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Hydrolysis of l -leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases
lnrrrnurmml Journalfor Prrn1rd in Grrrrt Eirrrlm
Pwosmlo~y
Vol.
22. No.
6. pp.
71 I-711.
1992 SC-B 1992
Auscrulrun
002&7519/92 $5.00 + 0.00 Pergmon Press Lid Si~cir~yfor Pmmrolog~v
HYDROLYSIS OF L-LEUCINE METHYL ESTER BY LEISHMANIA MEXICANA MEXICANA AMASTIGOTE CYSTEINE PROTEINASES CHRISTOPHERA.HUNTER,*BARBARAC.LOCKWOOD~~~GRAHAMH. Laboratory
of Biochemical
Parasitology,
Department
ofZoology,
UniversityofGlasgow,
COOMBS~ GlasgowG12
8QQ, Scotland,
U.K.
(Received 9 April 199 I ; accepted 1 I May 1992)
C.A., LOCKWOODB.C. and COOMEISG.H. 1992. Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases. International Journal,for Parasitology 22: 71 l-717. The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatinsodium dodecyl sulphatee polyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite’s cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.
Abstract-HUNTER
INDEX
KEY WORDS:
Leishmaniu
mexicana mexicanu; proteinases;
INTRODUCTION RABINOVITCH,
originally several
ZILBERFARB
reported r-amino
the
acid
&
RAMAZEILLES
antileishmanial
esters,
notably
(1986)
activity L-leucine
of
methyl
towards parasites within macrophages. Subsequently, the compounds were shown to be active towards isolated amastigotes (Rabinovitch, Zilberfarb & Pouchelet, 1987). We found, however, that it is only amastigotes of species in the Leishmania mexicana complex that are highly susceptible to r-leucine methyl ester (Hunter, MacPherson & Coombs, 1989). The amastigotes of these species characteristically contain highly active cysteine proteinase activities (Pupkis & Coombs, 1984) located within unusual lysosome-like organelles termed megasomes (Pupkis, Tetley & Coombs, 1986). It has been proposed that these enzymes account for the peculiar sensitivity of the parasites to amino acid esters (Hunter et al., 1989; Rabinovitch, 1989; Alfieri, Shaw, Zilberfarb & Rabinovitch, 1989). These esters also disrupt preparations of rat liver lysosomes, causing swelling and lysis (Goldman & Kaplan, 1973). This is thought to be due to the ester,
hydrolysis
of the esters
by lysosomal
enzymes
and
leucine methyl ester; megasomes.
consequent accumulation of amino acids causing osmotic stress (Reeves, 1979). It has been suggested that the antileishmanial activity of the compounds is mediated in a similar way, with the cysteine proteinases in megasomes being responsible for the hydrolysis. This hypothesis is based upon an assortment of findings including the lysosomal nature of megasomes (Pupkis et al., 1986) the positive correlation between the presence of megasomes containing cysteine proteinases and susceptibility to the esters (Hunter et al., 1989; Tetley, Hunter, Coombs & Vickerman, 1989) and also the protection against the esters afforded by inhibitors of cysteine proteinases (Alfieri, Ramazei!les, Zilberfarb, Galpin, Norman & Rabinovitch, 1988). In this paper we describe the copurification of the amastigote cysteine proteinases with a methyl esterase activity and describe some properties of the two activities. The results provide direct evidence that cysteine proteinases are responsible for the hydrolysis of L-leucine methyl ester in amastigotes and provides further support for the proposed mechanism of action of the L-amino acid esters against L. m. mexicana.
the
MATERIALSANDMETHODS address: Palo Alto Medical Research * Present Foundation, 860 Bryant Street, Palo Alto, CA 94301, U.S.A. t To whom all correspondence should be addressed.
Parasites. L. m. mexicuna (MNYC/BZ/62/M379) was maintained by serial subpassage in female CBA mice (Department of Zoology, University of Glasgow). Amastigotes were purified by the method described by Hart, 71 1
C. A. HURTER ef ui.
712
(cl
I
/I
34
56
30
20
Fraction
2
number
FIG. la
FIG. IL‘ I
2
3
4
5
6
7
8
FIG. 1. Separation of proteinases of L. m. m~xicarzn amastigotes by gel filtration. a. The cell extract was fractionated by gel ~ltration using a Superose 12 column. The eiuted protein, measured as A,,, (-), and the change in absorbance caused by the proteinase activity towards BzPFRNan (-•-) are shown. b. Samples (25 ~1) of the eluant fractions were analysed by gelatinSDSPAGE using a 5% (w/v) acrylamide mini-gel. Tracks l-8 correspond to fractions 1219. The low molecular weight enzymes ran as a single band at the dye front (tracks 668). The positions of molecular weight markers (kDa) are shown. c. Samples (50 ~1) of the eluant fractions were analysed by gelatin-SDS-PAGE using a 7.5% (w/v) acrylamide gel. Tracks I-6 correspond to alternate fractions from 17-27.
