Hostname: page-component-848d4c4894-xfwgj Total loading time: 0 Render date: 2024-06-16T02:40:26.788Z Has data issue: false hasContentIssue false
  • English
  • Français

Characterization of proteolytic enzymes from larval and adult Nippostrongylus brasiliensis

Published online by Cambridge University Press:  06 April 2009

J. Healer ,
F. Ashall  and
R. M. Maizels
J. Healer
Affiliation:
Wellcome Research Centre for Parasitic Infections, Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
F. Ashall
Affiliation:
Wellcome Research Centre for Parasitic Infections, Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
R. M. Maizels
Affiliation:
Wellcome Research Centre for Parasitic Infections, Department of Biology, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
Get access Rights & Permissions [Opens in a new window]

Abstract

Proteases from infective larval (L3) and adult stages of Nippostrongylus brasiliensis were investigated with a combination of techniques involving gelatin degradation and cleavage of fluorogenic substrates. Analysis of L3 excretory–secretory (ES) products revealed enzymes of Mr 51, 58, 79, ~ 150 and ~ 250 kDa. Inhibition profiles indicate that the major 51 kDa protease is a metallo-enzyme. Significantly, little activity was present in larval somatic extracts, suggesting the synthesis of zymogens or precursor forms prior to secretion. Adult ES contained a distinct enzyme, of 50 kDa, and a number of other proteases were detected in somatic extracts of this stage, ranging from 51 to > 300 kDa. The largest of these adult somatic enzymes is also a putative metallo-protease. While nearly all enzymes from both L3 and adult are heat labile, incubation at 100 °C generated a previously unobserved activity at 20 kDa. Furthermore, a protease of similar size may be found in uninfected rat intestinal tissue, suggesting specific uptake of a host-associated enzyme by the parasite in the form of an inactive, heat-labile complex.

Keywords

nematodeproteinasessubstrate gelsfluorogenic substratesmetallo-proteases
Type
Research Article
Information
Parasitology , Volume 103 , Issue 2 , October 1991 , pp. 305 - 314
Copyright
Copyright © Cambridge University Press 1991

