Monoclonal antibodies against a 60 kDa phenothiazine-binding protein from Trypanosoma brucei can discriminate between different trypanosome species

Molecular and Biochemical Parasitology, 21 (1986) 37-45 Elsevier

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MBP 00708

Monoclonal antibodies against a 60 kDa phenothiazine-binding protein from Trypanosoma brucei can discriminate between different trypanosome species J o s e p h Stieger* a n d T h o m a s S e e b e c k * " Institut far Allgemeine Mikrobiologie, University of Bern, Baltzerstrasse 4, 3012 Bern, Switzerland (Received 14 November 1985; accepted 17 May 1986)

The dominant structure of the cytoskeleton of the Trypanosomatidae consists of a tight array of singlet pellicular microtubules, which surround the entire cell body. These microtubules are in close and stable contact with the cellular membrane. These contacts can be selectively disrupted by the action of phenothiazine drugs, which are potent trypanocides in vitro. Phenothiazineaffinity chromatography of detergent solubilized proteins from Trypanosoma brucei has resulted in the isolation of a protein of an apparent molecular weight of 60 000. Polycional antibodies raised against this protein (p60) have been used to investigate the presence of similar proteins in other protozoa. No such crossreacting proteins have been observed outside the family Trypanosomatidae. Within this family, a strong crossreactivity was observed with Crithidia fasciculata, while only a marginal reaction was seen with two species of Leishmania and, quite unexpectedly, also with the stercorarian trypanosomes T. cruzi and T. rangeli. Different monoclonal antibodies against p60 are able to clearly distinguish different subgenera of salivarian trypanosomes, and most notably to differentiate between various isolates of T. congolense. Therefore, these antibodies may prove valuable for diagnostic and epidemiological applications. Key words: Trypanosoma brucei; Trypanosoma congolense; Monoclonal antibodies; Phenothiazine-binding protein; Species-specific antibodies

Introduction

The cytoskeleton of the Trypanosomatidae essentially consists of microtubule-based structures [1]. Trypanosomal microtubules and tubulin proteins are rather similar to those of other eukaryotes, both when analyzed biochemically [2, 3] and on the genetic level [4--6]. However, their supramolecular arrangement in the cell body is unique to hemoflagellates. A tight, helical array of singlet microtubules covers the entire cell body [7-9] and is in close and stable contact with the * Present address: Haematologisches Zentrallabor, Inselspital, 3010 Bern, Switzerland. ** To whom all correspondence should be addressed.

Abbreviations: DTT, dithiothreitol; EGTA, ethyleneglycolbis-(2-aminoethyl)-tetraacetic acid; kDa, kilodaltons; MOPS, morpholinopropione-sulfonic acid; NCS, N-chloro-succinimide; PEG, polyethyleneglycol; SDS, sodium dodecyl sulfate.

overlaying cell membrane [10, 11]. The microtubules in this array are laterally interconnected and they also form tight membrane contacts, the detailed nature of which remains to be understood. The overall architecture of this microtubule/membrane complex is well conserved throughout the family of the Trypanosomatidae and 'also, in African trypanosomes, throughout the entire life cycle. We have earlier observed that a number of drugs, most notably among them the phenothiazines [12], disrupt the contact between the pellicular microtubules and the membrane at micromolar concentrations [13]. Concomitantly with the disruption of the microtubule/membrane contacts, a rapid loss of cell motility and subsequent cell death occur [13-15]. A similar high and selective toxicity of phenothiazines and other tricyclic compounds has been described for Leishmania [16, 17] and also for Plasmodium fal-

0166-6851/86/$03.50 ~ 1986 Elsevier Science Publishers B.V. (Biomedical Division)

38

ciparum [18]. A more detailed understanding of the mechanisms of phenothiazine toxicity in protozoa is expected to provide valuable clues for the future design of anti-protozoal drugs. However, a straightforward interpretation of the observed disruptive effects of phenothiazines is not possible at present. Phenothiazines exert a large number of actions within living cells, such as blocking the action of calmodulin [19, 20], blocking a-adrenergic and muscarinic receptors [21] and inhibiting adenosine uptake [22], changing membrane polarization [23], interfering with membrane Ca 2÷ fluxes [24], inhibiting phospholipid-dependent protein kinases [25], interacting with tubulin [26] and actin [27] and generating radicals [28]. In addition, a number of cytosolic phenothiazine-binding proteins other than calmodulin have been isolated from brain by affinity-chromatography [29], though their functions are still unknown. We have initiated a study to explore the mode of peUicular microtubule/membrane interaction and the mechanism by which it is disrupted by phenothiazines. Phenothiazine affinity chromatography of total proteins solubilized by detergent from procyclic Trypanosoma brucei brucei has revealed a major protein of apparent molecular weight 60000 (p60). Monoclonal antibodies against this protein recognize exclusively p60 in total cell lysates of T. brucei, p60 has been detected only in trypanosomatids; it is present neither in other protozoa, nor in higher eukaryotes, including mammals. Interestingly, the p60 protein is not well conserved even between trypanosome subgenera. In consequence, monoclonal antibodies to p60 can readily distinguish between different trypanosome species and, hence, may be valuable tools for diagnosis and epidemiology of the trypanosomiases. Materials and Methods

Cell culture. Procyclic T. b. brucei, stock STIB 366 were grown at 26°C in SDM-79 medium containing 5% fetal calf serum as described [2]. Small cultures were grown in 25 cm 2 and 75 cm 2 tissue culture flasks, while for larger preparation, 0.5 1 batches of culture were grown in Fernbach flasks, maintained with constant gentle agitation at 26°C.

