The biosynthesis, processing, and immunolocalization of Leishmania pifanoi amastigote cysteine...

ELSEVIER Molecular and Biochemical Parasitology 68 (1994) 119-132

MOIZC~LAR AND BIOCHEMICAL PARASITOLOGY

The biosynthesis, processing, and immunolocalization of Leishmania pifanoi amastigote cysteine proteinases

S. Monroe Duboise a, Marcos A. Vannier-Santos b, Diamar Costa-Pinto c, Luis Rivas a,1, Alfred A. Pan a,2, Yara Traub-Cseko c, Wanderley De Souza b,

Diane McMahon-Pratt "'* " Yale University School of Medicine, Department of Epidemiology and Public Health, P.O. Box 208034, 60 College Street, New Haven, CT

06510-8034 USA b Departamento de Parasitologia e Biofisica Celular, lnstituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro

Rio de Janeiro, R.J. Brazil c Department of Biochemistry and Molecular Biology, Funda~o Oswaldo Cruz Rio de Janeiro, R.J. Brazil

Received 19 May 1994; accepted 2 August 1994

Abstract

Biosynthesis, enzymatic processing, and immunocytochemical localization of an abundant developmentally regulated cysteine proteinase of Leishmania pifanoi, Lpcys2, were investigated employing axenic cultured amastigotes and monoclonal antibodies specifically recognizing either the mature proteinase or the carboxy-terminal extension domain. Pulse labeling and protein sequence data indicated that a 45-kDa precursor is processed to a 40-kDa intermediate, which is further cleaved to generate the 27-kDa mature enzyme and a 15-kDa COOH-terminal domain. Evidence indicates that proteolytic activity is associated with the intermediate form as well as the mature proteinase. Treatment with selected cysteine but not aspartic acid proteinase inhibitors arrested proteolytic processing of Lpcys2 in vivo and inhibited parasite cell division. Electron microscopic immunolocalization of both catalytic and COOH-terminal domains in L. pifanoi and Leishmania amazonensis amastigotes showed intense labeling of megasomes, indicating that cleavage of the COOH-terminal domain probably occurs in the megasome. A low level of the mature proteinase was also associated with the flagellar pocket and plasma membrane; consistent with this observation, low level secretion of Lpcys2 into the culture medium was detected. Lpcysl, a related, less abundant amastigote-specific cysteine proteinase lacking a comparable COOH-terminal domain, was localized to the flagellar pocket and megasomes. Consequently, enzyme sorting to megasomes does not appear to depend upon the COOH-terminal domain; hence this region of Lpcys2 may not be essential for its intracellular targeting.

Keywords: Enzyme processing; Lysosomal enzyme; Megasome; Flagellar pocket; SubceUular targeting; Protease

Abbreviations: MAb, monoclonal antibody; PCR, polymerase chain reaction

1 Present address: Centro de Investigaciones Biologicas, 28006 Madrid, Spain

2 Present address: Abbott Laboratories, Abbott Park, IL 60064, USA.

* Corresponding author. Tel:(203) 785-2907; Fax:(203) 737- 2921; e-mail: [email protected].

1. Introduct ion

Abundant developmentally regulated cysteine proteinase activity is characteristic of the intracellular amastigote form of Leishmania pifanoi and other species of the Leishmania mexicana complex [1-4]. Cysteine proteinases appear to be essential to para-

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120 S.M. Duboise et al. / Molecular and Biochemical Parasitology 68 (1994) 119-132

site growth [3,5] and have been proposed as potential chemotherapeutic targets; others have sought to ex- ploit these enzymes for hydrolytic activation of leishmanicidal agents, such as L-leucine methyl ester [4,61.

The association of amastigote cysteine proteinases with large lysosomes called megasomes which are unique to amastigotes of the L. mexicana complex has been reported [7]. Two distinct cysteine proteinase genes of L. pifanoi, Lpcysl and Lpcys2, have been reported [8]; corresponding genes in L. mexicana have also been described [9,10], and related genes have been detected in other Leishmania species, including Leishmania braziliensis, Leish- mania major and Leishmania donovani [8]. The Lpcys2 gene is present in multiple tandemly arrayed copies, while the Lpcysl gene has only one or two copies [8-10].

The genomic nucleotide sequence of the Lpcys2 gene predicts a long, cysteine-rich COOH-terminal domain, as has been noted for cysteine proteinases of Trypanosoma cruzi and Trypanosoma brucei [11,12], but not for other well-characterized lysosomal proteinases. In contrast, the Lpcysl gene of L. pifanoi [13] and the L. mexicana homolog of Lpcysl [9] lack a COOH-terminal extension. The biological function of the COOH-terminal domain is unknown. The reported absence of a mannose-6-phosphate targeting signal on a cysteine proteinase of T. cruzi [14] has led to the suggestion that the long COOH-terminal domain may function in the sorting of such enzymes to a lysosomal compartment. Other possible roles are participation in proper folding and regulation of proteinase activity [15-17].

