Substrate specificity and inhibitor sensitivity of a Trypanosoma cruzi ...

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Feb 27, 1990 - to organisms of the phylogenetic order, Kinetoplastida, or to the family, Trypanosomatida. We have examined the alkaline peptidase of T. cruzi.


866 enzyme is a true cathepsin L. No enzyme with properties of cathepsin B were detected in T. cnrzi, although the enzyme described by Cazzulo and co-workers [3-51 has some features in common with cathepsin B. The enzyme that cleaves Ala-Ala-pNA is inhibited by E64, iodoacetic acid and leupeptin, but is not affected by inhibitors of serine, metallo- or aspartic peptidases. It does not release p-nitroaniline from Boc-Ala-Ala-pNA or from Ala-Ala-Ala-pNA, and therefore has properties of a cysteine-type dipeptidylaminopeptidase. We are examining the possibility that this enzyme is related to cathepsin C described in other systems. Another enzyme occurs in T. cruzi that cleaves Boc-Ala-Ala-pNA and this enzyme is inhibited by diisopropylfluorophosphate but not by phenylmethanesulphonylfluoride, leupeptin, pepstatin A, ophenanthroline, iodoacetic acid or E64. This serine peptidase is a major activity in epimastigote extracts.

However, of all the enzymes that we have detected in T. cmzi epimastigotes, the alkaline peptidase, which characteristically readily cleaves Bz-Arg-pNA, is the most abundant activity. Indeed, this enzyme apparently occurs as a major activity in other trypanosomatids [ 2 ] .Table 1 summarizes the peptidases that we have detected in T. cruzi epimastigotes using chromogenic and fluorogenic substrates. Fluoromethanes were kindly given by Dr Elliott Shaw. 1. Greig, S. & Ashall, F. ( 1 990) Mol. Biochem. I’arasitol. 39.3 1-38 2. Ashall, F. ( 1 990) Mol. Biochem. Parasitol. 38,77-88 3. Bontempi, E. et al. (1984) Comp. Biochem. Physiol. 77B, 599-604 4. Cazzulo, J. J. et al. ( I 989) Moi. Biochem. Parasitof. 33.33-42 5 . Bontempi, E. et al. (1989) Mol. Biochem. I’arasitol. 33,43-48 ~

Received 27 February 1990

Substrate specificity and inhibitor sensitivity of a Typunosomu cruzi alkaline peptidase HELEN ROBERTS: NlAMH HEALY,* ELLIOTT SHAWt and FRANK ASHALL*$ *Department of Pure and Applied Biology, Imperial College, South Kensington, London SW 7 ZRB, U.K. and tFriedrich Miescher Institute, Basle, Switzerland

at p2 at substrate concentrations below about 5 p ~Because . we have failed to obtain cleavage of intact proteins by the alkaline peptidase [ l ] , we propose that this enzyme is involved in cleavage of a restricted number of peptide or protein substrates in trypanosomatids. Indeed, peptidases with restricted protein substrate specificity that cleave on the carboxyl side of pairs of basic residues occur widely in biological systems and are frequently involved in protein processing [ 21. The alkaline peptidase is sensitive to Tos-Lys-CH,CI, leupeptin, diisopropylfluorophosphate (DFP)and to peptidyldiazomethanes containing arginine or lysine at P 1. Since DFP generally inhibits serine peptidases, whereas diazo-