Cysteine
proteinases
Vickerman & Coombs (1981) and modified by Mottram & Coombs (1985). Cell lysates produced by resuspension of IO9 parasites in 0.5 ml 0.25 M-sucrose containing 0.25% Triton X-100 were centrifuged at 240,000 x g for 1hat 4’C and the supernate used as the source of enzyme. These preparations were either used immediately or stored at -70°C until needed. Partial purification of proteinuses. Purification of the cysteine proteinases was carried out essentially as described by Pupkis & Coombs (1984) but using a Fast Protein Liquid Chromatography (FPLC) system. One millilitre of the cell lysate supernatant was applied to a Superose 12 column (I x 40 cm) previously equilibrated with 0.15 M-sodium phosphate buffer, pH 6.0, and eluted with the same buffer. Fractions (1 ml) of eluant containing the proteinases were pooled, concentrated using an Amicon Ultrafiltration Cell with a PM 10 membrane filter and applied in 1 or 2 ml volumes to a Mono Q column (1 ml) which had been equilibrated with 0.025 M-sodium phosphate buffer, pH 6.0. Unbound protein was eluted with 5 ml ofequilibration buffer and elution of bound proteins was carried out using a salt gradient of O-O.35 M-NaCI in the same phosphate buffer. Fractions (I ml) containing proteinase activity were pooled and concentrated as before. Electrophoresis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gelatin-SDS-PAGE were carried out as previously described (Lockwood, North, Mallinson & Coombs, 1987) with either 5, 7.5 or 10% (w/v) acrylamide in the separating gel and 5% (w/v) in the stacking gel. Samples were run under reducing conditions but were not boiled unless stated. Protein was detected either by Coomassie blue or silver staining (Oakley, Kirsch & Morris. 1980). Meusurement of proteinuse activity. Chromatographic fractions were assayed for proteinase activity using N-benzoylproline-phenylalanine-arginine p-nitroanilide (BzPFRNan). The substrate was used at a final concentration of 0.075 mM in a I IO ~1 reaction volume containing 1 mM-dithiothreitol (DTT) and 100 ~1 of each fraction. The change in absorbance at 405 nm over 10 min incubation at room temperature was measured using a Titertek Multiscan MCC/340. A change in absorbance of 1.O was given a value of 100 units of proteinase activity. To determine the specific activities of samples towards BzPFRNan at 37°C assays were carried out in 0.15 M-sodium phosphate buffer, pH 6.0, containing 0.1 mMBzPFRNan and 1 mM-DTT in a total reaction volume of I .2 ml. The reaction mixture was preincubated at 37°C for 10 min before the addition of DTT and BzPFRNan to initiate the reaction. The change in absorbance was monitored at 405 nm and the molar extinction coefficient of 4-nitroaniline was taken as 9500 I mol ‘cm-’ (Pupkis & Coombs. 1984). Protein estimation was carried out using the method of Sedmak & Grossberg (1977).with bovine serum albumin as standard. Detection of enzymatic hydrolysis of L-Ieucine methyl ester. Paper electrophoresis was used according to the method of Goldman & Kaplan (1973) to separate the products of the hydrolysis of t_-leucine methyl ester from the parent compound. The incubation mixture comprised 0.15 Msodium phosphate buffer, pH 6.0, I mM-DTT and 20 mM of L-
of Leishmania
713
leucine methyl ester. For determining the pH optimum of the activity the following buffers were used: 0.1 M-citric acid, pH
3-6 and 0.1 M-sodium phosphate, pH 6-8. The proteinase inhibitors antipain, chymostatin and E-64 were also included in some experiments as indicated. Antipain and E-64 were dissolved in water whilst chymostatin was solubihzed in dimethylsuiphoxide. Controls containing DMSO were carried out where appropriate. In all experiments reported, the amount of enzyme activity used was that which with BzPFRNan as substrate and under the standard conditions described above would have resulted in a change of absorbance mini’ of 0.1 (i.e. the release of 12.9 nmoles pnitroaniline min ‘). The reaction mixture was incubated for 3 h at 37°C and 40 ~1 aliquots then applied immediately to strips (3 x 25 cm) of Whatman 3MM paper. Electrophoresis was carried out at 8 V cm ’ for 3 h using a pyridine acetate buffer, pH 3.5 (glacial acetic acid: pyridine: water/ 10: 1:89,by volume). Subsequently the paper was air dried and stained by immersion in a solution of 0.2% ninhydrin in 75% (v/v) acetone for 30 min. The reaction products were quantified by eluting them with 10 ml of 0.4% cadmium acetate in water: glacial acetic acid: ethanol (1:5:4, by volume) and measuring the absorbance at 505 nm of the solution. The method was calibrated using leucine as standard. Non-enzymatic breakdown of L-leucine methyl ester was taken into account. LLeucine methyl ester, proteinase inhibitors, DTT and other biochemicals were purchased from Sigma. RESULTS Partial
purtjication
of proteinases
filtration resulted in the separation of several lower molecular weight proteinases from the majority of the larger proteinases. This was clearly shown by the bands of activity detected using gelatin-SDS-PAGE (Fig. I). The application of this technique also demonstrated that there was a partial separation of the various enzymes of higher molecular mass (Fig. 1b), although interestingly their order of elution from the Superose column did not correlate precisely with their apparent size as revealed by gelatin-SDS-PAGE. In Gel
I
20
IO
Fraction
number
FIG. 2a
30
C. A. HUNTERer ~1.
714
(b)
12345
6
789
36
24
FIG. 2b.
I
2
3
4
5
6
7
8
9
IO
- 36
FIG.2. The partial separation by ion exchange chromatography of the lower molecular weight proteinases of L. m. nw~icunu amastigotes. a. Fractions containing the lower molecular weight proteinases separated on gel filtration were pooled, concentrated and then submitted to ion exchange chromatography. The eluted protein, measured as A,,,, (-), and the change in absorbance caused by the proteinase activity towards BzPFRNan (-•-) are shown. The salt gradient (’ ~ .) is also shown (see methods for details). b. Samples (75 ,uI) from fractions 14-22 (tracks l-9) were analysed by gelatin-SDS-PAGE using a 10% (w/v) acrylamide gel. The positions ofmolecular weight markers (kDa) are shown. c. Samples (75 111)from fractions 1422 (tracks l-9) separated using a 10% (w/v) acrylamide gel and silver stained for protein.
Cysteine
2
proteinases
3
--
36 )
24 )
FIG. 3. Effect of boiling on the migration of proteinases during SDS-PAGE. The pooled fractions (14-24) from ion exchange chromatography were analysed using a 10% (w/v) acrylamide gel. Key to tracks: I, molecular weight markers; 2 and 3, 75 ,uI of pooled fractions (14-24) from ion exchange chromatography; the sample in lane 3 had been boiled for 2 min after the addition of sample buffer.
