Production and processing of a recombinant Fasciola hepatica cathepsin B-like enzyme (FhcatB1) reveals potential processing mechanisms in the parasite.
The liver fluke, Fasciola hepatica, apparently uses a number of cysteine proteases during its life cycle, most likely for feeding, immune evasion and invasion of tissues. A cathepsin B-like enzyme (herein referred to as FhcatB1) appears to be a major enzyme secreted by the invasive, newly excysted juvenile flukes of this parasite. To examine the processing mechanisms for this enzyme, a recombinant form was expressed in Pichia pastoris and purified to yield a homogenous pool of the enzyme. The purified enzyme could be autoactivated at low pH via a bi-molecular mechanism, a process that was greatly accelerated by the presence of large, negatively charged molecules such as dextran sulfate. The enzyme could also apparently be processed to the correct size by an asparaginyl endopeptidase via cleavage in an unusual insertion N-terminal to the normal cleavage site used to yield the active form of the enzyme. Thus, there appear to be a number of ways in which this enzyme can be processed to its optimally active form prior to secretion by F. hepatica.
Beckham
Simone A
SA
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Law
Ruby H-P
RH
Smooker
Peter M
PM
Quinsey
Noelene S
NS
Caffrey
Conor R
CR
McKerrow
James H
JH
Pike
Robert N
RN
Spithill
Terry W
TW
D000818:Animals; D001665:Binding Sites; D002401:Cathepsin B / Q000187:drug effects / Q000378:metabolism*; D018360:Crystallography, X-Ray; D016264:Dextran Sulfate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005210:Fasciola hepatica / Q000201:enzymology* / Q000469:parasitology*; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013329:Structure-Activity Relationship; D013997:Time Factors
D011994:Recombinant Proteins; D006493:Heparin; D016264:Dextran Sulfate; D002401:Cathepsin B