In the studies reported here we have exploited a well-characterized axenic culture system for L. pifanoi amastigotes [3,18-20], monoclonal antibodies (MAbs) specific either for a determinant present on the mature proteinase or an epitope on the COOH- terminal extension of the Lpcys2 proteinase, and cell permeant proteinase inhibitors [21] to investigate the biosynthesis, proteolytic processing, post-transla- tional modification, and subcellular localization of the abundant Lpcys2 amastigote cysteine proteinase. Comparative immunolocalization studies of the subcellular localization of Lpcys2 and Lpcysl, suggest that the proteinase COOH-terminal extension, although found in the megasome compartment, may

not be required for sorting to this lysosomal or- ganelle. In addition, the Lpcys2 proteinase appears to be released/secreted at low levels by the cultured amastigotes, suggesting a possible extracellular role for this abundant lysosomal enzyme.

2. Materials and methods

2.1. Parasite culture

Leishmania pifanoi strain MHOM/VE/60 /L t rod axenically amastigotes were cultured at 31°C in the Medium 199 based F-29 medium as previously described [3,22]. In preparations for proteinase purifica- tions amastigotes were grown in roller bottles to a density of approximately 5 X 107 parasites m1-1. For large scale preparations enriching for precursor forms of the proteinase, 1011 logarithmically growing amastigotes were placed in roller bottles containing 300 ml of fresh medium supplemented with 20 /xM antipain; after 24 h an additional 200 ml of fresh medium with 20 /zM antipain was added to help sustain viability and proteinase inhibition for an additional 12 h of growth.

2.2. Gel electrophoresis, zymography, and immunoblot analysis

SDS-PAGE was performed according to the method of Laemmli [23] using 15% polyacrylamide gels; samples were reduced with 2-mercaptoethanol unless otherwise indicated. Gels containing radiolabeled proteins were processed for fluorography by treatment with Autofluor (National Diagnostics) fol- lowed by drying and exposure at -70°C to Kodak X-Omat film.

Zymography to determine proteolytic activity associated with various molecular intermediates of Lp- cys2 was performed employing unheated and nonre- duced immunoaffinity purified proteinase, resolved on 15% acrylamide gels containing 0.1% gelatin (Sigma) using low-voltage (50 V) electrophoresis as previously described [24]. Proteinase activity was detected at pH 6.1 using a 0.1 M glycine buffer containing 2 mM CaCI 2. Molecular weight markers (Pharmacia LKB, Piscataway, N J) were visible on the background of stained gelatin when used in

S.M. Duboise et al. /Molecular and Biochemical Parasitology 68 (1994) 119-132 121

5-fold excess of the manufacturer's recommenda- tions.

For sequencing and Western blot analyses, protein samples resolved by SDS-PAGE were electrophoret- ically transferred to membranes using established conditions [25] and employing either nitrocellulose (0.2 /zm, BA83; Schleicher and Schuell), or, in the case of microsequencing experiments, polyvinylidene fluoride (PVDF) membranes (Immobilon-P; Millipore Corp.). Western blot analyses were performed as previously described [3,19] using indicated antibodies and either 12SI-radiolabeled anti-mouse immunoglobulin (ICN Biomedicals, Inc.) or donkey anti-rabbit IgG (Amersham) as probe.

2.3. Monoclonal and polyclonal antibodies

The production and characterization of the P-2 and A-2 MAbs used in these studies has been described previously [8,19]; these monoclonal antibodies were shown to recognize amastigote-specific antigenic determinants.

Rabbit polyclonal antibodies were prepared against recombinant Lpcysl and Lpcys2 fusion proteins. Briefly, Lpcysl and Lpcys2 PCR-amplified DNA fragments [8] were cloned into the pATHll vector [26] and preparation of inclusion bodies were performed as described [27]. Rabbits were inoculated intramuscularly with approximately 100 /zg of isolated fusion protein using Freund's complete adju- vant. Two weeks later animals were boosted with 100/xg protein; a month afterwards, the rabbits were bled.

2.4. Metabolic radiolabeling and pulse chase studies

For metabolic labeling, logarithmically growing L. pifanoi amastigotes (5 × 108) were harvested and resuspended in 5 ml of modified M199 based medium (F-29) made without trypticase and methionine but supplemented with 10% dialyzed heat-inactivated fe- tal bovine serum. Cells were initially incubated for 30 min in methionine-free medium before addition of the radiolabeled methionine (0.5 mCi; Amersham, Arlington Heights, IL). Cells were labeled for times indicated and then washed twice in phosphate- buffered saline (PBS), pelleted, and stored at - 70°C. In pulse-chase experiments, organisms were labeled

and then placed in complete medium containing cold methionine; aliquots were removed at the indicated time intervals, washed twice with PBS and stored frozen at -70°C pending analysis.

Radioimmune precipitation experiments were performed as previously described [19] employing radiolabeled ceils solubilized at 0°C (in 20 mM Tris, pH 7.3 buffer, containing 40 mM sodium chloride, 10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride (PMSF)/1 mM iodoacetamide/1 mM 1,10- phenanthroline/l% Nonidet P-40 (NP-40)), and clarified by centrifugation. Radiolabeled antigens were immunoprecipitated using ascites fluid containing MAb and MAb-Sepharose CL-2B immunoaffinity resins (P-2 or A-2). Proteins were eluted by boiling for 5 min in SDS gel loading buffer and then analyzed by SDS-PAGE.