TABLE I-PURIFICATION OF L. m. mexicam AMASTKOTE LOWER MOLFCULAR
Stage
Lysate Pellet Supernatant Gel filtration Ionexchange
WEIGHT
PROTEINASES*
Recovery of activity (%)t 100 5* 2 103 * 14 57f 17 35 f 18
Proteinase
176 26 534 930 1511
activity:
f f * * *
35 8 185 362 348
*The figures are the means (+ standard deviations) of seven to nine purifications carried out on separate occasions. Fractions 17-27 from gel filtration were pooled and concentrated using ultrafiltration. Fractions 14-24 from ion exchange were pooled and. if necessary, concentrated using ultrafiltration. tThe loss of activity during ultrafiltration was ignored in calculating the % recovery of activity. IThe activity towards BzPFRNan is in nmoles mini ’ (mg protein) ‘.
of Leishmanicr
715
contrast, the enzymes of lower molecular mass coeluted (Fig lb, c). These latter enzymes were partially separated by ion exchange chromatography on the Mono Q column (Fig. 2). The proteinases that were eluted first were totally separated from those that bound most strongly. However, none of the enzymes was completely purified: at least two proteinases were present in all fractions (Fig. 2b). The higher molecular weight proteinases were not detected in any of these fractions. Silver staining revealed that the profile of eluted proteins resembled that of gelatinase activity but that some other proteins were also present (Fig. 2~). There was insufficient protein in the fractions to allow detection with Coomassie blue. When the fractions (14-24) containing proteinase activity were pooled, boiled and analysed by SDSPAGE, Coomassie blue staining revealed a single band. More bands became apparent after silver staining. and the pattern was significantly different from that of the unboiled sample (Fig. 3). Some bands were noticeably more intense. whilst others were less prominent. As it was not possible to isolate the individual lower molecular weight proteinases completely, it was decided to pool the ion exchange fractions containing these enzymes (fractions 14-24) and to study them as a group. The overall purification and recovery of activity to this stage is given in Table I. The partially purified proteinases were totally inhibited by E-64 (10 pg ml-‘) and leupeptin (25 pg ml-‘) and stimulated five-fold by I rnr+DTT (data not shown) and so had the characteristics of cysteine proteinases (North, Mottram & Coombs, 1990). Hydrolysis of L-leucine methyl ester by the purtiall~~ pur$iedproteinases The separation of amino acids and their esters by paper electrophoresis formed the basis of the method developed for following hydrolysis of L-leucine methyl ester. Incubation of L-leucine methyl ester with L. m. mexicana amastigote lysate resulted in the production of leucine and leucyl-leucine (Fig. 4B). The dipeptide, which is thought to be an intermediate in the hydrolysis (Goldman & Kaplan, 1973) ran as a distinct band in advance of leucine. Similar results were obtained using the pooled fractions containing proteinase activity from the ion exchange column. Elution and quantification of the two hydrolysis products together provided a means of determining the rates of hydrolysis. It was confirmed that the reaction was linear under the conditions used. The use of the cysteine proteinase inhibitor E-64 prevented the hydrolysis of L-leucine methyl ester to leucine by a lysate of amastigotes (Fig. 4C). Amastigote lysates hydrolysed L-leucine methyl
C:. A. HUNTER
716
et 01.
FIG. 4. The hydrolysis of L-leucine methyl ester by L. m. me\-iccmcr amastigote
lysate. Paper electrophoresis was carried out as at the origin (0). Key: track A. leucine: track B, mixture resulting from the incubation of Lleucine methyl ester with L. nr. mrricanu amastigote lysate. The resultant leucine and leucyl leucine are arrowed, leucyl leucinc runs slightly ahead of leucine (Goldman & Kaplan. 1973): track C. mixture resulting from the same incubation as in B but with E-64 (100 pg ml ‘): track D. 1..leucine methyl ester.
described.
Samples were applied
ester at a specific activity of 97 f 9 nmoles min ’ (mg protein)-‘. The activity of the partially purified proteinase preparation towards L-leucine methyl ester was 1354 i 364 nmoles mini’ (mg protein))‘. Thus the activity towards L-leucine methyl ester apparently co-purified with the cysteine proteinases. The activity of the partially purified enzymes towards r-leucine methyl ester was optimal at pH 6.0, totally inhibited by E-64 at the lowest concentration tested (25 pg ml- ‘) by chymostatin (100 pg ml-‘) and by antipain (100 pg ml-‘). The activity towards r-leucine methyl ester was also stimulated three-fold by I mM-DTT. Thus the enzyme activity towards L-leucine methyl ester had characteristics of cysteine proteinases. Using the paper electrophoresis method developed, in a semi-quantitative manner, amastigote homogenates of both L. m. me.ricanu and L. donovani were shown to hydrolyse a variety of other L-amino acid methyl esters with the relative activities towards the different substrates being leucine > tyrosine > tryptophan > arginine = lysinc = isoleucine. The partially purified proteinase preparation, however, was active towards only L-leucine methyl ester and
L-leucinamide amides tested.
