Molecular cloning and functional characterisation of a cathepsin L-like proteinase from the fish kinetoplastid parasite Trypanosoma carassii.
Trypanosoma carassii is a fish kinetoplastid parasite that belongs to the family Trypanosomatida. In the present study we cloned a cathepsin L-like proteinase from T. carassii. The nucleotide sequence of 1371bp translated into a preproprotein of 456 amino acids. The preproprotein contained the oxyanion hole (Gln), the active triad formed by Cys, His and Asn and the conserved ERFNIN-like, GNFD and GCNGG motifs, characteristic for cathepsin L proteinases. Phylogenetic analysis showed that the T. carassii cysteine proteinase clustered with other cathepsin L-like proteinases from the Trypanosomatida. We produced a recombinant T. carassii cysteine proteinase in Escherichia coli and demonstrated that it has cathepsin L activity. Immunization of common carp (Cyprinus carpio L.) with the recombinant protein induced a very high increase in proteinase-specific antibodies but only slightly lowered parasitaemia. Our findings suggest that the T. carassii cysteine proteinase is highly conserved within the Trypanosomatida with respect to structure and activity but is not a major protective antigen in carp.
Ruszczyk
Aleksandra
A
Cell Biology and Immunology Group, Wageningen Institute of Animal Sciences, Wageningen University, Wageningen, The Netherlands.
Forlenza
Maria
M
Savelkoul
Huub F J
HF
Wiegertjes
Geert F
GF
D000595:Amino Acid Sequence; D000818:Animals; D000913:Antibodies, Protozoan / Q000097:blood; D002347:Carps / Q000276:immunology* / Q000469:parasitology; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics*; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases; D018076:DNA, Complementary / Q000737:chemistry; D018105:DNA, Kinetoplast / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005393:Fish Diseases / Q000276:immunology* / Q000469:parasitology; D008969:Molecular Sequence Data; D010802:Phylogeny; D015800:Protozoan Proteins / Q000008:administration & dosage / Q000032:analysis / Q000276:immunology; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000032:analysis / Q000276:immunology; D016415:Sequence Alignment / Q000662:veterinary; D013997:Time Factors; D014345:Trypanosoma / Q000201:enzymology / Q000235:genetics*; D014352:Trypanosomiasis / Q000662:veterinary*; D014611:Vaccination / Q000662:veterinary
D000913:Antibodies, Protozoan; D018076:DNA, Complementary; D018105:DNA, Kinetoplast; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L