We are investigating the peptidases of Trypanosoma cmzi, the protozoan parasite that causes South American trypanosomiasis. All stages of the life cycle of this parasite express a peptidase that cleaves on the carboxyl side of arginine and lysine residues of p-nitroanilide (pNA) and amidomethylcoumarin (MCA) substrates at alkaline pH [I]. In the absence of added reducing agents the alkaline peptidase is the major enzyme in detergent extracts of T. cruzi epimastigotes that cleaves these substrates at pH 8.0. We have detected, on the basis of pH profile, substrate specificity and 1 2 3 4 5 6 7 8 9 inhibitor sensitivity as well as electrophoretic mobility, a similar enzyme in 15 other species of trypanosomatid, including African trypanosomes and Leishmania [I].Because we have not detected a similar enzyme in any nontrypanosomatid protozoa or mammalian cells that we have tested, we hypothesize that the alkaline peptidase is specific to organisms of the phylogenetic order, Kinetoplastida, or to the family, Trypanosomatida. We have examined the alkaline peptidase of T. cruzi epimastigotes for its ability to cleave peptidyl-MCA substrates and for its sensitivity to a range of irreversible and reversible inhibitors. Purified T. cruzi alkaline peptidase was found to cleave Bz-Arg-MCA with a K , of about 12 p ~ . However, when a second basic residue is added to the arginine residue, as in Cbz-Arg-Arg-MCA, the K, falls by an order of magnitude to about 1 p ~All . substrates examined Fig. 1. Effects ofpeptidase inhibitors on the activity of T. cmzi that contained two basic residues (Cbz-Arg-Arg-MCA, alkaline peptidase in polyacrylamide gels Cbz-Gly-Arg-Arg-MCA, Cbz-Leu-Gly-Arg-MCA, CbzA detergent extract (1% Nonidet P-40) of T. cmzi Y strain Asn-Arg-Arg-MCA, Cbz-Gly-Lys-Arg-MCA and Cbz-LeuLys-Arg-MCA) had a K , value of about 1 p ~ whereas , all epimastigotes was electrophoresed and gel strips were pre. Vma. treated with various inhibitors for 10 min at 25°C. Strips other substrates had a K, greater than 10 p ~ The values for the substrates containing two basic residues are were then incubated at 25”C, pH 8, with 100 pM-Bz-Argalso lower than those for other substrates. From the data, it is MCA for 30 min and photographed on a fluorescent light apparent that the alkaline peptidase prefers arginine o r lysine box. Lane 1, no inhibitor; lane 2, 1 mwphenylmethanesulphonyl fluoride; lane 3, 50 p ~ - E 6 4 ;lane 4, 50 p ~ Abbreviations used: pNA, p-nitroanilide; MCA, amidomethylleupeptin; lane 5, 50 pwpepstatin A; lane 6, 50 courmarin; Bz, benzoyl; Cbz, benzyloxycarbonyl; Tos, tosyl; DFP, ~M-Tos-L~s-CH~CI; lane 7, 1 mM-o-phenanthroline; lane 8, diisopropylfluorophosphate. 50 pM-Cbz-Leu-Lys-diazomethane; lane 9 , 5 0 pM-Cbz-LeuMet-diazomethane. $To whom correspondence should be addressed. 1990

634th MEETING, BATH methanes are considered to be cysteine peptidase inhibitors 131, the class of peptidase to which the enzyme belongs is unclear and must await sequence data on the enzyme protein. Unlike most cysteine peptidases, the enzyme is not inhibited by E64 or iodoacetic acid. Certain organic bases, and especially those resembling arginine or lysine side-chains, also inhibit the T. cnizi alkaline peptidase. Thus, n-butylamine and agmatine inhibit the enzyme. Indeed, hirudonin and the anti-protozoal drug, pentamidine, are effective reversible inhibitors of the peptidase and, in agreement with the substrate preference for pairs of basic residues, these compounds resemble two arginine residues joined together. Figure I shows the T. cruzi alkaline peptidase as detected in polyacrylamide gels using the fluorogenic substrate, BzArg-MCA. A single band of fluoresence occurs in total detergent extracts of T. cnrzi, in agreement with our hypothesis that the parasite has one major enzyme that cleaves

867 this substrate [ l ] .The intensity of this band is reduced by leupeptin, Tos-Lys-CHzCI and Cbz-Leu-Lys-diazomethane, which agrees exactly with the inhibitor profile of the purified enzyme as determined using solution assays. The occurrence of the trypanosomatid alkaline peptidase as a major enzyme in T. cruzi and other trypanosomatids suggests that it is fundamental to these organisms. Its study should contribute to our knowledge of peptidases and to our understanding of organisms that cause some of the most devastating maladies of man. 1. Ashall, F. ( 1990) Mo/. Biochem. I’circlsird. 38, 77-XX 2. Fisher, J. M. & Scheller. R. H. (19x8) J . Hiol. Cham. 263. 16515-16518 3. Green, G. D. J. & Shaw, E. ( 1981) J. Biol. (’hem. 256, 1923-1928