and not the other
methyl
esters
and
DISCUSSION
The major tinding of this study is that the L-leucinc methyl ester-hydrolysing activity and cysteine proteinases of amastigotes of L. 112. me.xic,ana coeluted. This provides the first direct evidence that these proteinases are responsible for the hydrolysis ofamino acid esters in this form of the parasite. These results also suggest that the great susceptibility of amastigotes of L. m. mexicanu to L-leucine methyl ester may be due to the fact that cysteine proteinases are characteristically abundant in these forms of parasite (Pupkis & Coombs, 1984). Whilst the amastigote homogenates hydrolysed other methyl esters to degrees which correlated with the antileishmanial activity reported by Rabinovitch cr al. (1986) the finding that the partially purified enzymes did not hydrolyse some of the other methyl esters was surprising. However, amastigotes of L. donovani, which lacks these low molecular weight cysteine proteinascs (Lockwood er ul., 1987) and which is not susceptible to L-leucine
Cysteine
proteina: ;es of Leishmania
methyl ester (Hunter et al., 1989), were also able to hydrolyse L-leucine methyl ester as well as a variety of other methyl esters. This implies that the ability to hydrolyse these esters does not by itself confer susceptibility to them but rather that the presence of the cysteine proteinases within megasomes is central to the high sensitivity of L. m. mexicana amastigotes to Lleucine methyl ester. This study has also provided new information on the proteinases of L. m. mexicana. The finding that the cysteine proteinases can be partially separated by ion exchange chromatography shows that they differ in more than apparent, molecular weight. The observation that the mobilities of the proteins were modified by boiling has several implications. It confirms that the apparent molecular weight data derived from studies using gelatin-SDS-PAGE have to be treated with caution. It also explains why in a previous study, in which these proteinases were purified to apparent homogeneity, the pure samples, which were boiled before SDS-PAGE, contained only a single band or two close running bands of protein (Pupkis & Coombs, 1984). This work has also demonstrated that some of the individual cysteine proteinases are very similar in molecular mass and that their differing mobilities in gelatin-SDS-PAGE are probably due to other differences in properties such as the degree of glycosylation or 3D-structure. Work by Koehler & Ho (1988) has shown that similar differences between multiple cysteine proteinases of barley are due to minor differences in the N-terminal sequence. In studies building upon the findings reported in this paper it has been shown that there are at least three groups of cysteine proteinases in L. m. mesicana amastigotes (Robertson & Coombs, 1990). It will be necessary to characterize these enzymes further in order to elucidate the precise ways in which they differ and gain a greater insight into their functional significance to the parasites. Acknow/edgmen/.r~We thank Dr M. J. North for helpful discussions during this work. This study was supported by the Wellcome Trust.
Acta 318: 205-216.
HART D. T., VICKERMANK. & COOMBSG. H. 1981. A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers. Parasitology 83: 529-541. HuNrEk C. A., MACPHERSON L. M. & COOMBS G. H. 1989. Antileishmanial activity of L-leucine methyl ester and Ltryptophanamide. In: Leishmaniasis. The Current Status and New Strategies,for Control(Edited by HART D. T. ), pp. 741-747. Plenum Press, New York. KOEHLER S. & Ho T. D. 1988. Purification and characterisation of gibberellic acid-induced cysteine endoproteases in barley aleurone layers. Plant Physiology 87: 95-103. LCCKWWD B. C., NORTHM. J., MALLINSON D. J. & COOMBSG. H. 1987. Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 6% kDa activity. FEMS Microbiology Letters 48: 345-350. MOTTRAMJ. C. & COOMBSG. H. 1985. Leishmania mexicana: subcellular distribution of enzymes in amastigotes and promastigotes. Experimental Parasitology 59: 265-274. NORTH M. J., MOTTRAMJ. C. & COOMBSG. H. 1990. Cysteine proteinases of parasitic protozoa. Parasitology Today 6: 270-275. OAKLEY B. R.,
KIRSCH D. R. & MORRIS N. R. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry 105: 361363. PUPKIS M. F. & COOMBS G. H. 1984. Purification and characterisation of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes. Journal
qf General Microbiology
130: 2375-2383.
PUPKIS M. F., TETLEY L. & COOMBSG. H. 1986. Leishmania mexicana: the occurrence of amastigote hydrolases in unusual organelles. E.uperimental Parasitology 62: 29-39. RABINOVITCHM., ZILBERFARB V. & RAMAZEILLESC. 1986. Destruction of Leishmania mexicana amazonensis amastigotes within macrophages by lysosomotrophic amino acid esters. Journal of E.xperimental Medicine 163: 520-535. RABINOVIKH M.,
Destruction American
ZILBERFARB V. & POUCHELET M. 1987. of isolated amastigotes by amino acid esters. Journal of Tropical Medicine and Hygiene 36:
288-293.
RABINOVITCHM. 1989. Leishmanicidal activity of amino acid and peptide esters. Parasitology Today 5: 299-30 1. REEVES J. P. 1979. Accumulation of amino acids by lysosomes incubated with amino acid methyl esters. Journal
of‘Biologica1
Chemistry
254: 89 14-8921.
ROBERTSONC. D. & COOMBSG. H. 1990. Characterisation three groups of cysteine proteinases in the amastigotes
REFERENCES ALFIERI S. C.. RAMALEILLES C., ZILBERFARB V., GALPIN I., NORMAN S. E. & RABINOVITCHM. 1988. Proteinase
Biophysics
717
inhibfrom
itors protect Lei.~hmania amazonensis amastigotes destruction by amino acid esters. Molecular and Biochemical Parasitology 29: 191-202. ALFIERI S. C., SHAW E., ZILBERFARBV. & RABINOVITCHM. 1989. Leishmania amazonensls: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester. Experimental Parasitology 68: 42343 1. GOLDMAN R. & KAPLAN A. 1973. Rupture of rat liver lysosomes mediated by L-amino acid esters. Biochemica et
Leishmania Biochemical
mexicana Parasitology
mexicana.
Molecular
of of and
41: 269-276.
SEDMAKJ. J. & GROSSBERGS. E. 1977. A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue. Analytical
Biochemistry
79: 544-552.
TETLEY L., HUNTER C. A., COOMBSG. H. & VICKERMA~ K. 1989. Generation of megasomes during the promastigoteamastigote transformation of Leishmania mexicana mexicana in vitro. In: Leishmaniasis. The Current Status andNew Strategiesfor Control (Edited by HART D. T. ), pp. 449455. Plenum Press, New York.
Pwosmlo~y
Vol.
22. No.
6. pp.
71 I-711.
1992 SC-B 1992
Auscrulrun
002&7519/92 $5.00 + 0.00 Pergmon Press Lid Si~cir~yfor Pmmrolog~v
HYDROLYSIS OF L-LEUCINE METHYL ESTER BY LEISHMANIA MEXICANA MEXICANA AMASTIGOTE CYSTEINE PROTEINASES CHRISTOPHERA.HUNTER,*BARBARAC.LOCKWOOD~~~GRAHAMH. Laboratory
of Biochemical
Parasitology,
Department
ofZoology,
UniversityofGlasgow,
COOMBS~ GlasgowG12
8QQ, Scotland,
U.K.