Cells were harvested during exponential growth, i.e. at densities of about 5 x 106 to 1 x 107 per ml.

Purification of p60. The whole purification procedure was carried out at 4°C. 2 ml of packed cells were lysed in MM-S buffer containing 10 mM morpholinopropione-sulfonic acid (MOPS) pH 6.8, 1 mM MgC12, 10 mM CaCI2, 0.5 mM dithiothreitol (D'I-T), 0.05% Triton X-100, 20% glycerol, 1 ~g m1-1 Leupeptin, 0.1 mM phenylmethylsulfonyl fluoride by sonication for 3 x 15 s bursts at 50 W with a Branson sonifier B12. After half an hour on ice, the lysate was centrifuged for 1 h at 100000 × g at 4°C. The supernatant was removed and applied to a 5 ml AffiGel Phenothiazine column (Bio Rad Laboratories, Richmond, CA, Cat. No 153-6143), preequilibrated with MM-W buffer (10 mM MOPS pH 6.8, 1 mM MgCI 2, 0.1 mM CaCI 2, 0.5 mM DTF, 0.05% Triton X-100, 100 mM glucose). The column was then washed with 4 vol of MM-W buffer and the bound protein was finally eluted with MM-E buffer (10 mM MOPS pH 6.8, 1 mM MgCI2, 5 mM ethyleneglycol-bis-(2-aminoethyl)tetraacetic acid (EGTA), 0.5 mM DTT, 0.05% Triton X-100, 5 mM chlorpromazine hydrochloride. p60 was further purified by preparative sodium dodecyl sulfate (SDS)-gel electrophoresis on a 6-20% polyacrylamide gradient gel. After staining in 1 M KC1, the translucent protein band was excised and eluted electrophoretically. The purity of this preparation was then checked by SDS-PAGE. Monoclonal antibody production. BALB/c mice were immunized with electrophoretically purified p60. The first injection was done with 20 Ixg p60 in 125 p,l phosphate-buffered saline (PBS) and 125 ixl complete Freund's adjuvant. Thereafter, the mice were boosted four times at intervals of two weeks with 10 p,g p60 in 125 la,l PBS and 125 p,1 incomplete Freund's adjuvant. Serum activity against p60 protein was determined by an enzyme-linked immunosorbent assay as described below. Serum titers were considered satisfactory at 1:10000. On the last four days before sacrifice, 20 ixg p60 in 250 p,l PBS were injected daily via the intraperitoneal route. Spleen lymphocytes of this mouse were fused with hypoxanthine-ami-

39 nopterin-thymidine (HAT)-sensitive myeloma cells in the presence of polyethyleneglycol (PEG) 4000. Cells were seeded in six 96-well tissue culture plates at dilutions of either 100 000, 50 000 or 25 000 lymphocytes well -1. Hybridomas, defined by growth in HAT-containing medium, developed in 90%, 65% or 30% of the wells, respectively. Hybridoma lines were selected on the basis of a dot-immunobinding assay and Western blotting and were finally subcloned.

Screening of hybridoma cells. Clones producing antibodies to p60 were detected in a modified dotimmunobinding assay according to [30]. Briefly, 50-100 ng of p60 in 1 p,1 TBS pH 7.4 containing 5% horse serum were dotted on nitrocellulose filters of 4 mm diameter, which were then placed on the bottom of a 96-well dish. Filters were airdried for 1 h, and remaining free binding sites were blocked by incubating in a solution of 5% horse serum in TBS pH 7.4 for 1 h. 100 ~1 aliquots of individual culture supernatants were incubated in these wells, and bound antibodies were detected by anti-mouse antibody conjugated to horseradish peroxidase as described below. Each incubation step was carried out for 2-4 h on a rotary shaker, and was followed by extensive washing of the wells with TBS containing 0.05% Tween 20.

Polyclonal antiserum. Polyclonal antiserum was produced from the mouse from which the spleen was taken for hybridoma production. The antiserum was entirely monospecific for p60 and was used without further purification. lmmuno-blotting. Proteins separated on a 6-20% polyacrylamide SDS-gel were transferred electrophoretically (80 V, 0.4 A, 2 h at room temperature) onto nitrocellulose membranes. Nonspecific binding sites on the filter were blocked by incubation with 5% horse serum in TBS pH 7.4 for 2 h at room temperature. The blot was then incubated with either polyclonal antiserum (dilution 1:2500) or hybridoma culture supernatant (dilution 1:100 - 1:1000) in TBS containing 5% horse serum overnight at 4°C on a rotatory shaker. Bound antibodies were detected by incubation with anti-mouse IgG conjugated to horseradish

peroxidase (DAKOpatts a/s Denmark, No. P260, dilution 1:500) for 4 h at room temperature. Each incubation step was followed by thoroughly washing the filter with TBS containing 0.05% Tween 20. Peroxidase activity was visualized with a freshly prepared solution of 0.5 mg m1-1 diamino benzidine, 4 mM H202 in TBS pH 7.4. The reaction was terminated after 1-5 min by washing with water.

