Fasciola hepatica: Characterization and Cloning of the Major Cathepsin B Protease Secreted by Newly Excysted Juvenile Liver Fluke
| Autor: | Terry W. Spithill, Michael Panaccio, L Wilson, Robert T. Good, Richard Mark Sandeman, Gene Wijffels |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
DNA
Complementary Cathepsin L medicine.medical_treatment Molecular Sequence Data Immunology Immunoglobulins Polymerase Chain Reaction Cathepsin B Substrate Specificity Hemoglobins Cathepsin O Complementary DNA Endopeptidases parasitic diseases medicine Animals Fasciola hepatica Protease Inhibitors Amino Acid Sequence Cloning Molecular Cathepsin Sheep Protease Sequence Homology Amino Acid biology Serum Albumin Bovine General Medicine DNA Helminth Hydrogen-Ion Concentration biology.organism_classification Cathepsins Molecular biology Cysteine protease Rats Cysteine Endopeptidases Infectious Diseases Biochemistry biology.protein Electrophoresis Polyacrylamide Gel Parasitology RNA Helminth |
| Zdroj: | ResearcherID |
| ISSN: | 0014-4894 |
| DOI: | 10.1006/expr.1998.4234 |
| Popis: | Proteolytic activity present in the excreted/secreted (ES) material of newly excysted juvenile (NEJ) Fasciola hepatica was biochemically analyzed. By gelatin substrate SDS-PAGE, only one region of activity was observed in the NEJ ES material at a molecular mass of 29 kDa. Both the secreted cathepsin L from adult fluke and the 29-kDa proteolytic activity of NEJ ES show a common pH optimum of 7.5, a cysteine protease inhibition profile, and preference for the N-benzyloxycarbonyl (Z)-Phe-Arg-NHMec fluorogenic substrate over Z-Arg-Arg-NHMec and Z-Arg-NHMec. In vitro analysis revealed that the NEJ protease activity digested sheep immunoglobulin heavy chain and bovine serum albumin but not bovine hemoglobin. Amino-terminal protein sequence analysis of the 29-kDa NEJ protease band revealed two sequences with homology to the cathepsin B family of proteases. Using degenerate oligonucleotides designed from the N-terminal sequence, reverse transcriptase polymerase chain reaction with NEJ RNA amplified a cDNA sequence encoding the first 236 amino acids of mature cathepsin B. Using this cDNA fragment an overlapping cDNA was isolated from a LambadaZAP cDNA library constructed with poly(A)+ RNA from immature 5-week-old liver fluke. Together with the N-terminal sequence, these cDNAs predict a mature cathepsin B sequence of 254 amino acids which shows 48-51% sequence identity to mammalian and Schistosoma mansoni cathepsin B. We conclude that, in contrast to the major proteases released by adult fluke, the major secreted protease of NEJ of F. hepatica is of the cathepsin B class. |
| Databáze: | OpenAIRE |
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