Hostname: page-component-586b7cd67f-2plfb Total loading time: 0 Render date: 2024-11-22T23:20:56.588Z Has data issue: false hasContentIssue false
  • English
  • Français

Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels

Published online by Cambridge University Press:  06 April 2009

A. McGinty ,
M. Moore ,
D. W. Halton  and
B. Walker
A. McGinty
Affiliation:
Divisions of Biochemistry and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
M. Moore
Affiliation:
Divisions of Biochemistry and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
D. W. Halton
Affiliation:
Divisions of Cell and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
B. Walker
Affiliation:
Divisions of Biochemistry and Experimental Biology, School of Biology and Biochemistry, Medical Biology Centre, The Queen's University of Belfast, Belfast BT7 1NN, Northern Ireland
Get access Rights & Permissions [Opens in a new window]

Summary

The excreted/secreted proteinases of adult and juvenile Fasciola hepatica maintained in vitro were found to hydrolyse the fluorogenic substrates Cbz-Phe-Arg- and Cbz-Arg-Arg-NHMec. This activity was demonstrated to have a classical cysteine proteinase inhibitor profile, with turn-over of both substrates being blocked by pre-incubation with E64 and peptidyl diazomethanes. The Cbz-Arg-Arg-NHMec hydrolysing activity of the mature fluke exhibited an alkaline stability not characteristic of its mammalian lysosomal counterparts. Further, the biotinylated affinity reagents biotin-Phe-Ala- CHN2 and biotin-Phe-Cys(SBzyl)-CHN2 were used to label and characterize these cysteine proteinases in terms of apparent molecular weight and subsite specificity. Adult fluke media were found to contain four species of molecular weights 66, 58, 50 and 25–26 kDa; juvenile media contained three species of molecular weights 66, 54 and 25–26 kDa. The major 25–26 kDa cysteine proteinase common to both stages was shown to have a subsite specificity similar to that of mammalian cathepsin B.

Keywords

Fasciola hepaticacysteine proteinasebiotinylated affinity labels
Type
Research Article
Information
Parasitology , Volume 106 , Issue 5 , June 1993 , pp. 487 - 493
Copyright
Copyright © Cambridge University Press 1993

