mammalian cathepsin K, adult F. hepatica FhCL2 and the plant zingipain can also cleave collagen and substrates with Pro in P2 position, but only FhCL3 and zingipain hydrolyze these substrates with the highest efficiency. Molecular modeling and structural comparisons of the collagen cleaving cathepsins indicated that the strong substrate selectivity observed might be due to steric restrictions imposed by bulky aromatic residues at the S2-S3 subsites. The remarkable similarities of the active site clefts highlight the evolutive constrains acting on enzyme function. The presence of a collagen cleaving enzyme in F. hepatica juvenile stages is suggestive of a role in tissue invasion, an essential feature for the establishment of the parasites in their host.

Cysteine proteases are widespread in all life kingdoms, being central to diverse physiological processes based on a broad range of substrate specificity. Paralogous Fasciola hepatica cathepsin L proteases are essential to parasite invasion, tissue migration and reproduction. In spite of similarities in their overall sequence and structure, these enzymes often exhibit different substrate specificity. These preferences are principally determined by the amino acid composition of the active site's S2 subsite (pocket) of the enzyme that interacts with the substrate P2 residue (Schetcher and Berger nomenclature). Although secreted FhCL1 accommodates aliphatic residues in the S2 pocket, FhCL2 is also efficient in cleaving proline in that position. To understand these differences, we engineered the FhCL1 S2 subsite at three amino acid positions to render it identical to that present in FhCL2. The substitutions did not produce the expected increment in proline accommodation in P2. Rather, they decreased the enzyme's catalytic efficiency toward synthetic peptides. Nonetheless, a change in the P3 specificity was associated with the mutation of Leu67 to Tyr, a hinge residue between the S2 and S3 subsites that contributes to the accommodation of Gly in S3. Molecular dynamic simulations highlighted changes in the spatial distribution and secondary structure of the S2 and S3 pockets of the mutant FhCL1 enzymes. The reduced affinity and catalytic efficiency of the mutant enzymes may be due to a narrowing of the active site cleft that hinders the accommodation of substrates. Because the variations in the enzymatic activity measured could not be exclusively allocated to those residues lining the active site, other more external positions might modulate enzyme conformation, and, therefore, catalytic activity.