Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis.
Inhibitor sensitivity assays using azocaesin and FTC-caesin as substrates showed that the excretory/secretory (E/S) products of the infective-stage larvae of Trichinella spiralis contained serine, metallo-, cysteine and aspartic proteinases. The activity of the metallo-proteinase was zinc ion dependent (within a range of ZnSO(4) concentrations). Gelatin-substrate gel electrophoresis revealed two bands of molecular mass 48 and 58 kDa which were sensitive to the metallo-proteinase inhibitor EDTA. The former peptide was probably a cleavage product of the latter. The authenticity of the 58 kDa metallo-proteinase as an E/S product was confirmed by immunoprecipitation. Using PCR and RACE reactions, a complete nucleotide sequence of the metallo-proteinase gene was obtained. It comprised 2,223 bp with an open reading frame encoding 604 amino acid residues. The 3' untranslated region consisted of 352 bp, including a polyadenylation signal AATAA. A consensus catalytic zinc-binding motif was present. The conserved domains suggest that the cloned metallo-proteinase belongs to the astacin family and occurs as a single copy gene with 11 introns and 10 exons. Cluster analysis showed that the sequence of the metallo-proteinase gene of T. spiralis resembles those of Caenorhabdites elegans and Strongyloides stercoralis.
Department of Zoology, 5S-13, Kadoorie Biological Science Building, The University of Hong Kong, Pokfulam Road, Hong Kong, China.
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D000595:Amino Acid Sequence; D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D000947:Antigens, Helminth / Q000187:drug effects / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D015801:Helminth Proteins / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D007814:Larva / Q000201:enzymology; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D010802:Phylogeny; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D016415:Sequence Alignment; D017160:Trichinella spiralis / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D014235:Trichinellosis / Q000469:parasitology; D015032:Zinc / Q000494:pharmacology
D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D015801:Helminth Proteins; C097419:excretory-secretory antigen, Trichinella; D004492:Edetic Acid; D008666:Metalloendopeptidases; D015032:Zinc