(Received 9 April 199 I ; accepted 1 I May 1992)
C.A., LOCKWOODB.C. and COOMEISG.H. 1992. Hydrolysis of L-leucine methyl ester by Leishmania mexicana mexicana amastigote cysteine proteinases. International Journal,for Parasitology 22: 71 l-717. The main cysteine proteinases of the amastigote form of Leishmania mexicana mexicana were partially purified by gel filtration and ion exchange chromatography. The latter procedure resulted in the separation of some individual cysteine proteinases, as demonstrated by gelatinsodium dodecyl sulphatee polyacrylamide gel electrophoresis. Fractions containing the partially purified proteinases rapidly hydrolysed L-leucine methyl ester to leucine. The activity towards this compound co-eluted with and resembled the parasite’s cysteine proteinase activity. The results suggest that amastigotes of L.m.mexicana are susceptible to L-leucine methyl ester because this compound is rapidly hydrolysed by cysteine proteinases that occur in abundance in the megasomes of this stage.
Abstract-HUNTER
INDEX
KEY WORDS:
Leishmaniu
mexicana mexicanu; proteinases;
INTRODUCTION RABINOVITCH,
originally several
ZILBERFARB
reported r-amino
the
acid
&
RAMAZEILLES
antileishmanial
esters,
notably
(1986)
activity L-leucine
of
methyl
towards parasites within macrophages. Subsequently, the compounds were shown to be active towards isolated amastigotes (Rabinovitch, Zilberfarb & Pouchelet, 1987). We found, however, that it is only amastigotes of species in the Leishmania mexicana complex that are highly susceptible to r-leucine methyl ester (Hunter, MacPherson & Coombs, 1989). The amastigotes of these species characteristically contain highly active cysteine proteinase activities (Pupkis & Coombs, 1984) located within unusual lysosome-like organelles termed megasomes (Pupkis, Tetley & Coombs, 1986). It has been proposed that these enzymes account for the peculiar sensitivity of the parasites to amino acid esters (Hunter et al., 1989; Rabinovitch, 1989; Alfieri, Shaw, Zilberfarb & Rabinovitch, 1989). These esters also disrupt preparations of rat liver lysosomes, causing swelling and lysis (Goldman & Kaplan, 1973). This is thought to be due to the ester,
hydrolysis
of the esters
by lysosomal
enzymes
and
leucine methyl ester; megasomes.
consequent accumulation of amino acids causing osmotic stress (Reeves, 1979). It has been suggested that the antileishmanial activity of the compounds is mediated in a similar way, with the cysteine proteinases in megasomes being responsible for the hydrolysis. This hypothesis is based upon an assortment of findings including the lysosomal nature of megasomes (Pupkis et al., 1986) the positive correlation between the presence of megasomes containing cysteine proteinases and susceptibility to the esters (Hunter et al., 1989; Tetley, Hunter, Coombs & Vickerman, 1989) and also the protection against the esters afforded by inhibitors of cysteine proteinases (Alfieri, Ramazei!les, Zilberfarb, Galpin, Norman & Rabinovitch, 1988). In this paper we describe the copurification of the amastigote cysteine proteinases with a methyl esterase activity and describe some properties of the two activities. The results provide direct evidence that cysteine proteinases are responsible for the hydrolysis of L-leucine methyl ester in amastigotes and provides further support for the proposed mechanism of action of the L-amino acid esters against L. m. mexicana.
the
MATERIALSANDMETHODS address: Palo Alto Medical Research * Present Foundation, 860 Bryant Street, Palo Alto, CA 94301, U.S.A. t To whom all correspondence should be addressed.
Parasites. L. m. mexicuna (MNYC/BZ/62/M379) was maintained by serial subpassage in female CBA mice (Department of Zoology, University of Glasgow). Amastigotes were purified by the method described by Hart, 71 1
C. A. HURTER ef ui.
712
(cl
I
/I
34
56
30
20
Fraction
2
number
FIG. la
FIG. IL‘ I
2
3
4
5
6
7
8
FIG. 1. Separation of proteinases of L. m. m~xicarzn amastigotes by gel filtration. a. The cell extract was fractionated by gel ~ltration using a Superose 12 column. The eiuted protein, measured as A,,, (-), and the change in absorbance caused by the proteinase activity towards BzPFRNan (-•-) are shown. b. Samples (25 ~1) of the eluant fractions were analysed by gelatinSDSPAGE using a 5% (w/v) acrylamide mini-gel. Tracks l-8 correspond to fractions 1219. The low molecular weight enzymes ran as a single band at the dye front (tracks 668). The positions of molecular weight markers (kDa) are shown. c. Samples (50 ~1) of the eluant fractions were analysed by gelatin-SDS-PAGE using a 7.5% (w/v) acrylamide gel. Tracks I-6 correspond to alternate fractions from 17-27.
