title: Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.
abstract: BACKGROUND
  The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL) correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3), liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2.
  
  
  RESULTS
  Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y) domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains). Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp)-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains.
  
  
  CONCLUSIONS
  These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.
authors.affiliation: Infection, Immunity and Innovation (i3) Institute, University of Technology Sydney, Sydney, New South Wales, Australia. mark.robinson@uts.edu.au
authors.forename: Mark W
authors.initials: MW
authors.lastname: Robinson
authors.forename: Ileana
authors.initials: I
authors.lastname: Corvo
authors.forename: Peter M
authors.initials: PM
authors.lastname: Jones
authors.forename: Anthony M
authors.initials: AM
authors.lastname: George
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Padula
authors.forename: Joyce
authors.initials: J
authors.lastname: To
authors.forename: Martin
authors.initials: M
authors.lastname: Cancela
authors.forename: Gabriel
authors.initials: G
authors.lastname: Rinaldi
authors.forename: Jose F
authors.initials: JF
authors.lastname: Tort
authors.forename: Leda
authors.initials: L
authors.lastname: Roche
authors.forename: John P
authors.initials: JP
authors.lastname: Dalton
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D056668:Cathepsin L / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism*; D005210:Fasciola hepatica / Q000201:enzymology* / Q000472:pathogenicity*; D015801:Helminth Proteins / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity; D014774:Virulence; D037521:Virulence Factors / Q000378:metabolism*
chemical_list: D015801:Helminth Proteins; D037521:Virulence Factors; D003094:Collagen; D056668:Cathepsin L