Received 27 February I990

Mapping of B-cell epitopes on the Leishmania donovuni 70 kDa heat-shock protein G. R. WALLACE, J. MACFARLANE,A. E. BALL, J. L. CLARKE, M. A. MILES and J. M. KELLY Department of Medical Parasitology, London School of Il-vgieneund Tropical Medicine, Keppel Street, London WC’IE 7flT, U . K . The kinetoplastid protozoan Leishmuniu donovuni is the causative agent of visceral leishmaniasis (VL), a disease widespread in tropical and subtropical regions of the world. A llgt I I cDNA library was prepared from L. dono~ani mRNA and screened with sera from VL-infected patients. Inserts from positive cDNA clones were used to isolate the corresponding genomic DNA clones from a L-EMBL3 library, which were then analysed to determine the sequence [ I ] . This protocol led to the identification of a member of the 70 kDa heat-shock protein family (Hsp7O) as a major immunogen of the parasite. A series of recombinant deletion constructs was prepared, in the expression vector Bluescript (Stratagene).These were screened by Western blotting using sera from VL patients to determine regions containing B cell epitopes. An immunodominant epitope was identified in the (’-terminal region of the protein which was recognized by 90% o f sera tested. To map this epitope further, the region Lys-495-Ser-634, was synthesized as a set of overlapping peptides (10 mer overlapped by 5 ) on the Pepscan system (Cambridge Research Biochemicals) [ 21. The peptides are synthesized o n to polyetheylene pins which have been coated with polyacrylic acid and derivatized with hexamethylene diamine. Deprotection of the pins by piperidine/dimethylformamide (DMF), followed by washing with DMF and methanol, precedes the attachment of the first Fmoc amino acids 131. The cycle is repeated until the desired peptide is synthesized. At this stage, ihe side-chain protecting groups are removed and the peptides can be screened with antibodies in a simple ELISA system using 2,2-azino-bis(3-ethyIbenzthiazoline-6sulphonic acid) as the substrate. The peptides can be screened repeatedly on removal of bound antibody- by. sonication. Abbreviations used: VL, visceral leishmaniasis; Hsp, heat-shock protein; DMF, dimethylformamide.

Vol. 18

Amino acid number 523 D A M K A M K M K K







539 A A A A A A A




A,,, 0.20 1.93 2.35 1.80 1.63 1.51 0.43 0.10

Fig. 1. Identificurion of EADDRA us the immiinodominant epitope of the C-terminal region of Hsp7Ofrom L. donovuni Enzyme-linked immunosorbent assay results from peptides screened with T F sera

Peptides encompassing the C-terminal region of Hsp7O were reacted with serum from a patient ( T F ) with VL. Two peaks of activity were identified Met-525-Glu-534 and Ser615-Pro-624. A second set of peptides were synthesized covering the 525-534 region ( 10 mer overlapping by 9) and again tested with TF serum. The results (Fig. 1 ) clearly demonstrate that the epitope is EADDRA (528-533), as adjacent peptides which lose any of the above amino acids d o not give a positive signal. This epitope is found in a poorly conserved region o f the protein and may have an application in the diagnosis of VL. The second epitope identified by the peptide screening is currently being evaluated against a panel of sera from VLinfected patients to determine what role it may play in the humoral response to L. donovuni infection. I . MacFarlane, J., Blaxter, M. L., Bishop, R. P., Miles, M. A. & M, Hiochem,in the press 2, ceysen, H, M., R, H, & ~ ~ ~S, J,~(19x4) ~ I+.0c, l i N ~ , , /~. ~sci. U.S.A. ~ ~ 81.3998-4oo2 d . 3. Carnino. L. A. & Han. G. Y. 11072) J . ow. (%c,rn. 37. 34d4-3409 J,

J ,

Received 6 March 1990