Cysteine
proteinases
Vickerman & Coombs (1981) and modified by Mottram & Coombs (1985). Cell lysates produced by resuspension of IO9 parasites in 0.5 ml 0.25 M-sucrose containing 0.25% Triton X-100 were centrifuged at 240,000 x g for 1hat 4’C and the supernate used as the source of enzyme. These preparations were either used immediately or stored at -70°C until needed. Partial purification of proteinuses. Purification of the cysteine proteinases was carried out essentially as described by Pupkis & Coombs (1984) but using a Fast Protein Liquid Chromatography (FPLC) system. One millilitre of the cell lysate supernatant was applied to a Superose 12 column (I x 40 cm) previously equilibrated with 0.15 M-sodium phosphate buffer, pH 6.0, and eluted with the same buffer. Fractions (1 ml) of eluant containing the proteinases were pooled, concentrated using an Amicon Ultrafiltration Cell with a PM 10 membrane filter and applied in 1 or 2 ml volumes to a Mono Q column (1 ml) which had been equilibrated with 0.025 M-sodium phosphate buffer, pH 6.0. Unbound protein was eluted with 5 ml ofequilibration buffer and elution of bound proteins was carried out using a salt gradient of O-O.35 M-NaCI in the same phosphate buffer. Fractions (I ml) containing proteinase activity were pooled and concentrated as before. Electrophoresis. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gelatin-SDS-PAGE were carried out as previously described (Lockwood, North, Mallinson & Coombs, 1987) with either 5, 7.5 or 10% (w/v) acrylamide in the separating gel and 5% (w/v) in the stacking gel. Samples were run under reducing conditions but were not boiled unless stated. Protein was detected either by Coomassie blue or silver staining (Oakley, Kirsch & Morris. 1980). Meusurement of proteinuse activity. Chromatographic fractions were assayed for proteinase activity using N-benzoylproline-phenylalanine-arginine p-nitroanilide (BzPFRNan). The substrate was used at a final concentration of 0.075 mM in a I IO ~1 reaction volume containing 1 mM-dithiothreitol (DTT) and 100 ~1 of each fraction. The change in absorbance at 405 nm over 10 min incubation at room temperature was measured using a Titertek Multiscan MCC/340. A change in absorbance of 1.O was given a value of 100 units of proteinase activity. To determine the specific activities of samples towards BzPFRNan at 37°C assays were carried out in 0.15 M-sodium phosphate buffer, pH 6.0, containing 0.1 mMBzPFRNan and 1 mM-DTT in a total reaction volume of I .2 ml. The reaction mixture was preincubated at 37°C for 10 min before the addition of DTT and BzPFRNan to initiate the reaction. The change in absorbance was monitored at 405 nm and the molar extinction coefficient of 4-nitroaniline was taken as 9500 I mol ‘cm-’ (Pupkis & Coombs. 1984). Protein estimation was carried out using the method of Sedmak & Grossberg (1977).with bovine serum albumin as standard. Detection of enzymatic hydrolysis of L-Ieucine methyl ester. Paper electrophoresis was used according to the method of Goldman & Kaplan (1973) to separate the products of the hydrolysis of t_-leucine methyl ester from the parent compound. The incubation mixture comprised 0.15 Msodium phosphate buffer, pH 6.0, I mM-DTT and 20 mM of L-
of Leishmania
713
leucine methyl ester. For determining the pH optimum of the activity the following buffers were used: 0.1 M-citric acid, pH
3-6 and 0.1 M-sodium phosphate, pH 6-8. The proteinase inhibitors antipain, chymostatin and E-64 were also included in some experiments as indicated. Antipain and E-64 were dissolved in water whilst chymostatin was solubihzed in dimethylsuiphoxide. Controls containing DMSO were carried out where appropriate. In all experiments reported, the amount of enzyme activity used was that which with BzPFRNan as substrate and under the standard conditions described above would have resulted in a change of absorbance mini’ of 0.1 (i.e. the release of 12.9 nmoles pnitroaniline min ‘). The reaction mixture was incubated for 3 h at 37°C and 40 ~1 aliquots then applied immediately to strips (3 x 25 cm) of Whatman 3MM paper. Electrophoresis was carried out at 8 V cm ’ for 3 h using a pyridine acetate buffer, pH 3.5 (glacial acetic acid: pyridine: water/ 10: 1:89,by volume). Subsequently the paper was air dried and stained by immersion in a solution of 0.2% ninhydrin in 75% (v/v) acetone for 30 min. The reaction products were quantified by eluting them with 10 ml of 0.4% cadmium acetate in water: glacial acetic acid: ethanol (1:5:4, by volume) and measuring the absorbance at 505 nm of the solution. The method was calibrated using leucine as standard. Non-enzymatic breakdown of L-leucine methyl ester was taken into account. LLeucine methyl ester, proteinase inhibitors, DTT and other biochemicals were purchased from Sigma. RESULTS Partial
purtjication
of proteinases
filtration resulted in the separation of several lower molecular weight proteinases from the majority of the larger proteinases. This was clearly shown by the bands of activity detected using gelatin-SDS-PAGE (Fig. I). The application of this technique also demonstrated that there was a partial separation of the various enzymes of higher molecular mass (Fig. 1b), although interestingly their order of elution from the Superose column did not correlate precisely with their apparent size as revealed by gelatin-SDS-PAGE. In Gel
I
20
IO
Fraction
number
FIG. 2a
30
C. A. HUNTERer ~1.
714
(b)
12345
6
789
36
24
FIG. 2b.
I
2
3
4
5
6
7
8
9
IO
- 36
FIG.2. The partial separation by ion exchange chromatography of the lower molecular weight proteinases of L. m. nw~icunu amastigotes. a. Fractions containing the lower molecular weight proteinases separated on gel filtration were pooled, concentrated and then submitted to ion exchange chromatography. The eluted protein, measured as A,,,, (-), and the change in absorbance caused by the proteinase activity towards BzPFRNan (-•-) are shown. The salt gradient (’ ~ .) is also shown (see methods for details). b. Samples (75 ,uI) from fractions 14-22 (tracks l-9) were analysed by gelatin-SDS-PAGE using a 10% (w/v) acrylamide gel. The positions ofmolecular weight markers (kDa) are shown. c. Samples (75 111)from fractions 1422 (tracks l-9) separated using a 10% (w/v) acrylamide gel and silver stained for protein.
Cysteine
2
proteinases
3
--
36 )
24 )
FIG. 3. Effect of boiling on the migration of proteinases during SDS-PAGE. The pooled fractions (14-24) from ion exchange chromatography were analysed using a 10% (w/v) acrylamide gel. Key to tracks: I, molecular weight markers; 2 and 3, 75 ,uI of pooled fractions (14-24) from ion exchange chromatography; the sample in lane 3 had been boiled for 2 min after the addition of sample buffer.