2.5. In vivo proteinase inhibition studies

The ability of various proteinase inhibitors to block parasite growth and/or Lpcys2 processing in vivo was examined. For these studies, growth medium, as described above for metabolic labeling studies, was supplemented with various concentrations of selected proteinase inhibitors as indicated; processing was examined after 7 h of culture. Leu- peptin, antipain, and pepstatin A were obtained from Boehringer-Mannheim. Prototek, Enzyme Systems Products (Dublin, CA) supplied the cysteine proteinase specific inhibitor Z-Phe-AIa-CHzF. Viability of cells was assessed using erythrosin B exclusion [28]; viability and cell numbers were evaluated throughout the time course (84 h) employed.

2.6. Proteinase purification

Purification of the Lpcys2 proteinase was performed using immunoaffinity chromatography employing either A-2 or P-2 monoclonal antibody cova- lently coupled to CNBr-activated Sepharose 2B (Pharmacia). Briefly, washed amastigotes were resuspended in lysis buffer (20 mM Tris, pH 7.3/40 mM NaC1/10 mM EDTA/2 mM PMSF/1 mM 1,10-phenanthroline/1 mM iodoacetamide). When enzymatically active Lpcys2 proteinase was required, iodoacetamide was replaced with 2 mM 2,2'-di- thiodipyridine in the lysis buffer. Disruption of para-


sites was accomplished by nitrogen cavitation in a Parr bomb (equilibration conditions: 1800 psi, 20 min, 4°C). Cell lysates were clarified by centrifugation, first at 2000 X g for 5 min and then twice for 30 rain each time at 39 000 X g. The soluble fraction was loaded onto the affinity column at 4°C and the resin was then washed extensively using PBS. Pro- teinase was then eluted from the affinity resin using a 0.1 M sodium formate buffer, pH 3.5. Eluates were neutralized with 1 M Tris pH 9.0. Antigenic activity during immunoaffinity purification was determined by an indirect radioimmune binding assay using the A-2 monoclonal antibody, [12SI]F(ab~) rabbit anti- mouse immunoglobulin and glutaraldehyde-fixed L. pifanoi amastigotes attached to poly-L-lysine-coated round-bottom microtiter plates (polyvinyl chloride; Falcon), as targets. Protein concentrations were esti- mated as indicated by Bradford's assay (Biorad) using BSA as a standard or by UV adsorption at 280 nm. Antigenic protein fractions were pooled, and concentrated (Centricon-10 microconcentrators; Am- icon). Assessment of protein recovery and antigenic activity by competitive RIA indicated that Lpcys2 cysteine proteinase products represent approximately 1% of the total cellular protein of the axenically cultivated L. pifanoi amastigotes.

2. 7. Protein sequence determination

The Lpcys2 proteinase, 40-kDa precursor, and cleavage fragments isolated by immunoaffinity chromatography were further fractionated by SDS-PAGE and transferred (as indicated above) to PVDF membranes (Immobilon-P; Millipore). Amino acid analyses and protein microsequencing of proteins blotted to PVDF were performed by the Yale University School of Medicine Protein and Nucleic Acid Chem- istry Facility using either a Applied Biosystems Model 470 or 477 protein/peptide sequencer and methods previously described [29].

2.8. Immunoelectron microscopic proteinase localization

Polyclonal antisera and MAbs were used to determine the subcellular localization of Lpcysl and Lp- cys2 cysteine proteinases within L. pifanoi axenic amastigotes and L. amazonensis lesion-derived amastigotes using methods previously described for

immunogold cytochemical labeling [30]. Lesion derived amastigotes were isolated from infected ham- ster tissue according to methods previously described [31]. Briefly, the parasites were gently fixed in freshly prepared 4% paraformaldehyde/l% glutaraldehyde in sodium cacodylate buffer, pH 7.3. Samples were dehydrated and embedded at progressively lowered temperatures in lowicryl K4M. Thin sections were first reacted with an anti-proteinase antibody and then with either gold-labeled goat anti-mouse or anti-rabbit immunoglobulin antibody according to standard methods, previously described [30]. Under these conditions, controls reacted with secondary gold-labeled antibody alone (anti-mouse or anti-rabbit immunoglobulin) showed little, if any, background reaction.

2.9. Detection of released, extracellular Lpcys2

Evaluation of Lpcys2 secretion into the amastigote growth medium was examined using immunoprecipitation of radiolabeled proteinase with MAb A-2. Amastigotes were radiolabeled for 8 h with [35S]methionine (as described above), washed, and then resuspended in fresh unlabeled F-29 medium and cultured for an additional 8 h before harvesting the culture medium. All culture media samples were centrifuged first at 3000 X g for 10 min to remove amastigotes and then twice for 1 h each time at approximately 40 000 X g to remove any particulate material. Harvested culture medium was then concentrated 10-fold using Centricon-10 microconcentrators (Amicon) before analysis by immunoprecipitation.

