title: Cathepsin L1, the major protease involved in liver fluke (Fasciola hepatica) virulence: propetide cleavage sites and autoactivation of the zymogen secreted from gastrodermal cells.
abstract: The secretion and activation of the major cathepsin L1 cysteine protease involved in the virulence of the helminth pathogen Fasciola hepatica was investigated. Only the fully processed and active mature enzyme can be detected in medium in which adult F. hepatica are cultured. However, immunocytochemical studies revealed that the inactive procathepsin L1 is packaged in secretory vesicles of epithelial cells that line the parasite gut. These observations suggest that processing and activation of procathepsin L1 occurs following secretion from these cells into the acidic gut lumen. Expression of the 37-kDa procathepsin L1 in Pichia pastoris showed that an intermolecular processing event within a conserved GXNXFXD motif in the propeptide generates an active 30-kDa intermediate form. Further activation of the enzyme was initiated by decreasing the pH to 5.0 and involved the progressive processing of the 37 and 30-kDa forms to other intermediates and finally to a fully mature 24.5 kDa cathepsin L with an additional 1 or 2 amino acids. An active site mutant procathepsin L, constructed by replacing the Cys(26) with Gly(26), failed to autoprocess. However, [Gly(26)]procathepsin L was processed by exogenous wild-type cathepsin L to a mature enzyme plus 10 amino acids attached to the N terminus. This exogenous processing occurred without the formation of a 30-kDa intermediate form. The results indicate that activation of procathepsin L1 by removal of the propeptide can occur by different pathways, and that this takes place within the parasite gut where the protease functions in food digestion and from where it is liberated as an active enzyme for additional extracorporeal roles.
authors.affiliation: School of Biotechnology, Dublin City University, Dublin 9, Republic of Ireland.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Collins
authors.forename: Colin M
authors.initials: CM
authors.lastname: Stack
authors.forename: Sandra M
authors.initials: SM
authors.lastname: O'Neill
authors.forename: Sean
authors.initials: S
authors.lastname: Doyle
authors.forename: Thecla
authors.initials: T
authors.lastname: Ryan
authors.forename: Gerard P
authors.initials: GP
authors.lastname: Brennan
authors.forename: Angela
authors.initials: A
authors.lastname: Mousley
authors.forename: Michael
authors.initials: M
authors.lastname: Stewart
authors.forename: Aaron G
authors.initials: AG
authors.lastname: Maule
authors.forename: John P
authors.initials: JP
authors.lastname: Dalton
authors.forename: Sheila
authors.initials: S
authors.lastname: Donnelly
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002417:Cattle; D003545:Cysteine / Q000737:chemistry; D003546:Cysteine Endopeptidases; D003553:Cystine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004063:Digestion; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000737:chemistry; D004847:Epithelial Cells / Q000473:pathology; D005210:Fasciola hepatica / Q000472:pathogenicity*; D005753:Gastric Mucosa / Q000378:metabolism; D005822:Genetic Vectors; D005998:Glycine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
chemical_list: D018076:DNA, Complementary; D004792:Enzyme Precursors; D010455:Peptides; D011994:Recombinant Proteins; D003553:Cystine; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C063905:procathepsin L; C534281:CTSL protein, human; D056668:Cathepsin L; D003545:Cysteine; D005998:Glycine