TABLE I-PURIFICATION OF L. m. mexicam AMASTKOTE LOWER MOLFCULAR
Stage
Lysate Pellet Supernatant Gel filtration Ionexchange
WEIGHT
PROTEINASES*
Recovery of activity (%)t 100 5* 2 103 * 14 57f 17 35 f 18
Proteinase
176 26 534 930 1511
activity:
f f * * *
35 8 185 362 348
*The figures are the means (+ standard deviations) of seven to nine purifications carried out on separate occasions. Fractions 17-27 from gel filtration were pooled and concentrated using ultrafiltration. Fractions 14-24 from ion exchange were pooled and. if necessary, concentrated using ultrafiltration. tThe loss of activity during ultrafiltration was ignored in calculating the % recovery of activity. IThe activity towards BzPFRNan is in nmoles mini ’ (mg protein) ‘.
of Leishmanicr
715
contrast, the enzymes of lower molecular mass coeluted (Fig lb, c). These latter enzymes were partially separated by ion exchange chromatography on the Mono Q column (Fig. 2). The proteinases that were eluted first were totally separated from those that bound most strongly. However, none of the enzymes was completely purified: at least two proteinases were present in all fractions (Fig. 2b). The higher molecular weight proteinases were not detected in any of these fractions. Silver staining revealed that the profile of eluted proteins resembled that of gelatinase activity but that some other proteins were also present (Fig. 2~). There was insufficient protein in the fractions to allow detection with Coomassie blue. When the fractions (14-24) containing proteinase activity were pooled, boiled and analysed by SDSPAGE, Coomassie blue staining revealed a single band. More bands became apparent after silver staining. and the pattern was significantly different from that of the unboiled sample (Fig. 3). Some bands were noticeably more intense. whilst others were less prominent. As it was not possible to isolate the individual lower molecular weight proteinases completely, it was decided to pool the ion exchange fractions containing these enzymes (fractions 14-24) and to study them as a group. The overall purification and recovery of activity to this stage is given in Table I. The partially purified proteinases were totally inhibited by E-64 (10 pg ml-‘) and leupeptin (25 pg ml-‘) and stimulated five-fold by I rnr+DTT (data not shown) and so had the characteristics of cysteine proteinases (North, Mottram & Coombs, 1990). Hydrolysis of L-leucine methyl ester by the purtiall~~ pur$iedproteinases The separation of amino acids and their esters by paper electrophoresis formed the basis of the method developed for following hydrolysis of L-leucine methyl ester. Incubation of L-leucine methyl ester with L. m. mexicana amastigote lysate resulted in the production of leucine and leucyl-leucine (Fig. 4B). The dipeptide, which is thought to be an intermediate in the hydrolysis (Goldman & Kaplan, 1973) ran as a distinct band in advance of leucine. Similar results were obtained using the pooled fractions containing proteinase activity from the ion exchange column. Elution and quantification of the two hydrolysis products together provided a means of determining the rates of hydrolysis. It was confirmed that the reaction was linear under the conditions used. The use of the cysteine proteinase inhibitor E-64 prevented the hydrolysis of L-leucine methyl ester to leucine by a lysate of amastigotes (Fig. 4C). Amastigote lysates hydrolysed L-leucine methyl
C:. A. HUNTER
716
et 01.
FIG. 4. The hydrolysis of L-leucine methyl ester by L. m. me\-iccmcr amastigote
lysate. Paper electrophoresis was carried out as at the origin (0). Key: track A. leucine: track B, mixture resulting from the incubation of Lleucine methyl ester with L. nr. mrricanu amastigote lysate. The resultant leucine and leucyl leucine are arrowed, leucyl leucinc runs slightly ahead of leucine (Goldman & Kaplan. 1973): track C. mixture resulting from the same incubation as in B but with E-64 (100 pg ml ‘): track D. 1..leucine methyl ester.
described.
Samples were applied
ester at a specific activity of 97 f 9 nmoles min ’ (mg protein)-‘. The activity of the partially purified proteinase preparation towards L-leucine methyl ester was 1354 i 364 nmoles mini’ (mg protein))‘. Thus the activity towards L-leucine methyl ester apparently co-purified with the cysteine proteinases. The activity of the partially purified enzymes towards r-leucine methyl ester was optimal at pH 6.0, totally inhibited by E-64 at the lowest concentration tested (25 pg ml- ‘) by chymostatin (100 pg ml-‘) and by antipain (100 pg ml-‘). The activity towards r-leucine methyl ester was also stimulated three-fold by I mM-DTT. Thus the enzyme activity towards L-leucine methyl ester had characteristics of cysteine proteinases. Using the paper electrophoresis method developed, in a semi-quantitative manner, amastigote homogenates of both L. m. me.ricanu and L. donovani were shown to hydrolyse a variety of other L-amino acid methyl esters with the relative activities towards the different substrates being leucine > tyrosine > tryptophan > arginine = lysinc = isoleucine. The partially purified proteinase preparation, however, was active towards only L-leucine methyl ester and
L-leucinamide amides tested.
