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. 2000 Dec;9(12):2567–2572. doi: 10.1110/ps.9.12.2567

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.

P M Smooker 1, J C Whisstock 1, J A Irving 1, S Siyaguna 1, T W Spithill 1, R N Pike 1
PMCID: PMC2144524  PMID: 11206078

Abstract

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.

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Selected References

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  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barrett A. J., Kirschke H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol. 1981;80(Pt 100):535–561. doi: 10.1016/s0076-6879(81)80043-2. [DOI] [PubMed] [Google Scholar]
  3. Berasain P., Carmona C., Frangione B., Dalton J. P., Goñi F. Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced. Exp Parasitol. 2000 Feb;94(2):99–110. doi: 10.1006/expr.1999.4479. [DOI] [PubMed] [Google Scholar]
  4. Berti P. J., Storer A. C. Alignment/phylogeny of the papain superfamily of cysteine proteases. J Mol Biol. 1995 Feb 17;246(2):273–283. doi: 10.1006/jmbi.1994.0083. [DOI] [PubMed] [Google Scholar]
  5. Carmona C., Dowd A. J., Smith A. M., Dalton J. P. Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles. Mol Biochem Parasitol. 1993 Nov;62(1):9–17. doi: 10.1016/0166-6851(93)90172-t. [DOI] [PubMed] [Google Scholar]
  6. Chapman C. B., Mitchell G. F. Proteolytic cleavage of immunoglobulin by enzymes released by Fasciola hepatica. Vet Parasitol. 1982 Nov;11(2-3):165–178. doi: 10.1016/0304-4017(82)90039-5. [DOI] [PubMed] [Google Scholar]
  7. Coulombe R., Grochulski P., Sivaraman J., Ménard R., Mort J. S., Cygler M. Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. EMBO J. 1996 Oct 15;15(20):5492–5503. [PMC free article] [PubMed] [Google Scholar]
  8. Dowd A. J., McGonigle S., Dalton J. P. Fasciola hepatica cathepsin L proteinase cleaves fibrinogen and produces a novel type of fibrin clot. Eur J Biochem. 1995 Aug 15;232(1):241–246. doi: 10.1111/j.1432-1033.1995.tb20805.x. [DOI] [PubMed] [Google Scholar]
  9. Dowd A. J., Smith A. M., McGonigle S., Dalton J. P. Purification and characterisation of a second cathepsin L proteinase secreted by the parasitic trematode Fasciola hepatica. Eur J Biochem. 1994 Jul 1;223(1):91–98. doi: 10.1111/j.1432-1033.1994.tb18969.x. [DOI] [PubMed] [Google Scholar]
  10. Dowd A. J., Tort J., Roche L., Ryan T., Dalton J. P. Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae. Mol Biochem Parasitol. 1997 Sep;88(1-2):163–174. doi: 10.1016/s0166-6851(97)00090-x. [DOI] [PubMed] [Google Scholar]
  11. Drenth J., Kalk K. H., Swen H. M. Binding of chloromethyl ketone substrate analogues to crystalline papain. Biochemistry. 1976 Aug 24;15(17):3731–3738. doi: 10.1021/bi00662a014. [DOI] [PubMed] [Google Scholar]
  12. Heussler V. T., Dobbelaere D. A. Cloning of a protease gene family of Fasciola hepatica by the polymerase chain reaction. Mol Biochem Parasitol. 1994 Mar;64(1):11–23. doi: 10.1016/0166-6851(94)90130-9. [DOI] [PubMed] [Google Scholar]
  13. Klebe R. J., Harriss J. V., Sharp Z. D., Douglas M. G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene. 1983 Nov;25(2-3):333–341. doi: 10.1016/0378-1119(83)90238-x. [DOI] [PubMed] [Google Scholar]
  14. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  15. McKerrow J. H., Sun E., Rosenthal P. J., Bouvier J. The proteases and pathogenicity of parasitic protozoa. Annu Rev Microbiol. 1993;47:821–853. doi: 10.1146/annurev.mi.47.100193.004133. [DOI] [PubMed] [Google Scholar]
  16. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411–2423. doi: 10.1002/pro.5560041120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Perrière G., Gouy M. WWW-query: an on-line retrieval system for biological sequence banks. Biochimie. 1996;78(5):364–369. doi: 10.1016/0300-9084(96)84768-7. [DOI] [PubMed] [Google Scholar]
  18. Roche L., Dowd A. J., Tort J., McGonigle S., MacSweeney A., Curley G. P., Ryan T., Dalton J. P., McSweeney A. Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae. Eur J Biochem. 1997 Apr 15;245(2):373–380. doi: 10.1111/j.1432-1033.1997.t01-1-00373.x. [DOI] [PubMed] [Google Scholar]
  19. Sali A., Blundell T. L. Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol. 1993 Dec 5;234(3):779–815. doi: 10.1006/jmbi.1993.1626. [DOI] [PubMed] [Google Scholar]
  20. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tkalcevic J., Ashman K., Meeusen E. Fasciola hepatica: rapid identification of newly excysted juvenile proteins. Biochem Biophys Res Commun. 1995 Aug 4;213(1):169–174. doi: 10.1006/bbrc.1995.2112. [DOI] [PubMed] [Google Scholar]
  22. Tort J., Brindley P. J., Knox D., Wolfe K. H., Dalton J. P. Proteinases and associated genes of parasitic helminths. Adv Parasitol. 1999;43:161–266. doi: 10.1016/s0065-308x(08)60243-2. [DOI] [PubMed] [Google Scholar]
  23. Turk D., Guncar G., Podobnik M., Turk B. Revised definition of substrate binding sites of papain-like cysteine proteases. Biol Chem. 1998 Feb;379(2):137–147. doi: 10.1515/bchm.1998.379.2.137. [DOI] [PubMed] [Google Scholar]
  24. Wijffels G. L., Panaccio M., Salvatore L., Wilson L., Walker I. D., Spithill T. W. The secreted cathepsin L-like proteinases of the trematode, Fasciola hepatica, contain 3-hydroxyproline residues. Biochem J. 1994 May 1;299(Pt 3):781–790. doi: 10.1042/bj2990781. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wilson L. R., Good R. T., Panaccio M., Wijffels G. L., Sandeman R. M., Spithill T. W. Fasciola hepatica: characterization and cloning of the major cathepsin B protease secreted by newly excysted juvenile liver fluke. Exp Parasitol. 1998 Feb;88(2):85–94. doi: 10.1006/expr.1998.4234. [DOI] [PubMed] [Google Scholar]
  26. Yamasaki H., Aoki T. Cloning and sequence analysis of the major cysteine protease expressed in the trematode parasite Fasciola sp. Biochem Mol Biol Int. 1993 Nov;31(3):537–542. [PubMed] [Google Scholar]

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