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

Ashall, F. (1990). Characterization of alkaline peptidases of Trypanosoma cruzi and other trypanosomatids. Molecular and Biochemical Parasitology 38, 77–88.CrossRefGoogle ScholarPubMed
Ashall, F., Healy, N., Greig, S., Kiderlen, A., Curry, A. & Blackwell, j. (1990). Detection of an alkaline peptidase in leishmania amastigotes and promastigotes. Biochemical Society Transactions 18, 864–5.CrossRefGoogle Scholar
Barrett, A. J. & Kirschke, H. (1981). Cathepsin B, cathepsin H and cathepsin L. Methods in Enzymology 80, 535–61.CrossRefGoogle ScholarPubMed
Chapman, c. B. & Mitchell, G. F. (1982). Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica. Veterinary Parasitology 11, 165–78.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
Dalton, J. P. & Heffernan, M. (1989). Thiol proteinases released in vitro by Fasciola hepatica. Molecular and Biochemical Parasitology 35, 161–6.CrossRefGoogle ScholarPubMed
Fairweather, I., Anderson, H. R. & Baldwin, T. M. A. (1987). Fasciola hepatica – Tegumental surface alterations following treatment in vitro with the deacetylated (amine) metabolite of diamphenethide. Zeitschrift für Parasitenkunde 73, 99–106.Google ScholarPubMed
Green, G. D. J. & Shaw, E. (1981). Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases. Journal of Biological Chemistry 256, 1923–8.CrossRefGoogle ScholarPubMed
Heffernan, M., Smith, A., Curtain, D., Mcdonnell, S., Ryan, J. & Dalton, J. P. (1991). Characterization of a cathepsin B proteinase released by Fasciola hepatica (liver fluke). Biochemical Society Transactions 19, 278.CrossRefGoogle ScholarPubMed
Itow, s. & Plessman-Camargo, E. (1977). Proteolytic activities in cell extracts of Trypanosoma cruzi. Journal of Protozoology 24, 591–5.CrossRefGoogle ScholarPubMed
Kirschke, H., Wikstrom, P. & Shaw, E. (1988). Active center differences between cathepsin L and cathepsin B – the S, binding region. FEBS Letters 228, 128–30.CrossRefGoogle Scholar
Laemlli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 277, 680–5.CrossRefGoogle Scholar
La Rochelle, W. J. & Froehner, S. C. (1986). Immunochemical detection of proteins biotinylated on nitrocellulose replicas. Journal of Immunological Methods 92, 65–71.CrossRefGoogle ScholarPubMed
Lockwood, B. C., North, M. J., Scott, K. I., Bremner, A. F. & Coombs, G. H. (1987). The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads. Molecular and Biochemical Parasitology 24, 89–95.CrossRefGoogle ScholarPubMed
Lushbaugh, W. B., Hofbauer, A. F. & Pittman, F. E. (1985). Entamoeba histolytica – Purification of cathepsin B. Experimental Parasitology 59, 328–36.CrossRefGoogle ScholarPubMed
Mason, R. W., Johnson, D. A., Barrett, A. J. & Chapman, H. A. (1986). Elastinolytic activity of human cathepsin L. Biochemical Journal 233, 925–7.CrossRefGoogle ScholarPubMed
Neale, K. A. & Alderete, J. F. (1990). Analysis of the proteinases of representative Trichomonas vaginalis isolates. Infection and Immunity 58, 157–62.CrossRefGoogle ScholarPubMed
Pamer, E. G., So, M. & Davis, c. E. (1989). Identification of a developmentally regulated cysteine protease of Trypanosoma brucei. Molecular and Biochemical Parasitology 33, 27–32.CrossRefGoogle ScholarPubMed
Rege, A. A., Herrera, P. R., Lopez, M. & Dresden, M. H. (1989). Isolation and characterization of a cysteine proteinase from Fasciola hepatica adult worms. Molecular and Biochemical Parasitology 35, 89–95.CrossRefGoogle ScholarPubMed
Robertson, C. D., North, M. J., Lockwood, B. C. & Coombs, G. H. (1990). Analysis of the proteinases of Trypanosoma brucei. Journal of General Microbiology 136, 921–5.CrossRefGoogle ScholarPubMed
Rupova, L. & Keilova, H. (1979). Isolation and some properties of an acid protease from Fasciola hepatica. Zeitschrift für Parasitenkunde 61, 83–91.CrossRefGoogle ScholarPubMed
Shaw, E., Wikstrom, P. & Ruscica, J. (1983). An exploration of the primary specificity site of cathepsin B. Archives of Biochemistry and Biophysics 222, 424–9.CrossRefGoogle ScholarPubMed
Simpkin, K. G., Chapman, C. R. & Coles, G. C. (1980). Fasciola hepatica: a proteolytic digestive enzyme. Experimental Parasitology 49, 281–7.CrossRefGoogle ScholarPubMed
Smith, M. A. & Clegg, J. A. (1981). Improved culture of Fasciola hepatica in vitro. Zeitschrift für Parasitenkunde 66, 9–15.CrossRefGoogle ScholarPubMed
Walker, B., Cullen, B. M., Kay, G., Halliday, I. M., McGinty, A. & Nelson, J. (1992). The synthesis, kinetic characterization and application of a novel biotinylated affinity label for cathepsin B. Biochemical Journal 283, 449–53.CrossRefGoogle ScholarPubMed
Yamasaki, H., Aoki, T. & Oya, H. (1989). A cysteine proteinase from the liver fluke Fasciola sp.: purification, characterization, localization and application to immunodiagnosis. Japanese Journal of Parasitology 38, 373–84.Google Scholar