Hostname: page-component-586b7cd67f-t7czq Total loading time: 0 Render date: 2024-11-28T04:17:18.467Z Has data issue: false hasContentIssue false
  • English
  • Français

Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases

Published online by Cambridge University Press:  06 April 2009

W. R. McMaster ,
C. J. Morrison ,
M. H. Macdonald  and
P. B. Joshi
W. R. McMaster
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
C. J. Morrison
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
M. H. Macdonald
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
P. B. Joshi
Affiliation:
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
Get access Rights & Permissions [Opens in a new window]

Summary

The major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63000 Mr present on promastigotes and amastigotes from diverse species of Leishmania. GP63 shares several characteristics with the members of the matrix metalloproteinase family including degradation of at least one component of the extracellular matrix, location at the cell surface, requirement for Zn2+ for proteinase activity and inhibition of the proteinase activity by chelating agents and α2–macroglobulin. Site-directed mutagenesis of the cloned L. major GP63 genes was carried out to determine whether the proposed active site of Leishmania GP63 was homologous to those of other zinc metalloproteinases. The codon encoding the catalytic glutamic acid was modified to encode an aspartic acid and when expressed in COS–7 cells the resulting mutant GP63 had no demonstrable proteinase activity compared to wild type GP63. GP63 was predicted to be synthesized as a precursor protein containing a pro region at the NH2–terminus of GP63 implicated to be involved with the regulation of proteinase activity. As with many other proteinases, including matrix metalloproteinases, these enzymes are synthesized as latent proteinases that require activation for full proteinase activity. L. major recombinant GP63 (rGP63) has been produced in the baculovirus expression system where rGP63 was secreted as a latent proteinase. To study the activation of baculovirus rGP63, purified rGP63 was incubated with the mercurial compound, HgCl2, at concentrations previously shown to result in activation of other latent matrix degrading metalloproteinases and resulted in a significant enhancement of GP63 proteinase activity. The similarity of GP63 to the family of matrix-degrading proteinases suggests that the proteinase activity of GP63 maybe involved with the pathology of lesion formation in the mammalian host and may also be involved with the promastigote life stage in the sandfly vector. To study the functional role of GP63 proteinase, mutant strains of L. major, deficient in the expression of GP63, are currently being derived by targeted gene deletion. Using this strategy results have demonstrated the deletion of an entire L. major GP63 locus, containing in total six GP63 genes. Strategies to delete the second GP63 gene locus are developed and will determine whether deletion of both loci results in viable promastigotes. L. major strains deficient in the expression of GP63 may then be used to address the function of GP63 glycoprotein in the life cycle of Leishmania.

Keywords

active site mutationLeishmaniasurface metalloproteinaseGP63
Type
Research Article
Information
Parasitology , Volume 108 , supplement S1: Symposia of the British Society for Parasitology Volume 31:Functional molecules on the surface of protozoan parasites , March 1994 , pp. S29 - S36
Copyright
Copyright © Cambridge University Press 1994

