Cloning and expression of the major secreted cathepsin B-like protein from juvenile Fasciola hepatica and analysis of immunogenicity following liver fluke infection.
The functions of the cathepsin B-like proteases in liver flukes are unknown and analysis has been hindered by a lack of protein for study, since the protein is produced in small amounts by juvenile flukes. To circumvent this, we isolated and characterized a cDNA encoding the major secreted cathepsin B from Fasciola hepatica. The predicted preproprotein is 339 amino acids in length, with the mature protease predicted to be 254 amino acids long, and shows significant similarity to parasite and mammalian cathepsin B. Only one of the two conserved histidine residues required for cathepsin B exopeptidase activity is predicted to be present. Recombinant preproprotein was produced in yeast, and it was shown that the recombinant proprotein can undergo a degree of self-processing in vitro to the mature form, which is active against gelatin and synthetic peptide substrates. The recombinant protein is antigenic in vaccinated rats, and antibodies to the protein are detected early after infection of rats and sheep with F. hepatica. The kinetics of the response to cathepsin B and cathepsin L after infection of sheep and rats confirm the temporal expression of these proteins during the life cycle of the parasite.
Law
Ruby H P
RH
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
Smooker
Peter M
PM
Irving
James A
JA
Piedrafita
David
D
Ponting
Rebecca
R
Kennedy
Nicholas J
NJ
Whisstock
James C
JC
Pike
Robert N
RN
Spithill
Terry W
TW
D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000097:blood*; D002401:Cathepsin B / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D005210:Fasciola hepatica / Q000235:genetics / Q000254:growth & development*; D005211:Fascioliasis / Q000276:immunology / Q000469:parasitology / Q000517:prevention & control*; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012756:Sheep; D012757:Sheep Diseases / Q000469:parasitology; D014611:Vaccination; D014612:Vaccines; D019444:Vaccines, DNA
D000909:Antibodies, Helminth; D011994:Recombinant Proteins; D014612:Vaccines; D019444:Vaccines, DNA; D002401:Cathepsin B