Hostname: page-component-586b7cd67f-2plfb Total loading time: 0 Render date: 2024-11-22T22:59:17.890Z Has data issue: false hasContentIssue false
  • English
  • Français

Biosynthesis and glycosylation of serine/threonine-rich secreted proteins from Toxocara canis larvae

Published online by Cambridge University Press:  06 April 2009

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

Summary

Toxocara canis infective stage larvae continually produce excretory–secretory (TES) glycoproteins in long-term in vitro culture. The kinetics of synthesis and secretion were studied by metabolic labelling with radioactive [35S]methionine, [14C]serine and [14C]threonine. Maximal incorporation rates required overnight pre-incubation of parasites in medium depleted of the appropriate amino acid. Larvae rapidly incorporated isotope into their somatic tissues, but there was a minimum delay of 10 h before secretion of labelled antigens. Labelling with [14C]serine and [14C]threonine demonstrated a relative abundance of these amino acids in the major surface/secreted glycoproteins of this nematode (TES-32 and 120). Pulse-chase experiments suggested that TES-120 may be derived from a 58 kDa precursor, reflecting extensive post-translational glycosylation. Inhibition of N-glycosylation with tunicamycin and digestion with N-glycanase provided evidence of N-glycosylation in the lower molecular weight ES components (TES-32, 55 and 70). These agents had no effect on the higher molecular weight components (TES-120 and 400) implying that for these molecules glycosylation is predominantly O-linked. The largest ES component (TES-400) was unusual, in incorporating serine and threonine but not methionine, and by exhibiting increased apparent molecular weight following pronase digestion; it is suggested that this molecule is a proteoglycan.

Keywords

nematodeglycosylationexcretory–secretory antigens (TES)precursorspost-translational modification
Type
Research Article
Information
Parasitology , Volume 105 , Issue 2 , October 1992 , pp. 297 - 308
Copyright
Copyright © Cambridge University Press 1992