3. Results and discussion

3.1. Intracellular processing of the Lpcys2 amastigote cysteine proteinase and recognition of two distinct epitopes by the A-2 and P-2 monoclonal antibodies

As previously reported, analysis of amino terminal and internal amino acid sequences of the mature 27-kDa cysteine proteinase, purified using A-2 MAb immunoaffinity chromatography, indicated identity with the proteinase encoded by the Lpcys 2 gene of L. pifanoi [8]. Sequential immunoprecipitation (de-

S.M. Duboise et al. / Molecular and Biochemical Parasitology 68 (1994) 119-132 123

pletion) experiments indicated that an identical com- ponent, migrating at approximately 40 kDa (under reducing conditions), was jointly recognized by two monoclonal antibodies, A-2 and P-2 (data not shown). Further Western blot results, clearly indicated that the A-2 and P-2 mAbs both recognize an identical precursor form of the Lpcys2 amastigote proteinase (Fig. 1). Cysteine proteinase was purified using an A-2 MAb immunoaffinity column and resolved by SDS-PAGE under non-reducing conditions. It is apparent that MAb P-2 binds the renatured proteinase precursor but not the mature proteinase (Fig. 1B). Under non-reducing conditions, which are required for effective recognition of the antigen on im- munoblots by the MAb, the intermediate form(s) and mature proteinase migrate at 32-36 kDa and 21 kDa, respectively, rather than 40 kDa and 27 kDa as typically seen under reducing conditions (Fig. 1A); the differences in apparent molecular mass on SDS- PAGE are apparently due to changes in protein folding.

Pulse chase immunoprecipitation studies using MAbs A-2 and P-2, confirmed these results and indicated the in vivo biochemical processing of the Lpcys2 proteinase by the L. pifanoi amastigotes (Fig. 2). After 2 h of pulse labeling with [35S]methionine the predominant molecule, observed on SDS-PAGE analyses (reducing conditions) of im- munoprecipitations employing MAb A-2, is an approximately 40-kDa form of the proteinase. With longer labeling periods (6 h), a weak band at approximately 45 kDa was also evident in A-2 immunopre- cipitations (data not shown). During the subsequent chase period in the absence of radiolabel, the 40-kDa molecule is progressively converted to the 27-kDa proteinase (Fig. 2A). Similar results were found in metabolic labeling experiments employing L. amazonensis amastigote-infected macrophages (data not shown). Assuming an equivalent affinity of the monoclonal antibody for the 40-kDa intermediate and the 27-kDa enzyme, pulse-chase studies indicate an apparent half life of between 1 and 2 h for the 40-kDa form. Concurrent immunoprecipitation with MAb P-2 detects proteins of 45 kDa and 40 kDa appearing during the pulse labeling; subsequently, during the chase period, these are processed to a protein fragment of 15 kDa which is further degraded to a 12-kDa form. The 27-kDa proteinase recognized by

MAb A-2 was not detected by MAb P-2 (Fig. 2B). These results indicate that the P-2 and A-2 MAbs recognize distinct, non-overlapping epitopes of the Lpcys2 cysteine proteinase.

In the pulse-chase studies discussed above, it was not possible to distinguish whether a precursor product relationship exists between the 45-kDa and 40- kDa proteins immunoprecipitated by MAb P-2. To approach this question, cells were cultured in the presence of the cysteine proteinase inhibitor, antipain (20 /~M), in an attempt to block processing of precursor forms (Fig. 2C). In pulse-chase experiments, the increasing level of the 40-kDa intermediate coincides with a decreased detection of the 45-

A 1 2 3

B Mr

(kDa)

- 9 2 -

- 6 8 -

!!~ii ~ii~!!i~i!!i!iiii~i!ii!!~ii~il ~

- 3 0 -

- 2 0 . 1 -

- 1 4 . 2 -

Fig. 1. Shown are the results of SDS-PAGE and Western blot analysis of the Lpcys2 cysteine proteinase of Leishmania pifanoi. (A) Cysteine proteinase preparations, isolated using A-2 MAb immunoaffinity chromatography, resolved on 15% SDS-PAGE under either (2) reducing or (3) non-reducing conditions and stained for protein using Coomassie brilliant blue. Lane 1 repre- sents a control for background staining. (B) Shown are the results from immunoblot analysis employing P-2 MAb and Lpcys 2 cysteine proteinase, isolated using A-2 MAb immunoaffinity chromatography. Proteins were resolved on 15% SDS-PAGE under non-reducing conditions and immunoblot analysis performed as described in Materials and methods.

124 S.M. Duboise et al. /Molecular and Biochemical Parasitology 68 (1994) 119-132

kDa protein, suggesting that the 45-kDa molecule is a precursor of the 40-kDa protein. In addition, these experimental results suggest that potentially a cysteine proteinase may be in part responsible for in vivo processing of the Lpcys2 proteinase precursor into fully active enzyme. Weakly radiolabeled high Mr molecules (70-80 kDa) were not consistently observed under these experimental conditions; these molecules, which completely disappear within 2 h of the cold chase in the experiment shown, may represent multimers of the Lpcys2 cysteine proteinase.

These data indicate the biosynthesis of the Lpcys2 cysteine proteinase involves the production of a 45- kDa precursor which is subsequently processed to generate a 40-kDa molecule; the 40-kDa intermediate is cleaved to generate the mature enzyme and low molecular weight fragments. The A-2 MAb appears to recognize an epitope associated with the mature 27-kDa proteinase while the P-2 mAb recognizes an epitope associated with a 15-kDa cleavage product of the 40-kDa intermediate.