and not the other
methyl
esters
and
DISCUSSION
The major tinding of this study is that the L-leucinc methyl ester-hydrolysing activity and cysteine proteinases of amastigotes of L. 112. me.xic,ana coeluted. This provides the first direct evidence that these proteinases are responsible for the hydrolysis ofamino acid esters in this form of the parasite. These results also suggest that the great susceptibility of amastigotes of L. m. mexicanu to L-leucine methyl ester may be due to the fact that cysteine proteinases are characteristically abundant in these forms of parasite (Pupkis & Coombs, 1984). Whilst the amastigote homogenates hydrolysed other methyl esters to degrees which correlated with the antileishmanial activity reported by Rabinovitch cr al. (1986) the finding that the partially purified enzymes did not hydrolyse some of the other methyl esters was surprising. However, amastigotes of L. donovani, which lacks these low molecular weight cysteine proteinascs (Lockwood er ul., 1987) and which is not susceptible to L-leucine
Cysteine
proteina: ;es of Leishmania
methyl ester (Hunter et al., 1989), were also able to hydrolyse L-leucine methyl ester as well as a variety of other methyl esters. This implies that the ability to hydrolyse these esters does not by itself confer susceptibility to them but rather that the presence of the cysteine proteinases within megasomes is central to the high sensitivity of L. m. mexicana amastigotes to Lleucine methyl ester. This study has also provided new information on the proteinases of L. m. mexicana. The finding that the cysteine proteinases can be partially separated by ion exchange chromatography shows that they differ in more than apparent, molecular weight. The observation that the mobilities of the proteins were modified by boiling has several implications. It confirms that the apparent molecular weight data derived from studies using gelatin-SDS-PAGE have to be treated with caution. It also explains why in a previous study, in which these proteinases were purified to apparent homogeneity, the pure samples, which were boiled before SDS-PAGE, contained only a single band or two close running bands of protein (Pupkis & Coombs, 1984). This work has also demonstrated that some of the individual cysteine proteinases are very similar in molecular mass and that their differing mobilities in gelatin-SDS-PAGE are probably due to other differences in properties such as the degree of glycosylation or 3D-structure. Work by Koehler & Ho (1988) has shown that similar differences between multiple cysteine proteinases of barley are due to minor differences in the N-terminal sequence. In studies building upon the findings reported in this paper it has been shown that there are at least three groups of cysteine proteinases in L. m. mesicana amastigotes (Robertson & Coombs, 1990). It will be necessary to characterize these enzymes further in order to elucidate the precise ways in which they differ and gain a greater insight into their functional significance to the parasites. Acknow/edgmen/.r~We thank Dr M. J. North for helpful discussions during this work. This study was supported by the Wellcome Trust.
Acta 318: 205-216.
HART D. T., VICKERMANK. & COOMBSG. H. 1981. A quick, simple method for purifying Leishmania mexicana amastigotes in large numbers. Parasitology 83: 529-541. HuNrEk C. A., MACPHERSON L. M. & COOMBS G. H. 1989. Antileishmanial activity of L-leucine methyl ester and Ltryptophanamide. In: Leishmaniasis. The Current Status and New Strategies,for Control(Edited by HART D. T. ), pp. 741-747. Plenum Press, New York. KOEHLER S. & Ho T. D. 1988. Purification and characterisation of gibberellic acid-induced cysteine endoproteases in barley aleurone layers. Plant Physiology 87: 95-103. LCCKWWD B. C., NORTHM. J., MALLINSON D. J. & COOMBSG. H. 1987. Analysis of Leishmania proteinases reveals developmental changes in species-specific forms and a common 6% kDa activity. FEMS Microbiology Letters 48: 345-350. MOTTRAMJ. C. & COOMBSG. H. 1985. Leishmania mexicana: subcellular distribution of enzymes in amastigotes and promastigotes. Experimental Parasitology 59: 265-274. NORTH M. J., MOTTRAMJ. C. & COOMBSG. H. 1990. Cysteine proteinases of parasitic protozoa. Parasitology Today 6: 270-275. OAKLEY B. R.,
KIRSCH D. R. & MORRIS N. R. 1980. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Analytical Biochemistry 105: 361363. PUPKIS M. F. & COOMBS G. H. 1984. Purification and characterisation of proteolytic enzymes of Leishmania mexicana mexicana amastigotes and promastigotes. Journal
qf General Microbiology
130: 2375-2383.
PUPKIS M. F., TETLEY L. & COOMBSG. H. 1986. Leishmania mexicana: the occurrence of amastigote hydrolases in unusual organelles. E.uperimental Parasitology 62: 29-39. RABINOVITCHM., ZILBERFARB V. & RAMAZEILLESC. 1986. Destruction of Leishmania mexicana amazonensis amastigotes within macrophages by lysosomotrophic amino acid esters. Journal of E.xperimental Medicine 163: 520-535. RABINOVIKH M.,
Destruction American
ZILBERFARB V. & POUCHELET M. 1987. of isolated amastigotes by amino acid esters. Journal of Tropical Medicine and Hygiene 36:
288-293.
RABINOVITCHM. 1989. Leishmanicidal activity of amino acid and peptide esters. Parasitology Today 5: 299-30 1. REEVES J. P. 1979. Accumulation of amino acids by lysosomes incubated with amino acid methyl esters. Journal
of‘Biologica1
Chemistry
254: 89 14-8921.
ROBERTSONC. D. & COOMBSG. H. 1990. Characterisation three groups of cysteine proteinases in the amastigotes
REFERENCES ALFIERI S. C.. RAMALEILLES C., ZILBERFARB V., GALPIN I., NORMAN S. E. & RABINOVITCHM. 1988. Proteinase
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inhibfrom
itors protect Lei.~hmania amazonensis amastigotes destruction by amino acid esters. Molecular and Biochemical Parasitology 29: 191-202. ALFIERI S. C., SHAW E., ZILBERFARBV. & RABINOVITCHM. 1989. Leishmania amazonensls: involvement of cysteine proteinases in the killing of isolated amastigotes by L-leucine methyl ester. Experimental Parasitology 68: 42343 1. GOLDMAN R. & KAPLAN A. 1973. Rupture of rat liver lysosomes mediated by L-amino acid esters. Biochemica et
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41: 269-276.
SEDMAKJ. J. & GROSSBERGS. E. 1977. A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue. Analytical
Biochemistry
79: 544-552.
TETLEY L., HUNTER C. A., COOMBSG. H. & VICKERMA~ K. 1989. Generation of megasomes during the promastigoteamastigote transformation of Leishmania mexicana mexicana in vitro. In: Leishmaniasis. The Current Status andNew Strategiesfor Control (Edited by HART D. T. ), pp. 449455. Plenum Press, New York.