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

REFERENCES

Badley, J. E., Grieve, R. B., Bowman, D. D. & Glickman, L. T. (1987). Immune-mediated adherence of eosinophils to Toxocara canis infective larvae: the role of excretory-secretory antigens. Parasite Immunology 9, 133–43.CrossRefGoogle ScholarPubMed
Betschart, B., Rudin, W. & Weiss, N. (1985). The isolation and immunogenicity of the cuticle of Dipetalonema viteae (Filarioidea). Zeitschrift für Parasitenkunde 71, 87–95.CrossRefGoogle ScholarPubMed
Bradley, J. E., Gregory, W. F., Bianco, A. E. & Maizels, R. M. (1989). Biochemical and immunochemical characterisation of a 20-kilodalton complex of surfaceassociated antigens from adult Onchocerca gutturosa filarial nematodes. Molecular and Biochemical Parasitology 34, 197–208.CrossRefGoogle ScholarPubMed
Burke, T. M. & Roberson, E. L. (1985 a). Prenatal and lactational transmission of Toxocara canis and Ancylostoma caninum: experimental infection of the bitch before pregnancy. International Journal for Parasitology 15, 71–5.CrossRefGoogle ScholarPubMed
Burke, T. M. & Roberson, E. L. (1985 b). Prenatal and lactational transmission of Toxocara canis and Ancylostoma caninum: experimental infection of the bitch at midpregnancy and at parturition. International Journal for Parasitology 15, 485–90.CrossRefGoogle ScholarPubMed
Carlier, Y., Bout, D., Strecker, G., Debray, H. & Capron, A. (1980). Purification, immunochemical and biologic characterization of the Schistosoma circulating M antigen. Journal of Immunology 124, 2442–50.CrossRefGoogle ScholarPubMed
Caulfield, J. P., Cianci, C. M. I., McDiarmid, S. S., Suyemitsu, T. & Schmid, K. (1987). Ultrastructure, carbohydrate, and amino acid analysis of two preparations of the cercarial glycocalyx of Schistosoma mansoni. Journal of Parasitology 73, 514–22.CrossRefGoogle ScholarPubMed
De Savigny, D. H. (1975). In vitro maintenance of Toxocara canis larvae and a simple method for the production of Toxocara ES antigen for use in serodiagnosis test for visceral larva migrans. Journal of Parasitology 61, 781–2.CrossRefGoogle Scholar
Denkers, E. Y., Wassom, D. L. & Hayes, C. E. (1990). Characterization of Trichinella spiralis antigens sharing an immunodominant, carbohydrate-associated determinant distinct from phosphorylcholine. Molecular and Biochemical Parasitology 41, 241–50.CrossRefGoogle ScholarPubMed
Elbein, A. D. (1987). Inhibitors of the biosynthesis and processing of N-linked oligosaccharide chains. Annual Review of Biochemistry 56, 497–534.CrossRefGoogle ScholarPubMed
Fattah, D. I., Maizels, R. M., McLaren, D. J. & Spry, C. J. F. (1986). Toxocara canis: interaction of human eosinophils with the infective larvae. Experimental Parasitology 61, 421–33.CrossRefGoogle ScholarPubMed
Gaillard, J.-L., Berche, P., Frehel, C., Couin, E. & Cossart, P. (1991). Entry of L. monocytogenes into cells is mediated by internalin, a repeat protein reminiscent of surface antigens from Gram-positive cocci. Cell 65, 1127–41.CrossRefGoogle ScholarPubMed
Gillespie, S. H. (1988). The epidemiology of Toxocara canis. Parasitology Today 4, 180–2.CrossRefGoogle ScholarPubMed
Glickman, L. T. & Schantz, P. M. (1981). Epidemiology and pathogenesis of zoonotic toxocariasis. Epidemiologic Reviews 3, 230–50.CrossRefGoogle ScholarPubMed
Khoo, K-H., Maizels, R. M., Page, A. P., Taylor, G. W., Rendell, N. & Dell, A. (1991). Characterisation of nematode glycoproteins: the major O-glycans of Toxocara excretory secretory antigens are methylated trisaccharides. Glycobiology 1, 163–71.CrossRefGoogle ScholarPubMed
Kozarsky, K., Kingsley, D. & Kreiger, M. (1988). Use of a mutant cell line to study the kinetics and function of O-linked glycosylation of low density lipoprotein receptors. Proceedings of the National Academy of Sciences, USA 85, 4335–9.CrossRefGoogle Scholar
Kwan-Lim, G.-E., Gregory, W. F., Selkirk, M. E., Partono, F. & Maizels, R. M. (1989). Secreted antigens of filarial nematodes: survey and characterisation of in vitro excretory/secretory (E/S) products of adult Brugia malayi filarial parasites. Parasite Immunology 11, 629–54.CrossRefGoogle Scholar
Maizels, R. M., De Savigny, D. & Ogilvie, B. M. (1984). Characterization of surface and excretory–secretory antigens of Toxocara canis infective larvae. Parasite Immunology 6, 23–37.CrossRefGoogle ScholarPubMed
Maizels, R. M., Kennedy, M. W., Meghji, M., Robertson, B. D. & Smith, H. V. (1987 a). Shared carbohydrate epitopes on distinct surface and secreted epitopes of the parasitic nematode Toxocara canis. Journal of Immunology 139, 207–14.CrossRefGoogle ScholarPubMed
Maizels, R. M., Bianco, A. E., Burke, J., Flint, J. E., Gregory, W. F., Kennedy, M. W., Lim, G. E., Robertson, B. D. & Selkirk, M. E. (1987 b). Glycoconjugate antigens from parasitic nematodes. In Molecular Paradigms for Eradicating Helminthic Parasites UCLA Symposia on Molecular and Cellular Biology (ed. MacInnis, A. J.). pp. 267–79. New York: A. R. Liss.Google Scholar
