title: A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.
abstract: The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia.
authors.forename: P M
authors.initials: PM
authors.lastname: Smooker
authors.forename: J C
authors.initials: JC
authors.lastname: Whisstock
authors.forename: J A
authors.initials: JA
authors.lastname: Irving
authors.forename: S
authors.initials: S
authors.lastname: Siyaguna
authors.forename: T W
authors.initials: TW
authors.lastname: Spithill
authors.forename: R N
authors.initials: RN
authors.lastname: Pike
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002403:Cathepsins / Q000235:genetics* / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000378:metabolism; D005210:Fasciola hepatica / Q000201:enzymology*; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity / Q000235:genetics*
chemical_list: D015801:Helminth Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C426647:cathepsin L5