3.2. Amino terminal sequences of immunoaffinity purified Lpcys2 molecules

In order to localize the specific regions of Lpcys2 associated with epitopes recognized by the A-2 and

P-2 MAbs, the amino terminal protein sequences were determined for several of the Lpcys2 molecular forms occurring either during normal processing or autodegradation during purification in the absence of proteinase inhibitors. The amino terminal sequence of the 40-kDa intermediate, a 15-kDa proteolysis fragment isolated using the A-2 MAb and mature 27-kDa [8] are identical. The sequence was determined to be: Ala-Val-Pro-Asp-Ala-Val-Asp-Trp- Arg-Glu-Lys-Gly-Ala-Val-Thr-Pro-Val-Lys-Asp-Gln- Gly-Ala-Cys-Gly-Ser-Cys-Trp-Ala-Phe-Ser-Ala-Val- Gly-Asn-Ile-. Consequently, these data indicate that the epitope recognized by MAb A-2 is located in the amino-terminal half of the mature cysteine proteinase.

The amino-terminal amino acid sequence of the 15-kDa fragment isolated by immunoaffinity chromatography employing the MAb P-2 was determined to be Ala-Pro-Ala-Pro-Val-Met-Val-Glu-Gln-X-Ile- Cys-Phe-Asp-Lys- (X designates an undetermined residue). This sequence corresponds to the beginning of the carboxy-terminal extension region/domain of the enzyme deduced from Lpcys2 nucleotide sequence [8]. The third residue (Ala), however, differs from the cDNA-derived protein sequence and may reflect some heterogeneity present in the Lpcys2 gene family in the COOH-terminal extension region.

A B M r

(kDa) 0 0.5 h 1 h 2 h 3 h 4 h 0

9 7 -

6 8 -

4 6 -

3 0 -

1 4 -

C

0.5h l h 2h 3h 4h 0 l h 2h M F

4 h (kDa)

- 97

- 68

- 46

- - 3(3

- - 1 4

Fig. 2. Pulse-chase study of Lpcys2 biosynthesis and maturational processing. Results shown are SDS-PAGE analyses of radioimmune precipitations of [35 S]rnethionine pulse-labeled L. pifanoi amastigotes using developmental stage specific MAbs A-2 (shown in A) and P-2 (shown in B and C). Times of chase are as indicated for each lane. As results under normal culture conditions (shown in B) did not reveal a precursor-product relationship of 45-kDa and 40-kDa molecules, processing was studied further in the presence of 20 p.M antipain (shown in C).


The observed cleavage point occurs within the flexi- ble hinge region predicted between protein domains (proteinase-COOH extension) in the 40-kDa intermediate. These sequence data conclusively show that MAb P-2 recognizes an epitope found within the COOH-terminal extension domain.

3.3. Proteolytic activity of immunoaffinity-purified proteins

Based upon the proteolytic cleavage fragments of Lpcys2 generated during cellular processing, the proteinase activity of the various forms (intermediate, mature enzyme) was investigated. Using gelatin containing substrate gels, most of the proteolytic activity isolated employing the A-2 immunoaffinity resin migrates as a 22-kDa band (Fig. 3). Under the non-reducing gel conditions employed, the mobility of all molecular forms of the enzyme are increased relative to electrophoresis under reduced conditions (Fig. 1). Thus, the major proteinase activity corresponds well with that of the mature cysteine proteinase enzyme. Similarly, the proteolysis observed associated with components migrating between 30

Mr (kDa)

- 92 - 6 8 - 4 6

)ii!

- 3 0

-20.1

-14.2

Fig. 3. Proteinase activity of isolated Lpcys2 proteinase. Shown are the results experiments examining the proteinase activity associated with various forms of the Lpcys 2 proteinase. Briefly, enzymatically active Lpcys 2 cysteine proteinase was isolated as described in Materials and methods using MAb A-2 immunoaffinity chromatography and separated by SDS-PAGE using a 15% acrylamide gel containing 0.1% gelatin under non-reducing and relatively non-denaturing conditions as described [24]. Proteolytic activity was detected at pH 6.1.

and 36 kDa appears to be due to the intermediate form. Although further investigation is required to evaluate and characterize the enzymatic activity (substrate specificity, pH optimum) associated with the intermediate form, these data suggest that one mechanism for the generation of the mature proteinase molecule may be autocatalytic. The proteolytic activity detected at higher M r (70 kDa to 85 kDa) is likely due to self-association of the proteinase and/or precursor molecules, given the relatively gentle conditions used (no heating or reduction of the protein sample) for substrate gel separation.

3.4. In vivo inhibition of Lpcys2 processing and parasite growth

Several proteinase inhibitors, found in preliminary studies to be capable of in vivo inhibition of Lpcys2 processing, were further examined over a range of concentrations to determine their effects on both proteinase processing and amastigote growth. Results using the reversible cysteine/serine proteinase inhibitor, antipain, and the irreversible highly specific cysteine proteinase inhibitor, Z-Phe-Ala-CH2F, and the aspartic proteinase inhibitor, pepstatin A are shown in Fig. 4.