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Ansari, A. A., Khan, M. A. & Ghatak, S. (1976). Ascardia galli: trypsin and chymotrypsin inhibitors. Experimental Parasitology 39, 74–83.CrossRefGoogle Scholar
Barrett, A. J. & Kirschke, H. (1981). Cathepsin B, cathepsin H and cathepsin L. Methods in Enzymology 80, 535–61.CrossRefGoogle ScholarPubMed
Bolla, R. & Weinstein, P. P. (1980). Acid protease activity during development and aging of Nippostrongylus brasiliensis. Comparative Biochemistry and Physiology 66B, 475–81.Google Scholar
Bolton, A. E. & Hunter, W. M. (1973). The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent. The Biochemical Journal 133, 529–39.CrossRefGoogle ScholarPubMed
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–54.CrossRefGoogle ScholarPubMed
Chappell, C. L. & Dresden, M. H. (1986). Schistosoma mansoni: proteinase activity of ‘hemoglobinase’ from the digestive tract of adult worms. Experimental Parasitology 61, 160–7.CrossRefGoogle ScholarPubMed
Cox, G. N., Pratt, D., Hageman, R. & Boisvenue, R. J. (1990). Molecular cloning and primary sequence of a cysteine protease expressed by Haemonchus contortus adult worms. Molecular and Biochemical Parasitology 41, 25–34.CrossRefGoogle ScholarPubMed
Davis, A. H., Nanduin, J. & Watson, D. C. (1987). Cloning and gene expression of Schistosoma mansoni protease. Journal of Biological Chemistry 262, 12851–5.CrossRefGoogle ScholarPubMed
Dalton, J. P. & Heffernan, M. (1989). Thiol proteases released in vitro by Fasciola hepatica. Molecular and Biochemical Parasitology 35, 161–6.CrossRefGoogle ScholarPubMed
Dresden, M. H., Rege, A. A. & Murrell, K. D. (1985). Strongyloides ransomi: Proteolytic enzymes from larvae. Experimental Parasitology 59, 257–63.CrossRefGoogle ScholarPubMed
Gamble, H. R., Purcell, J. P. & Fetterer, R. H. (1989). Purification of a 44 kilodalton protease which mediates the ecdysis of infective Haemonchus contortus larvae. Molecular and Biochemical Parasitology 33, 49–58.CrossRefGoogle ScholarPubMed
Hotez, P. J. & Cerami, A. (1983). Secretion of a proteolytic anticoagulant by Ancylostoma hookworms. Journal of Experimental Medicine 157, 1594–603.CrossRefGoogle ScholarPubMed
Hotez, P. J., Trang, N. L., McKerrow, J. H. & Cerami, A. (1985). Isolation and characterization of a proteolytic enzyme from the adult hookworm Ancylostoma caninum. Journal of Biological Chemistry 260, 7343–8.CrossRefGoogle ScholarPubMed
Jennings, F. W., Mulligan, W. & Urquhart, G. M. (1963). Variables in X-ray ‘inactivation’ of Nippostrongylus brasiliensis larvae. Experimental Parasitology 13, 367–73.CrossRefGoogle ScholarPubMed
Juhasz, S. & Kassai, T. (1981). A protease inhibitor of Nippostrongylus brasiliensis. Molecular and Biochemical Parasitology 3, 83–90.CrossRefGoogle ScholarPubMed
Kassai, T. & Toth, B. L. (1969). Inhibitory effect of worm metabolic antigen on the proteolytic activity of intestinal fluid. Parasitologica Hungaria 2, 39–44.Google Scholar
Keene, W. C., Jeong, K. H., McKerrow, J. H. & Werb, Z. (1983). Degradation of extracellular matrix by larvae of S. mansoni. II. Degradation by newly transformed and developing schistosomula. Laboratory Investigation 49, 201–7.Google ScholarPubMed
Klinkert, M. Q., Felleisen, R., Link, G., Ruppel, A. & Beck, E. (1989). Primary structures of Sm31/32 diagnostic proteins of Schistosoma mansoni and their identification as proteases. Molecular and Biochemical Parasitology 33, 113–22.CrossRefGoogle ScholarPubMed
Knox, D. P. & Jones, D. G. (1990). Studies on the presence and release of proteolytic enzymes (proteinases) in gastro-intestinal nematodes of ruminants. International Journal for Parasitology 20, 243–9.CrossRefGoogle ScholarPubMed
Lackey, A., James, E. R., Sakanari, J. A., Resnick, S. D., Brown, M., Bianco, A. E. & McKerrow, J. H. (1989). Extracellular proteases of Onchocerca. Experimental Parasitology 68, 176–85.CrossRefGoogle ScholarPubMed
Lewart, R. M. & Lee, C. L. (1954). Studies on the passage of helminth larvae through host tissues. I. histochemical studies on the extracellular changes caused by penetrating larvae. II. Enzymatic activity of larvae in vitro and in vivo. Journal of Infectious Diseases 95, 13–51.CrossRefGoogle Scholar
