Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

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

  • 157 Accesses

  • 33 Citations


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.

This is a preview of subscription content, log in to check access.

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4. A
Fig. 5. A
Fig. 6.
Fig. 7.
Fig. 8.
Figs. 9A, B.
Fig. 10.


  1. Baramova E, Foidart JM (1995) Matrix metalloproteinase family. Cell Biol Int 19:239–242

  2. Breathnach R, Chambon P (1981) Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383

  3. Campbell WC (1983) Historical introduction. In: Campbell WC (ed) Trichinella and trichinellosis. Plenum Press, New York, pp 1–30

  4. Coombs GH, Mottram JC (1997) Parasite proteinases and amino acid metabolism: possibilities for chemotherapeutic exploitation. Parasitology 114:S61-S80

  5. Criado AF, Armas SC, Gimenez CP, Casado NE, Jimenez AG, Rodriguez FC (1992) Proteolytic enzymes from Trichinella spiralis larvae. Vet Parasitol 45:133–140

  6. De Armas-Serra C, Gimenez-Pardo C, Jimenez-Gonzalez A, Bernadina WE, Rodriguez-Caabeiro F (1995a) Purification and preliminary characterization of a protease from the excretion-secretion products of Trichinella spiralis muscle-stage larvae. Vet Parasitol 59:157–168

  7. De Armas-Serra C, Gimenes-Pardo C, Bernadina WE, Rodriguez-Caabeiro F (1995b) Antibody response to a protease secreted by Trichinella spiralis muscle larvae. Parasitol Res 81:540–542

  8. Gamble HR, Fetterer RH, Mansfield LS (1996) Developmentally regulated zinc metalloproteinases from third- and fourth-stage larvae of the ovine nematode Haemonchus contortus. J Parasitol 82:197–202

  9. Haffner A, Guilavogui AZ, Tischendorf FW, Brattig NW (1998) Onchocerca volvulus: Microfilariae secrete elastinolytic and males nonelastinolytic matrix-degrading serine and metalloproteinases. Exp Parasitol 90:26–33

  10. Hill DE, Gamble HR, Rhoads ML, Fetterer RH, Urban JF (1993) Trichuris suis: A zinc metalloprotease from culture fluids of adult parasites. Exp Parasitol 77:170–178

  11. Jiang W, Bond JS (1992) Families of metalloendopeptidases and their relationship. FEBS Lett 312:110–114

  12. Ko RC, Mak CH (1999) Trichinellosis as a model of new frontier research on parasitic infections. Int Med Res J 3:1–11

  13. Ko RC, Yeung MHF (1989) Specificity of ES antigens in detection of Trichinella spiralis antibodies in Chinese pigs. Trop Biomed 6:99–111

  14. Lai YY (1996) Identification and characterization of proteolytic enzymes in Trichinella spp. M.Phil thesis. Department of Zoology, Univerisity of Hong Kong, Hong Kong

  15. Lai YY, Ko RC (1994) A neutral metallo-protease from Trichinella spiralis infective larvae. Eighth International Congress of Parasitology, Izmir, Turkey, Abstract no. 192

  16. McKerrow JH (1989) Parasite proteinases. Exp Parasitol 68:111–115

  17. Moczon T, Wranicz M (1999) Trichinella spiralis: proteinases in the larvae. Parasitol Res 85:47–58

  18. Nagano I, Wu Z, Nakada T, Matsuo A, Takahashi Y (2001) Molecular cloning and characterization of a serine proteinase inhibitor from Trichinella spiralis. Parasitology 123:77–83

  19. Pei D, Weiss SJ (1995) Furin-dependent intracellular activation of the human stromelysin-3 zymogen. Nature 375:244–247

  20. Pendás AM, Knäuper V, Puente XS, Llano E, Mattei MG, Apte S, Murphy G, Lόpez-Otín C (1997) Identification and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location and tissue distribution. J Biol Chem 272:4281–4286

  21. Polzer M, Taraschewski H (1993) Identification and characterization of the proteolytic enzymes in the developmental stages of the eel-pathogenic nematode Anguillicola crassus. Parasitol Res 79:24–27

  22. Shapiro SD (1998) Matrix metalloproteinase degradation of extracellular matrix: biological consequences. Curr Opin Cell Biol 10:602–608

  23. Stöcker W, Zwilling R (1995) Astacin. Methods in Enzymol 248:305–325

  24. Stöcker W, Grams F, Baumann U, Reinemer P, Gomis-Ruth FX, Mckay DB, Bode W (1995) The metzincins. Topological and sequential relations between the astacins, adamalysins, serralysins and matrixins (collagenases) define a superfamily of zinc-peptidases. Protein Sci 4:823–840

  25. Takamoto M, Sugane K (1993) Trichinella spiralis infective L1 larvae secrete serine proteinase(s). Jpn J Parasitol 42:277–294

  26. Todorova VK (2000) Proteolytic enzymes secreted by larval stage of the parasitic nematode Trichinella spiralis. Folia Parasitol 47:141–145

  27. Todorova VK, Knox DP, Kennedy MW (1995) Proteinases in the excretory/secretory products (ES) of adult Trichinella spiralis. Parasitology 111:201–208

  28. Trap C, Le Rhun D, Roman T, Liu MY, Perret C, Boireau P (2000) Molecular cloning and analysis of a common stage Trichinella spiralis serine protease gene. Proceedings of the 10th International Conference on Trichinellosis, Fontainebleau, France, pp 39

  29. van Wart HE, Birkedal-Hansen H (1990) The cysteine switch: a principle of regulation of metalloproteinase gene family. Proc Natl Acad Sci U S A 87:5578-5582

  30. Yan L, Leontovich A, Fei K, Sarras MP (2000) Hydra metalloproteinase 1: a secreted astacin metalloproteinase whose apical axis expression is differentially regulated during head regeneration. Dev Biol 219:115–128

  31. Yang M, Murray MT, Kurkinen M (1997) A novel matrix metalloproteinase gene (XMMP) encoding vitronectin-like motifs is transiently expressed in Xenopus laevis early embryo development. J Biol Chem 272:13527–13533

  32. Yasumasu S, Yamada K, Adasaka K, Misunaga K, Iuchi I, Shimada H, Yamagami K (1992) Isolation of cDNAs for LCE and HCE, two constituent proteases of the hatching enzyme of Oryzlas latipes and concurrent expression of their mRNAs during development. Dev Biol 153:250–258

Download references


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.

Author information

Correspondence to R. C. Ko.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation


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