The 45-kDa proteinase precursor is quickly processed to yield a 40-kDa intermediate; this process is significantly retarded but, in contrast to the results for the 40-kDa intermediate, is not completely blocked in the presence of cysteine proteinase inhibitors. These data suggest that the early processing of the 45-kDa precursor may involve/utilize more than one class of proteinase. The precursor detected (45 kDa) is presumably the Lpcys2 proenzyme. The early proteolytic processing of lysosomal enzymes is thought to occur prelysosomally, and to be critical for the correct subcellular localization and folding; the pro-region peptide may inhibit enzymatic activity prior to maturation in an appropriate cellular compartment [32]. Other classes of proteinases including metalloproteinases and aspartic proteinases have been reported to be involved in the processing of lysosomal enzymes [33,34]. The potent aspartic proteinase inhibitor, pepstatin A, however, failed to effect Lp- cys2 processing (Fig. 4C); consequently, it is un- likely that aspartic proteinase participates in enzymatic processing. The reported expression [35] and

126 S.M. Duboise et aL /Molecular and Biochemical Parasitology 68 (1994) 119-132

localization of the gp63 metalloprotease in L. mexicana amastigotes [36], suggests a potential role in Leishmania lysosomal enzyme processing; however, further work is required to establish this point.

The processing of the 40-kDa proteinase intermediate to generate the mature 27-kDa proteinase and the 15-kDa carboxy terminal extension is completely blocked in the presence of cysteine proteinase inhibitors, suggesting that this process is predominantly dependent upon cysteine proteinase(s) and may be an autocatalytic event. Evidence indicating that both the intermediate form and mature enzyme have proteolytic activity is consistent with this possibility. Further, similar results have been reported for the autocatalytic processing of cruzipain [12,37].

Antipain substantially blocked processing of the 40-kDa intermediate and inhibited parasite growth at levels down to less than 5 ~M (Fig. 4A). The effect on growth appeared to correlate with inhibition of processing and was reversed when parasites were transferred to inhibitor free medium (data not shown). Parasite levels observed after 84 h of culture for the various concentrations of inhibitors are shown in Fig. 4; the relative levels are similar to observations of

cell growth at earlier time points. Z-Phe-AIa-CH2F also showed significant inhibition of Lpcys2 processing and amastigote growth but required a concentration of between 100 and 200 /xM to completely block Lpcys2 processing and growth (Fig. 4B). Ex- clusion of erythrosin B indicated that amastigotes were viable [28] after 24 and 48 h of cultivation in the presence of either antipain or Z-Phe-AIa-CH2F; consequently the effects of these inhibitors appeared static rather than leishmanicidal.

The developmental stage-specific expression of cysteine proteinases in various trypanosomatids [8,9,38] suggests that these enzymes have important roles in the life cycle of the parasites. As observed in other systems [39], the developmental regulation of these proteinases is presumably due to specific protein degradation or processing associated with physi- ological adaptation facilitating survival and growth. Cysteine proteinase inhibitor treatment has been reported to block developmental transformation in both L. mexicana [5] and in T. cruzi [40,41]. The inhibition of cell division found here to correlate with inhibition of the final processing step in Lpcys2 maturation, suggests that this enzyme may be essen-

A2 P2 c q [ I

Mr M r c c c (kDa) (kDa) ~ ~ ~ "~ ~=_ ~.

A -66 B 65 C .~ o o ~ ~ .~°° ~o ~ ~o_ ~J~ Mr

-46

-,6 ~ ~ m ~ CkDal

- 30 ~ 30

1 2 3 4 6 6 1 2 3 4 6 -36

100 100

60 , - -14

x 60 × 60

40 m 40

8 20 20

0 I I ( 0 I 25 12.5 6.25 3.125 1.56 0.781 200 100 25 6.25 1.56

[ Anlipain ] IIM [ z-Phe.Ala-Ch2F ] pM

Fig. 4. In vivo inhibition of Lpcys2 processing and amastigote growth. Shown are the results from radioimmune precipitation experiments employing the A-2 MAb. L. pifanoi amastigotes were metabolically labeled for 7 h using [35S]methionine in the presence of variable concentrations as indicated of (A) antipain; (B) Z-Phe-AIa-CH2F; or (C) pepstatin A. The corresponding growth after 84 H in the presence of each of the inhibitor/concentrations is indicated below and are representative of the relative levels observed at earlier time points. Organisms were monitored throughout the experiment for viability using erythrosin B [28]; under experimental conditions employed, none of the inhibitors appeared to be toxic. The level of growth appeared to correlate with the level of processing observed.

S.M. Duboise et a L / Molecular and Biochemical Parasitology 68 (1994) 119-132 127

tial to parasite growth. However, the abundance of Lpcys2 and consequent requirement to deliver relatively high intracellular inhibitor doses across multi- pie membranes and through a host cell environment rich in similar enzymes and the fact that the effect observed was static (and reversible after 24 h), may indicate limitations of Lpcys2 as a singular target for chemotherapeutic agents. The use of specific inhibitors in conjunction with other therapy nevertheless warrants further investigation. Alternatively, such an abundant enzyme as Lpcys2 may be useful for activating leishmanicidal agents, as has been clearly indicated from studies employing various amino acid esters [4,6].

confirmed and extended these observations (Figs. 6 and 7). Figs. 6A and B, showing L. pifanoi and L. amazonensis amastigotes, respectively, indicate that the COOH-terminal extension domain of Lpcys2, recognized by the P-2 monoclonal antibody is associated with the megasomes. In addition, smaller vesicles found throughout the cytoplasm including some near the flagellar pocket (Fig. 6E) were labeled by the P-2 antibody; the COOH-terminal extension, however, was not detected in the flagellar pocket or on the cell surface. Consequently, this unusual domain of unknown function clearly must be trans- ported with the proteinase domain to the lysosomal (megasomal) compartment. The final proteolytic pro-