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

Bordier, C. (1987). The promastigote surface protease of Leishmania. Parasitology Today 3, 151–3.CrossRefGoogle ScholarPubMed
Bouvier, J., Bordier, C., Vogel, H., Reichelt, R. & Etges, R. (1989). Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase. Molecular and Biochemical Parasitology 37, 235–46.CrossRefGoogle ScholarPubMed
Bouvier, J., Schneider, P., Etges, R. & Bordier, C. (1990). Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania. Biochemistry 29, 10113–19.CrossRefGoogle ScholarPubMed
Button, L. L. & McMaster, W. R. (1988). Molecular cloning of the major surface antigen of Leishmania. Journal of Experimental Medicine 167, 724–9; [Correction (1990), 171, 599.]CrossRefGoogle ScholarPubMed
Button, L. L., Russell, D. G., Klein, H. L., Medina-Acosta, E., Karess, R. E. & McMaster, W. R. (1989). Genes encoding the major surface glycoprotein in Leishmania are tandemly linked at a single chromosome locus and are constitutively transcribed. Molecular and Biochemical Parasitology 32, 271–84.CrossRefGoogle Scholar
Button, L. L., Reiner, N. E. & McMaster, W. R. (1991). Modification of GP63 genes from diverse species of Leishmania for expression of recombinant protein at high levels in Escherichia coli. Molecular and Biochemical Parasitology 44, 213–24.CrossRefGoogle ScholarPubMed
Button, L. L., Wilson, G., Astell, C. R. & McMaster, W. R. (1993). Recombinant Leishmania surface glycoprotein GP63 is secreted in the baculovirus expression system as a latent metalloproteinase. GENE 134, 75–81.CrossRefGoogle ScholarPubMed
Chaudhuri, G., Chaudhuri, M., Pan, A. & Chang, K.-P. (1989). Surface acid proteinase (gp63) of Leishmania mexicana: a metallo-enzyme capable of protecting liposome-encapsulated proteins from phagolysosomal degradation by macrophages. Journal of Biological Chemistry 264, 7483–9.CrossRefGoogle Scholar
Connell, N. D., Medina-Acosta, H., McMaster, W. R., Bloom, B. R. & Russell, D. G. (1993). Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guerin expressing the Leishmania surface proteinase gp63. Proceedings of the National Academy of Sciences, USA 90, 11473–7.CrossRefGoogle ScholarPubMed
Crabbe, T., Willenbrock, F., Eaton, D., Hynds, P., Carne, A. F., Murphy, G. & Docherty, A. J. P. (1992). Biochemical characterization of matrilysin. Activation conforms to the stepwise mechanisms proposed for other matrix metalloproteinases. Biochemistry 32, 8500–7.CrossRefGoogle Scholar
Cruz, A., Coburn, C. M., & Beverley, S. M. (1991). Double targeted gene replacement for creating null mutants. Proceedings of the National Academy of Sciences, USA 88, 7170–4.CrossRefGoogle ScholarPubMed
Frommel, T. O., Button, L. L., Fujikura, Y. & McMaster, W. R. (1990). The major surface glycoprotein (GP63) is present in both life stages of Leishmania. Molecular and Biochemical Parasitology 38, 25–32.CrossRefGoogle ScholarPubMed
Grant, G. A., Goldberg, G. I., Wilhelm, S. M., He, C. & Eisen, A. Z. (1992). Activation of extracellular metalloproteinases by proteases and organomercurials. Matrix (Suppl) 1, 217–23.Google ScholarPubMed
Heumann, D., Burger, D., Vischer, T., De Colmenares, M., Bouvier, J. & Bordier, C. (1989). Molecular interactions of Leishmania promastigote surface protease with human α2-macroglobulin. Molecular and Biochemical Parasitology 33, 67–72.CrossRefGoogle Scholar
Ilg, T., Harbecke, D. & Overath, P. (1993). The lysosomal gp63-related protein in Leishmania mexicana amastigotes is a soluble metalloproteinase with an acidic pH optimum. Federation of European Biochemical Societies 327, 103–7.CrossRefGoogle ScholarPubMed
Ip, H. S., Russell, D. G. & Cross, G. A. M. (1990). L. mexicana mexicana gp63 is a site-specific neutral endopeptidase. Molecular and Biochemical Parasitology 40, 163–72.CrossRefGoogle ScholarPubMed
Jardim, A., Alexander, J., Teh, H.-S., Ou, D. W. & Olafson, R. w. (1990). Immunoprotective Leishmania major synthetic T cell epitopes. Journal of Experimental Medicine 172, 645–8.CrossRefGoogle ScholarPubMed
Laban, A., Tobin, J. F., De Lafaille, M. A. C., & Wirth, D. F. (1990). Stable expression of the bacterial neor gene in Leishmania enriettii. Nature 343, 572–4.CrossRefGoogle ScholarPubMed
Lebowitz, J. H., Coburn, C. M., McMahon-Pratt, D., & Beverley, S. M. (1990). Development of a stable Leishmania expression vector and application to the study of parasite surface antigen genes. Proceedings of the National Academy of Sciences, USA 87, 9736–40.CrossRefGoogle Scholar
