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Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis

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Abstract

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 ZnSO4 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.

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Acknowledgements

This study was supported by a grant from the Hong Kong Research Grant Council (HKU 426/96 M) and the Conference and Research Grants of the University of Hong Kong to R.C.K. We are very grateful for the assistance of Ms. Y.Y.Y. Chung and Mr. R. Leung.

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Correspondence to R. C. Ko.

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Lun, H.M., Mak, C.H. & Ko, R.C. Characterization and cloning of metallo-proteinase in the excretory/secretory products of the infective-stage larva of Trichinella spiralis . Parasitol Res 90, 27–37 (2003). https://doi.org/10.1007/s00436-002-0815-0

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Keywords

  • Serine Proteinase
  • Proteolytic Activity
  • Deduce Amino Acid Sequence
  • Agmatine
  • Aspartic Proteinase