3.5. Subcellular localization of the amastigote cysteine proteinases

Immunolocalization studies employed L. pifanoi axenic amastigotes and amastigotes of L. amazonensis and both monoclonal antibodies to Lpcys2 as well as polyclonal rabbit antibodies against either Lpcysl or Lpcys2 recombinant fusion proteins, (Materials and methods). The specificities of the polyclonal sera were evaluated by Western blot analyses employing whole amastigote homogenates. As seen in Fig. 5, the polyclonal antibody to Lpcys2 recognizes the mature enzyme and precursor forms. The apparently weak band at 40 kDa most likely reflects the general abundance of this intermediate form within the whole amastigotes employed for these analyses. The antibody to Lpcysl recognizes components that are distinct in M r from those recognized by the Lpcys2 polyclonal antibody. These data indicate that no immunological cross-reaction occurs between the two polyclonal sera and their respective antigenic cysteine proteinases, and are consistent with the known level of homology between the two proteinases [8,13].

Most of the cysteine proteinase activity in amastigotes of the L, mexicana complex is associated with unique enlarged lysosomes called megasomes [7]. Earlier immunofluorescence localization of Lpcys2 in L. pifanoi axenic amastigotes was indicative of a megasomal localization [8] of the enzyme; electron microscopic immunolocalization experiments of both axenically grown L. pifanoi amastigote and L. amazonensis lesion amastigotes

M r (kDa)

6 8 -

4 6 -

1 2 3 4

3 0 -

1 4 -

Fig. 5. Specificities of the polyclonal anti-cysteine proteinase antibodies. Shown are the autoradiographic results from im- munobiot analyses of whole L. pifanoi amastigotes, resolved on SDS-PAGE electrophoresis and polyclonal rabbit antibodies against recombinant Lpcysl (lane 2), Lpcys 2 (lane 4) fusion proteins or control preimmune rabbit sera (lanes 1 and 3) under reducing conditions. Methods employed were as described in Materials and methods.


cessing step to remove the COOH-terminal domain from the catalytic domain presumably occurs either immediately prior to import or within the megasome.

The MAb A-2 recognizing the mature Lpcys2 proteinase also most extensively labeled the megasomes of both L. pifanoi axenic amastigotes (Fig. 6C) and L. amazonensis lesion-derived amastigotes (Fig. 6D), However, in a some cells low levels of the mature Lpcys2 proteinase were also detected in the flagellar pocket and at the cell surface (Fig. 6F). Polyclonal rabbit antiserum against a recombinant Lpcys2-fusion protein was also tested and showed a similar pattern with a clear predominance of megasomal labeling (Figs. 7A and 7B). In addition, Lpcysl another L. pifanoi amastigote cysteine proteinase [8] was found associated with the megasomes (Fig. 7C) using antiserum to a Lpcysl fusion protein. Lpcysl,

however, was also consistently found to be localized extensively in the flagellar pocket of L. pifanoi amastigotes (Fig. 7C and D). As the Lpcysl proteinase of L. pifanoi and the equivalent proteinase of L. mexicana complex parasites, lack the COOH- terminal extension found for Lpcys2 [9,13], these results illustrate that the presence of a COOH-terminal extension is not required for sorting to the megasomal compartment.

This is the first report of the subcellular location of a trypanosomatid cysteine proteinase COOH- terminal domain. Possible functions of the unusual COOH-terminal domain remain a matter for specula- tion [15,16]. These domains are not highly conserved in protein sequence, with the exception of the cysteine and to some extent glycine and proline residues; this sequence conservation pattern suggests that gen-

Fig. 6. Subcellular localization of the two domains of the Lpcys2 cysteine proteinase (COOH-terminal extension and mature proteinase) using monoclonal antibodies. Shown are the photographic results from immunogold electron microscopic studies employing the MAb P-2 (A,B,E) and A-2 (C,D,F) and either L. pifanoi axenic amastigotes (A,C); or L. amazonensis lesion-derived amastigotes (B,D,E,F). Both the mature proteinase and COOH-extension domains are localized to megasomal organelles; the mature proteinase also appears to be associated with the surface of the amastigote and flagellar pocket area. (P. flagellar pocket; N, nucleus).


eral structural features rather than specific sequence motifs may be critical to function [8]. It has been suggested that the COOH-terminal region may be important for the proteinase targeting, proper folding for enzymatic activity, a n d / o r the inhibition of proteinase activity. However, studies to date have not indicated a precise function of the COOH-terminal domain. A recombinant Trypanosoma brucei rhode-

siense enzyme deleted of its carboxy-terminal extension was enzymatically active, indicating that the carboxy terminal domain is not required for proper

folding for enzymatic activity [42]. It is clear from the absence of mannose-6-phosphate on cruzipain and the absence of metabolic pathways in T. cruzi

and Leishmania involved in the synthesis of mannose-6-phosphate [14], that alternate lysosomal targeting pathways are operative in these trypanosomatid parasites. Consequently, it has been suggested that the COOH-terminal extension of these trypanosomatid enzymes may serve as a membrane anchor to regulate the enzyme transport or dispersion [43] or may be needed for sorting to the lysosomal

Fig. 7. Subcellular localization of the Lpcysl and Lpcys2 cysteine proteinases employing polyclonal antibodies. Shown are the photographic results from immunogold electron microscopic studies employing L. pifanoi axenic amastigotes. In the case of Lpcys 2 (A,B), areas of external membrane, the flagellar pocket as well as intense megasomal labeling was observed. Subcellular localization of Lpcysl (C,D) indicated preferential association with vesicles and flagellum found within the flagellar pocket area as well as the megasomal compartment.

