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A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica

Published online by Cambridge University Press:  10 February 2001

PETER M. SMOOKER
Affiliation:
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
JAMES C. WHISSTOCK
Affiliation:
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
JAMES A. IRVING
Affiliation:
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
SUGEESHWARA SIYAGUNA
Affiliation:
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
TERRY W. SPITHILL
Affiliation:
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
ROBERT N. PIKE
Affiliation:
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia
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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.

Type
FOR THE RECORD
Copyright
© 2000 The Protein Society

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