A major cathepsin B protease from the liver fluke Fasciola hepatica has atypical active site features and a potential role in the digestive tract of newly excysted juvenile parasites.
The newly excysted juvenile (NEJ) stage of the Fasciola hepatica lifecycle occurs just prior to invasion into the wall of the gut of the host, rendering it an important target for drug development. The cathepsin B enzymes from NEJ flukes have recently been demonstrated to be crucial to invasion and migration by the parasite. Here we characterize one of the cathepsin B enzymes (recombinant FhcatB1) from NEJ flukes. FhcatB1 has biochemical properties distinct from mammalian cathepsin B enzymes, with an atypical preference for Ile over Leu or Arg residues at the P(2) substrate position and an inability to act as an exopeptidase. FhcatB1 was active across a broad pH range (optimal activity at pH 5.5-7.0) and resistant to inhibition by cystatin family inhibitors from sheep and humans, suggesting that this enzyme would be able to function in extracellular environments in its mammalian hosts. It appears, however, that the FhcatB1 protease functions largely as a digestive enzyme in the gut of the parasite, due to the localization of a specific, fluorescently labeled inhibitor with an Ile at the P(2) position. Molecular modelling and dynamics were used to predict the basis for the unusual substrate specificity: a P(2) Ile residue positions the substrate optimally for interaction with catalytic residues of the enzyme, and the enzyme lacks an occluding loop His residue crucial for exopeptidase activity. The unique features of the enzyme, particularly with regard to its specificity and likely importance to a vital stage of the parasite's life cycle, make it an excellent target for therapeutic inhibitors or vaccination.
Beckham
Simone A
SA
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Piedrafita
David
D
Phillips
Carolyn I
CI
Samarawickrema
Nirma
N
Law
Ruby H P
RH
Smooker
Peter M
PM
Quinsey
Noelene S
NS
Irving
James A
JA
Greenwood
Deanne
D
Verhelst
Steven H L
SH
Bogyo
Matthew
M
Turk
Boris
B
Coetzer
Theresa H
TH
Wijeyewickrema
Lakshmi C
LC
Spithill
Terry W
TW
Pike
Robert N
RN
D000818:Animals; D020134:Catalytic Domain; D002401:Cathepsin B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D002403:Cathepsins / Q000037:antagonists & inhibitors; D015891:Cystatins / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005210:Fasciola hepatica / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development*; D041981:Gastrointestinal Tract / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008018:Life Cycle Stages / Q000187:drug effects; D015335:Molecular Probes / Q000737:chemistry; D010271:Parasites / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development*; D021381:Protein Transport / Q000187:drug effects; D012756:Sheep; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000187:drug effects
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D015335:Molecular Probes; D002403:Cathepsins; C426647:cathepsin L5; D002401:Cathepsin B