Maizels, R. M., Gregory, W. F., Kwan-Lim, G.-E. & Selkirk, M. E. (1989). Filarial surface antigens: the major 29,000 mol. wt. glycoprotein and a novel 17,000–200,000 mol. wt. complex from adult Brugia malayi parasites. Molecular and Biochemical Parasitology 32, 213–27.CrossRefGoogle Scholar
Maizels, R. M., Robertson, B. D., Blaxter, M. L. & Selkirk, M. E. (1991). Parasite Antigens, Parasite Genes. A Laboratory Manual for Molecular Parasitology. Cambridge: Cambridge University Press.Google Scholar
Meghji, M. & Maizels, R. M. (1986). Biochemical properties of larval excretory–secretory (ES) glycoproteins of the parasitic nematode Toxocara canis. Molecular and Biochemical Parasitology 18, 155–70.CrossRefGoogle Scholar
Nyame, K., Cummings, R. D. & Damian, R. T. (1987). Schistosoma mansoni synthesizes glycoproteins containing terminal O-linked N-acetylglucosamine residues. Journal of Biological Chemistry 262, 7990–5.CrossRefGoogle ScholarPubMed
Nyame, K., Cummings, R. D. & Damian, R. T. (1988). Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula. Journal of Parasitology 74, 562–72.CrossRefGoogle Scholar
O'farrell, P. Z., Goodman, H. M. & O'farrell, P. H. (1977). High-resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133–42.CrossRefGoogle ScholarPubMed
Page, A. P., Hamilton, A. J. & Maizels, R. M. (1992 a). Toxocara canis: monoclonal antibodies to carbohydrate epitopes of secreted (TES) antigens localize to different secretion-related structures in infective larvae. Experimental Parasitology (in the Press).CrossRefGoogle ScholarPubMed
Page, A. P., Richards, D. T., Lewis, J. W., Omar, H. M. & Maizels, R. M. (1991). Comparison of isolates and species of Toxocara and Toxascaris by biosynthetic labelling of somatic and ES proteins from infective larvae. Parasitology 103, 451–64.CrossRefGoogle ScholarPubMed
Page, A. P., Rudin, W., Fluri, E., Blaxter, M. L. & Maizels, R. M. (1992 b). Toxocara canis: a labile antigenic surface coat overlying the epicuticle of infective larvae. Experimental Parasitology (in the Press).Google ScholarPubMed
Parsons, J. C., Bowman, D. D. & Grieve, R. B. (1986). Tissue localization of excretory–secretory antigens of larval Toxocara canis in acute and chronic murine toxocariasis. American Journal of Tropical Medicine and Hygiene 35, 974–81.CrossRefGoogle ScholarPubMed
Reddy, P., Caras, I. & Kreiger, M. (1989). Effects of 0- linked glycosylation on the cell surface expression and stability of decay-accelerating factor, a glycosphingolipid-anchored membrane protein. Journal of Biological Chemistry 264, 17329–36.CrossRefGoogle Scholar
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
Rockey, J. H., John, T., Donnelly, J. J., McKenzie, D. F., Stromberg, B. E. & Soulsby, E. J. L. (1983). In vitro interaction of eosinophils from ascarid-infected eyes with Ascaris suum and Toxocara canis larvae. Investigative Ophthalmology and Visual Science 24, 1346–57.Google ScholarPubMed
Segrest, J. P. & Jackson, R. L. (1972). Molecular weight determination of glycoproteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Methods in Enzymology 28, 54–63.CrossRefGoogle Scholar
Selkirk, M. E., Nielsen, L., Kelly, C., Partono, F., Sayers, G. & Maizels, R. M. (1989). Identification, synthesis and immunogenicity of cuticular collagens from the filarial nematodes Brugia malayi and B. pahangi. Molecular and Biochemical Parasitology 32, 229–46.CrossRefGoogle Scholar
Selkirk, M. E., Gregory, W. F., Yazdanbakhsh, M., Jenkins, R. E. & Maizels, R. M. (1990). Cuticular localisation and turnover of the major surface glycoprotein (gp29) of adult Brugia malayi. Molecular and Biochemical Parasitology 42, 41–4.CrossRefGoogle ScholarPubMed
Smith, H. V. (1991). Immune evasion and immunopathology in Toxocara canis infection. In Parasitic Nematodes–Antigens, Membranes and Genes (ed. Kennedy, M. W.) pp. 116–39. London: Taylor and Francis.Google Scholar
Sprent, J. F. A. (1958). Observations on the development of Toxocara canis (Werner, 1782) in the dog. Parasitology 48, 184–210.CrossRefGoogle ScholarPubMed
Sugane, K., Howell, M. J. & Nicholas, W. L. (1985). Biosynthetic labelling of the excretory and secretory antigens of Toxocara canis larvae. Journal of Helminthology 59, 147–51.CrossRefGoogle ScholarPubMed
Tarentino, A. L., Gómez, C. M. & Plummer, T. H. Jr (1985). Deglycosylation of asparagine-linked glycans by peptide: N-glycosidase F. Biochemistry 24, 4665–71.CrossRefGoogle ScholarPubMed
Taylor, M. R. H., Keane, C. T., O'connor, P., Mulvihill, E. & Holland, C. (1988). The expanded spectrum of toxocaral disease. Lancet 1, 692–5.CrossRefGoogle ScholarPubMed
Umemoto, J., Bhavanandan, V. P. & Davidson, E. A. (1977). Purification and properties of an endo-α-N-acetyl-D-galactosaminidase from Diplococcus pneumonzae. Journal of Biological Chemistry 252, 8609–14.CrossRefGoogle Scholar
Williamson, H. J. E., Allerdyce, R. A., Clemett, R. S. & Hidajat, R. R. (1990). Serum and neutrophils alter the rate of excretory/secretory antigen release by Toxocara canis infective larvae in vitro. Parasite Immunology 12, 175–87.CrossRefGoogle ScholarPubMed