Structural and functional relationships in the virulence-associated cathepsin L proteases of the parasitic liver fluke, Fasciola hepatica

Abstract

The helminth parasite Fasciola hepatica secretes cysteine proteases to facilitate tissue invasion, migration, and development within the mammalian host. The major proteases cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) were recombinantly produced and biochemically characterized. By using site-directed mutagenesis, we show that residues at position 67 and 205, which lie within the S2 pocket of the active site, are critical in determining the substrate and inhibitor specificity. FheCL1 exhibits a broader specificity and a higher substrate turnover rate compared with FheCL2. However, FheCL2 can efficiently cleave substrates with a Pro in the P2 position and degrade collagen within the triple helices at physiological pH, an activity that among cysteine proteases has only been reported for human cathepsin K. The 1.4-A three-dimensional structure of the FheCL1 was determined by x-ray crystallography, and the three-dimensional structure of FheCL2 was constructed via homology-based modeling. Analysis and comparison of these structures and our biochemical data with those of human cathepsins L and K provided an interpretation of the substrate-recognition mechanisms of these major parasite proteases. Furthermore, our studies suggest that a configuration involving residue 67 and the "gatekeeper" residues 157 and 158 situated at the entrance of the active site pocket create a topology that endows FheCL2 with its unusual collagenolytic activity. The emergence of a specialized collagenolytic function in Fasciola likely contributes to the success of this tissue-invasive parasite.

FIGURE 1.

Sequence alignment of the matureF. hepatica cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) with human cathepsin L (hCatL), human cathepsin K (hCatK), and papain was performed with ClustalW (EBI, EMBL). Residues within the S2 subsite of the active site involved in determining substrate specificity are indicated with arrows and numbers (see also Table 2).

FIGURE 2.

Activation of purified recombinant to FheproCL1 and FheproCL2. The 37-kDa zymogens were autocatalytically activated and processed to 24.5-kDa mature enzymes by incubation for 2 h at 37 °C in 0.1 m sodium citrate buffer, pH 5.0, containing 2 mm DTT and 2.5 mm EDTA. Reaction samples were analyzed by 4–20% SDS-PAGE; lanes 1–4, activation reaction of FheCL1 at 0, 30, 60, and 120 min; lanes 5– 8, activation reaction of FheCL2 at 0, 30, 60, and 120 min. Similar results were obtained with variant peptidases (not shown). MW, molecular mass markers.

FIGURE 3.

Profiling of the P1–P4 substrate specificity of FheCL1 and FheCL2 using positional scanning synthetic combinatorial libraries. The y axis represents activity against the substrates relative to the highest activity of the library, whereas the x axis presents the amino acids as represented by the one-letter code (n = norleucine).

FIGURE 4.

Comparison of the P2 specificities of recombinant wild type FheCL1, variant FheCL1 L205A (L205A), variant FheCL1 L67Y (L67Y), and wild type FheCL2 using positional scanning synthetic combinatorial libraries.

FIGURE 5.

Comparison of the collagen cleaving activities of wild type FheCL1, variant FheCL1 L205A, variant FheCL1 L67Y, and wild type FheCL2. A, type I collagen was incubated with FheCL1 and FheCL2 at pH 4.0 and 5.5 and at 28 °C for 3 h, and the reaction was analyzed by 4–20% SDS-PAGE: lane 1, collagen alone; lane 2 collagen plus FheCL1 pH 4.0; lane 3, collagen plus FheCL2 pH 4.0; lane 4, collagen alone; lane 5, collagen plus FheCL1, pH 5.5; lane 6, collagen plus FheCL2, pH 5.5. B, type I collagen was incubated with active recombinant peptidase (5.47 μm) at 28 °C at neutral pH (PBS, pH 7.3) for 20 h, and the reactions analyzed as above: lane 1, collagen alone (i.e. no peptidase added); lane 2, collagen plus FheCL1; lane 3, collagen plus FheCL1 L209A; lane 4, collagen plus FheCL1 L67Y; lane 5, collagen plus FheCL2. Molecular mass standards are indicated on the right, and collagen chains (α1, α2, β 11, β 12, and γ, see Ref. 44) are indicated on the left.

FIGURE 6.

A, bi-lobed mature FheproCL1 Gly²⁵ is shown as a schematic. The predominantly helical domain is at left, and the predominantly sheet domain is at right. The mutated active site residue Gly²⁵ lies in the cleft between the two domains and is indicated in red. B, structure of full-length FheproCL1 Gly²⁵ zymogen is shown, with the mature segment surface illustrated in blue and the prosegment as a schematic. The extended C-terminal portion of the prosegment runs through the active site cleft. The catalytic machinery of the enzyme is highlighted in pink. Figures were created with PyMol (42).

FIGURE 7.

A, surface representation of the active site region of FheproCL1 Gly²⁵. The S2 pocket of the enzyme is highlighted in pink, and key residues implicated in substrate preference, including the gatekeeper residues (Val¹⁵⁷ and Asn¹⁵⁸), are noted. B, representation of the active site region of FheproCL1 Gly²⁵ and FheCL2. The S2 pocket of both enzymes are highlighted. Gatekeeper residues of FheproCL1 Gly²⁵ are further indicated in pink stick representation and those of the modeled FheCL2 in yellow. Figures created with PyMol (42).

FIGURE 8.

Comparison of the substrate specificity of human cathepsin L (CL), human cathepsin K (CK), recombinant FheCL1, FheCL1 L205A (L205A), FheCL1 L67Y (L67Y), and FheCL2. Data shown as relative k_cat/K_m for the hydrolysis of the substrates Z-Phe-Arg-NHMec, Z-Phe-Leu-NHMec, and Tos-Gly-Pro-ArgNHMec. Asterisk indicates data for human CL and CK are taken from Lecaille et al. (44).