Letarte, M., Vera, S., Tran, R., Addis, J. B., Onizuka, R. J., Quackenbush, E. J., Jongeneel, C. V. & Mcinnes, R. R. (1988). Common acute lymphocytic leukemia antigen is identical to neutral endopeptidase. Journal of Experimental Medicine 168, 1247–53.CrossRefGoogle ScholarPubMed
Lopez, A. J., Reins, H.-A., Etges, R. J., Button, L. L., McMaster, W. R., Overath, P. & Klein, J. (1991). Genetic control of the immune response in mice to Leishmania mexicana surface protease. Journal of Immunology 146, 1328–34.CrossRefGoogle ScholarPubMed
Matthews, B. W. (1988). Structural basis of the action of thermolysin and related zinc metalloproteinases. Acc. Chem. Res. 21, 333–40.CrossRefGoogle Scholar
Matthews, B. W., Jansonius, J. N., Colman, P. M., Schoenborn, B. P. & Duporque, D. (1972). Three-dimensional structure of thermolysin. Nature (New Biology) 238, 37–41.CrossRefGoogle ScholarPubMed
Medina-Acosta, E., Karess, R. E., Schwartz, H. & Russell, D. G. (1989). The promastigote surface protease (gp63) of Leishmania is expressed but differentially processed and localized in the amastigote stage. Molecular and Biochemical Parasitology 37, 263–74.CrossRefGoogle ScholarPubMed
Medina-Acosta, E., Karess, R. E. & Russell, D. (1993). Structurally distinct genes for the surface protease (gp63) of Leishmania mexicana are developmentally regulated. Molecular and Biochemical Parasitology 57, 31–46.CrossRefGoogle ScholarPubMed
Miller, R. A., Reed, S. G. & Parsons, M. (1990). Leishmania gp63 molecule implicated in cellular adhesion lacks an Arg-Gly-Asp sequence. Molecular and Biochemical Parasitology 39, 267–74.CrossRefGoogle ScholarPubMed
Nagase, H., Barrett, A. J. & Woesdsner, J. F. (1992). Nomenclature and glossary of the matrix metalloproteinases. Matrix (Suppl) 1, 421–4.Google ScholarPubMed
Ramamoorthy, R., Donelson, J. E., Paetz, E. E., Maybodi, M., Roberts, S. C. & Wilson, M. E. (1992). Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form. Journal of Biological Chemistry 267, 1888–95.CrossRefGoogle Scholar
Russell, D. G. & Alexander, J. (1988). Effective immunization against cutaneous leishmaniasis with denned membrane antigens reconstituted into liposomes. Journal of Immunology 140, 1274–9.CrossRefGoogle Scholar
Russell, D. G. & wilhelm, H. (1986). The involvement of the major surface glycoprotein (gp63) of Leishmania promastigotes in attachment to macrophages. Journal of Immunology 136, 2613–20.CrossRefGoogle ScholarPubMed
Russo, D. M., Burns, J. M. Jr., Carvalho, E. M., Armitage, R. J., Grabstein, K. H., Button, L. L., McMaster, W. R. & Reed, S. G. (1991). Human T cell responses to gp63, a surface antigen of Leishmania. Journal of Immunology 147, 3575–80.CrossRefGoogle Scholar
Schneider, P., Rosat, J.-P., Bouvier, J., Louis, J. & Bordier, C. (1992). Leishmania major: differential regulation of the surface metalloprotease in amastigote and promastigote stages. Experimental Parasitology 75, 196–206.CrossRefGoogle ScholarPubMed
Springman, E. B., Angleton, E. L., Birkedal-Hansen, H. & Van Wart, H. E. (1990). Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a ‘cysteine switch’ mechanism for activation. Proceedings of the National Academy of Sciences, USA 87, 364–8.CrossRefGoogle Scholar
Steinkraus, H. B. & Langer, P. J. (1992). The protein sequence predicted from a Leishmania guyanensis gp63 major surface glycoprotein gene is divergent as compared with other Leishmania species. Molecular and Biochemical Parasitology 52, 141–4.CrossRefGoogle ScholarPubMed
Tobin, J. F., Laban, A., & Wirth, D. F. (1991). Homologous recombination in Leishmania enriettii. Proceedings of the National Academy of Sciences, USA 88, 864–8.CrossRefGoogle ScholarPubMed
Tzinia, A. K. & Soteriadou, K. P. (1991). Substrate-dependent pH optima of gp63 purified from seven strains of Leishmania. Molecular and Biochemical Parasitology 47, 83–90.CrossRefGoogle ScholarPubMed
Vallee, B. L. & Auld, D. s. (1992). Active zinc binding sites of zinc metalloproteinases. Matrix (Suppl) 1, 5–19.Google Scholar
Webb, J., Button, L. L. & McMaster, W. R. (1991). Heterogeneity of the genes encoding the major surface glycoprotein of Leishmania donovani. Molecular and Biochemical Parasitology 48, 173–84.CrossRefGoogle ScholarPubMed
Yang, D. M., Fairweather, N., Button, L. L., McMaster, W. R., Kahl, L. P. & Liew, F. Y. (1990) Oral Salmonella typhimurium (AroA−) vaccine expressing a major Leishmania surface protein (GP63) preferentially induces Th1 cells and protective immunity against Leishmaniasis. Journal of Immunology 145, 2281–5.CrossRefGoogle Scholar