Lewart, R. M. & Lee, C. L. (1956). Quantitative studies of the collagenase-like enzymes of cercariae of Schistosoma mansoni and the larvae of Strongyloides ratti. Journal of Infectious Diseases 99, 1–14.CrossRefGoogle Scholar
Maki, J., Furuhashi, A. & Yanagiswa, T. (1982). The activity of acid proteases hydrolysing haemoglobin in parasitic helminths with special reference to interspecific and intraspecific distribution. Parasitology 84, 137–47.CrossRefGoogle ScholarPubMed
Matthews, B. E. (1977). The passage of larval helminths through tissue barriers. Symposia of the British Society for Parasitology 15, 93–119.Google Scholar
Matthews, B. E. (1982). Skin penetration by Necator americanus larvae. Parasitology Research 68, 86–91.Google ScholarPubMed
McKerrow, J. H. (1989). Parasite proteases. Experimental Parasitology 68, 111–15.CrossRefGoogle ScholarPubMed
McKerrow, J. H., Jones, P., Sage, H. & Pino-Heiss, S. (1985 a). Proteinases from the invasive larvae of the trematode parasite Schistosoma mansoni degrade connective tissue and basement membrane macromolecules. The Biochemical Journal 231, 47–51.CrossRefGoogle ScholarPubMed
McKerrow, J. H., Pino-Heiss, S., Lindquist, R. & Werb, Z. (1985 b). Purification and characterization of an elastinolytic proteinase secreted by cercariae of Schistosoma mansoni. Journal of Biological Chemistry 260, 3703–7.CrossRefGoogle ScholarPubMed
McKerrow, J. H., Brindley, P., Brown, M., Gam, A. A., Staunton, C. & Neva, F. A. (1990). Strongyloides stercoralis: identification of a protease that facilitates penetration of skin by the infective larvae. Experimental Parasitology 70, 134–43.CrossRefGoogle ScholarPubMed
Newport, G. N., McKerrow, J. H., Hedstrom, R., Petitt, M., Barr, P. & Agabian, N. (1988). Cloning of the proteinase that facilitates invasion by schistosome parasites. Journal of Biological Chemistry 263, 13179–84.CrossRefGoogle ScholarPubMed
Ogilvie, B. M. & Jones, V. E. (1977). Nippostrongylus brasiliensis: a review of immunity and the host/parasite relationship. Experimental Parasitology 29, 138–77.CrossRefGoogle Scholar
Peanasky, R. J., Bentz, Y., Paulson, B., Graham, D. L. & Babin, D. R. (1984). The isoinhibitors of chymotrypsin/elastase from Ascaris lumbricoides: isolation by affinity chromatography and association with the enzymes. Archives of Biochemistry and Biophysics 232, 127–34.CrossRefGoogle ScholarPubMed
Pino-Heiss, S., Petitt, M., Beckstead, J. H. & McKerrow, J. H. (1986). Preparation of mouse monoclonal antibodies and evidence for a host immune response to the preacetabular gland proteinase of Schistosoma mansoni. American Journal of Tropical Medicine and Hygiene 35, 536–43.CrossRefGoogle Scholar
Robertson, B. D., Kwan-Lim, G. E. & Maizels, R. M. (1988). A sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin. Analytical Biochemistry 172, 284–7.CrossRefGoogle ScholarPubMed
Robertson, B. D., Bianco, A. E., McKerrow, J. H. & Maizels, R. M. (1989). Proteolytic enzymes secreted by larvae of the nematode Toxocara canis. Experimental Parasitology 69, 30–6.CrossRefGoogle Scholar
Rogers, W. P. & Brooks, F. (1978). Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus. International Journal for Parasitology 8, 449–52.CrossRefGoogle ScholarPubMed
Ruff, V., Desai, S., Dubrul, E. F. & Komuniecki, R. (1988). In vitro synthesis and processing of components of the Ascaris suum pyruvate dehydrogenase complex. Molecular and Biochemical Parasitology 29, 1–8.CrossRefGoogle ScholarPubMed
Sarkis, G. J., Kurpiewski, M. R., Ashcom, J. D., Jen-Jacobson, L. & Jacobson, L. A. (1988). Proteases of the nematode Caenorhabditis elegans. Archives of Biochemistry and Biophysics 261, 80–90.CrossRefGoogle ScholarPubMed
Tamashiro, W. K., Rao, M. & Scott, A. L. (1987). Proteolytic cleavage of IgG and other protein substrates by D. immitis microfilarial enzymes. Journal of Parasitology 73, 149–54.CrossRefGoogle Scholar
Verwaerde, C., Auriault, C., Neyrinck, J. L. & Capron, A. (1988). Properties of serine proteases of Schistosoma mansoni Schistosomula involved in the regulation of IgE synthesis. Scandinavian Journal of Immunology 27, 17–24.CrossRefGoogle ScholarPubMed