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Identification and characterization of the cysteine and serine proteinases of the trematode, Haplometra cylindracea and determination of their haemoglobinase activity

Published online by Cambridge University Press:  06 April 2009

S. J. Hawthorne ,
D. W. Halton  and
B. Walker
S. J. Hawthorne
Affiliation:
Divisions of Biochemistry, The Queen's University of Belfast, Belfast BT7 INN, Northern Ireland, UK
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 INN, Northern Ireland, UK
B. Walker
Affiliation:
Divisions of Biochemistry, The Queen's University of Belfast, Belfast BT7 INN, Northern Ireland, UK
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Summary

The excreted/secreted proteinases of Haplometra cylindracea maintained in vitro, were found to hydrolyse the fluorogenic substrates, Z-ArgArg-NHMec and Z-PheArg-NHMec. This activity was shown to be typically that of cysteine proteinases, as turn-over of both substrates could be blocked by pre-incubation with peptidyl diazomethyl ketones. The biotinylated affinity reagent, biotin-Phe Ala-DMK, used in combination with Z-PheTyr(OBut)-DMK, was employed for the labelling and characterization of these cysteine proteinase activities. Three cathepsin B-like species were detected, with molecular weights of 48, 22–23 and 14 kDa, together with a cathepsin L-like enzyme, with a molecular weight of 55 kDa. The proteinases were also found to have hydrolytic activity towards the substrate, Z-GlyGlyArg-NHMec, which could be blocked by pre-incubation with either of the serine proteinase-selective reagents, Z-ArgP(OPh)2, or biotin-LysP(OPh)2, showing the activity to be trypsin-like. Using the biotinylated affinity label to characterize the trypsin-like enzymes revealed two molecular species with molecular weights of 20 and 24 kDa.

Keywords

Haplometra cylindraceaTrematodaproteinasescysteine proteinasesserine proteinaseshaemoglobinase activity
Type
Research Article
Information
Parasitology , Volume 108 , Issue 5 , June 1994 , pp. 595 - 601
Copyright
Copyright © Cambridge University Press 1994

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References

REFERENCES

Apodaca, G. & McKerrow, J. H. (1989). Purification and characterisation of a 27000 Mr extracellular proteinase from Trichophyton rubrum. Infection and Immunity 57, 3072–80.CrossRefGoogle Scholar
Barrett, A. J. & Kirschke, H. (1981). Cathepsin B, cathepsin H and cathepsin L. Methods in Enzymology 80, 535–61.CrossRefGoogle ScholarPubMed
Bayliss, R. S., Knowles, J. R. & Wybrandt, G. B. (1969). An aspartic acid residue at the active site of pepsin. The Biochemical Journal 113, 377–86.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
Chavez-Olortegui, C., Resende, M. & Tavares, C. A. P. (1992). Purification and characterisation of a 47 kDa protease from Schistosoma mansoni cercarial secretion. Parasitology 105, 211–18.CrossRefGoogle ScholarPubMed
Cohen, F. E., Gregoret, L. M., Amiri, P., Aldape, K., Bailey, J. & McKerrow, J. H. (1991). Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling. Biochemistry 30, 11221–9,CrossRefGoogle ScholarPubMed
Cullen, B. M., Halliday, I., Kay, G., Nelson, J. & Walker, B. (1992). The application of a novel biotinylated affinity label for the detection of a cathepsin B-like precursor produced by breast-tumour cells in culture. The Biochemical Journal 283, 461–5.CrossRefGoogle 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
Halton, D. W. (1967). Observations on the nutrition of digenetic trematodes. Parasitology 57, 639–60.CrossRefGoogle ScholarPubMed
Hamilton, R., Walker, B. J. & Walker, B. (1993). A convenient synthesis of N-protected diphenyl phosphonate ester analogues of ornithine, lysine and homolysine. Tetrahedron Letters 34, 2847–50.CrossRefGoogle Scholar
Harth, G., Andrews, N., Mills, A. A., Engel, J. C., Smith, R. & McKerrow, J. H. (1993). Peptide-fluoromethyl ketones arrest intracellular replication and intracellular transmission of Trypanosoma cruzi. Molecular and Biochemical Parasitology 58, 17–24.CrossRefGoogle ScholarPubMed
Hawthorne, S. J., Hamilton, R., Walker, B. J. & Walker, B. (1994). The synthesis, kinetic characterization and application of biotinylated diphenyl phosphonate analogues of ornithine and lysine for the detection of trypsin-like serine proteinases. The Biochemical Journal (in the Press).Google Scholar
Kirschke, H., Wikstrom, P. & Shaw, E. (1988). Active center differences between cathepsin L and cathepsin B – the S1 binding region. FEBS Letters 228, 128–30.CrossRefGoogle ScholarPubMed
Knox, D. P., Redmond, D. L. & Jones, D. G. (1993). Characterization of proteinases in extracts of adult Haemonchus contortus, the ovine abomasal nematode. Parasitology 106, 395–404.CrossRefGoogle ScholarPubMed
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 277, 680–5.CrossRefGoogle Scholar
McGinty, A., Moore, M., Halton, D. W. & Walker, B. (1993). Characterization of the cysteine proteinases of the common liver fluke Fasciola hepatica using novel, active-site directed affinity labels. Parasitology 106, 487–93.CrossRefGoogle ScholarPubMed
Rege, A. A., Herrera, P. R., Lopez, M. & Dresden, M. H. (1989). Isolation and characterisation 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
Rosenthal, P. J., McKerrow, J. H., Rasnick, D. & Leech, J. H. (1989). Plasmodium falciparum: inhibitors of lysosomal cysteine proteinases inhibit a trophozoite proteinase and block parasite development. Molecular and Biochemical Parasitology 35, 177–84.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
Smyth, J. D. & Halton, D. W. (1983). The Physiology of Trematodes, 2nd Edn.Cambridge: Cambridge University Press.Google Scholar
Walker, B., Cullen, B. M., Kay, G., Halliday, I., McGinty, A. & Nelson, J. (1992). The synthesis, kinetic characterisation and application of a novel biotinylated affinity label for cathepsin B. The Biochemical Journal 283, 449–53.CrossRefGoogle ScholarPubMed
Yamasaki, H., Kominami, E. & Aoki, T. (1992). Immunocytochemical localisation of a cysteine protease in adult worms of the liver fluke Fasciola sp. Parasitology Research 78, 574–80.CrossRefGoogle ScholarPubMed