130 S.M. Duboise et aL /Molecular and Biochemical Parasitology 68 (1994) 119-132

compartment. However, both membrane association and promotion of correct enzyme localization are properties that have been associated with pro-peptides in other systems [44] and there are numerous precedents for lysosomal proteins being sorted properly to lysosomes independent of mannose-6-phosphate targeting [17]. In the case of Lpcys2, evidence indicates the presence of high mannose carbohydrate (S.M. Duboise, unpublished results); however, the potential role of alternate carbohydrate moieties in kinetoplas- tid lysosomal targeting remains to be determined. Although the results presented in the current study are consistent with the postulated targeting role of this domain of trypanosomatid cysteine proteinases, immunoelectron microscopic analyses of the Lpcysl cysteine proteinase, used as a control, suggest that the COOH-terminal domain is not required for megasomal targeting. This enzyme, although a similar cathepsin L-like, developmentally regulated proteinase of L. pifanoi [13], like that of the related L. mexicana enzyme [9], lacks a COOH-terminal domain and is nevertheless sorted to lysosomal com- partments. The higher comparative level of Lpcysl in contrast to Lpcys2 proteinase associated with the flagellar pocket area in comparison to the megasomes, however, may indicate either a difference in intracellular sorting (efficiency) of the enzymes or a difference in their retention in the megasomes or flagellar pocket.

Although the presence of an intact COOH-terminal domain in active crnzipain appears to argue against the possibility that this domain inactivates the enzyme, the COOH-terminal extension of cruzipain is refractory to proteolytic digestion [37]. The localization of both the mature Lpcys2 and its cleaved COOH-terminal domain in megasomes, considered together with results of the pulse-chase study using MAb P-2, indicates that degradation of the Lpcys2 COOH-terminal domain also occurs slowly. Thus, a role for the COOH-terminal extension within megasomes, even after release from the proteinase domain, cannot at present be excluded.

Low-level secretion of lysosomal enzymes has frequently been observed [17] and precedents exist for secretion of large amounts of a lysosomal enzyme, notably mammalian cathepsin L proenzyme which has also been called major excreted protein (MEP) [45]. Therefore, it was not surprising to detect a low level of Lpcys2 in L. pifanoi amastigote

1 2 3 4 Mr (kDa)

- 92 .5

- 6 9

- 4 6

- 3 0

-14.5

Fig. 8. Lpcys2 secretion. Shown are the autoradiographic results of immunoprecipitation experiments employing either MAb A-2 (Lanes 2 and 4) or MAb P-2 (lanes 1 and 3) and culture supernatants from [35S]methionine labeled L. pifanoi amastigotes grown in the absence (lanes 1 and 2) or presence of 20 /xM antipain (lanes 3 and 4). Methods employed for metabolic labeling and the isolation of culture supematants were as described in Materials and methods.

culture medium by immunoprecipitation of radiolabeled Lpcys2 with MAb A-2 (Fig. 8). It should be emphasized that concentration of the used growth medium was required before Lpcys2, which is highly abundant intracellularly (approximately 1% of the total cell protein), could be readily detected. Thus, the extracellular enzyme is a minor proportion of the total Lpcys2 synthesized. The mature enzyme, but not the Lpcys2 COOH-terminal extension, was detected at low levels in the flagellar pocket, on the amastigote plasma membrane, and released into the culture medium. Low level secretion of an abundant lysosomal enzyme due to missorting is not unusual [17], but even coincidentally secreted Lpcys2 could be significant to host-parasite interaction. Inhibition of the similar enzyme of Trypanosoma cruzi, cruzipain (GP57/51), has been shown to interfere with host cell invasion as well as parasite replication and intracellular development [40,41]. T. cruzi also has large pre-lysosomal organelles called reservosomes


[46] where cruzipain is predominantly localized. In- terestingly, cruzipain has also been detected in the flagellar pocket of trypomastigotes and on the surface of epimastigotes and amastigotes [38,47]. It has recently been suggested that the maturation process of the phagolysosomes of L. mexicana infected macrophages along with lack of release of amastigote antigens serve to limit presentation of parasite antigens [48]. The effect of a potent, cathepsin L-like proteolytic enzyme of broad specificity, may potentially serve to limit the availability of parasite-derived peptides for antigen presentation or otherwise indirectly modulate the host immune response to infection.

Acknowledgements

The authors would like to thank Dr. Ken Williams and Ms. Kathy Stone of the Yale University-W.M. Keck Foundation Biotechnology Resource Labora- tory for advice and protein sequence determinations, Dr. Peter Hotez for providing help with substrate gel procedures, Dr. Lynn Soong for helpful discussions on analyses and Dr. Norma Andrews for advice regarding peptide fluormethyl ketone inhibitors. This work was supported through grants from the NIH (AI27811) and Grants NIH, MacArthur Foundation for Molecular Parasitology,TWAS, PAPES-FIOCruz, Plan Nacional de Biotechnologia (CICyT) and the Rockefeller Biotechnology Career Development Fel- lowship Program.

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