Peptide mapping. Peptide mapping was performed as described [2, 31]. For NCS (N-chlorosuccinimide) cleavage, gel slices containing p60 were equilibrated in a solution of ureaJH20/ CH3COOH (1:1:1, w/v/v) followed by digestion with 0.015 M NCS in the same solution for 30 min at room temperature. After equilibration in electrophoresis sample buffer to remove the cleavage agent, the slices were loaded into the slots of a 15% polyacrylamide gel. For limited proteolytic digestions with Staphylococcus aureus V8, approximately 5 ~g of p60 in gel electrophoresis sample buffer (80 mM TrisHCI pH 6.8, 0.1 M 2-mercaptoethanol, 2% SDS, 10% glycerol) were digested with four different protease concentrations (1, 5, 15, 50 ~g m1-1) at 37°C for 30 min. The reaction was terminated by boiling the samples for 5 min and the resulting peptides were fractionated on 15% polyacrylamide gels. Results

Isolation of p60. Detergent soluble proteins were prepared as detailed in Materials and Methods. The gel-electrophoretic analysis of this detergentsolubilized fraction reveals a highly complex banding pattern (Fig. 1, lane 1). This fraction was applied to a phenothiazine affinity column and, after extensive washes, bound proteins were recovered by elution with 5 mM chlorpromazine. Analysis of this bound fraction demonstrated the presence of a single major protein migrating at an apparent molecular weight of 60 000 (Fig. 1, lane 2). In addition to this major band, a number of minor polypeptides of greater mobility were also detected. The relative amounts of these proteins vary from preparation to preparation. Immunoblotting experiments (see below) suggested that

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Fig. 1. Purificationof pr0. Lane 1: column input (total soluble proteins); lane 2: bound proteins eluted with 5 mM chlorpromazine; lane 3: electrophoreticallypurifiedp60. Numbers on the left indicate the molecularweight of marker proteins. they are not degradation products of p60. These minor proteins have not yet been studied any further. The binding of p60 to the phenothiazine ligand is apparently not dependent on Ca > ions, as replacing of Ca 2+ by E G T A in the elution buffer is not in itself sufficient to effectively elute the bound p60. Hence, retention of p60 by the phenothiazine ligand may be predominantly due to hydrophobic interactions by domains of the protein which are not significantly altered by the presence or absence of Ca z+ ions. Preliminary subcellular fractionation experiments suggest that p60 is associated with the microtubule/membrane complex. This is different from the distribution of calmodulin, which we find predominantly in the soluble cytoplasmic fraction (unpublished observations).

Monoclonal antibodies against p60. Initially, total eluate fractions (compare Fig. 1, lane 2) were used for immunization. However, some of the contaminating minor proteins apparently are highly immunogenic, and no hybridomas producing an-

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Fig. 2. Epitope mapping of monoclonal antibodies. (A) V8 peptides, (B) NCS peptides. Lanes 1: Prototype monoclonal antibody 2C B10-1; lanes 2: prototype monoclonalantibody 2D G3; lanes 3: polycionalp60 antiserum. tibodies against p60 could be isolated. Consequently gel-purified p60 (Fig. 1, lane 3) was then successfully used as an antigen. Culture supernatants of hybridoma clones were tested for their specificity by staining Western blots of total trypanosomal cell lysates. Antibodies were scored as positive only if they were completely specific for p60. Positive clones were further characterized by analyzing the epitope specificity of the secreted antibodies. For this analysis, p60 was digested with V8 protease or chemically cleaved with NCS, and Western blots of the resulting peptide fragments were then probed with the individual antibodies. All p60specific monoclonal antibodies tested displayed one or the other of two distinct staining patterns (exemplified in Fig. 2).

p60 is a trypanosomatid-specific protein. The antip60 antibodies were subsequently used to investigate the evolutionary conservation of the p60 protein. When lysates from a number of different cell types, both of mammalian and protozoal or-

41 igin, were probed with the polyclonal p60-antiserum, a clear pattern emerged. The polyclonal antibody recognizes a protein similar to p60 in different genera of hemoflagellates (T. brucei (control), Leishmania and Crithidia) (Fig. 3). In contrast, it does not react with any of the other protozoa tested (Tetrahymena pyriformis, Dictyostelium discoideum, Giardia lamblia, Pseudomicrothorax dubius, Nassula aurea, Eimeria acervulina, Chlamydomonas reinhardtii and Entamoeba histolytica). Also, no reaction was detected with mammalian cells (results not shown). This recognition pattern clearly indicates that p60 is a trypanosomatid-specific protein. A p60 homologue has actually been isolated from Crithidia fasciculata by phenothiazine affinity chromatography (unpublished results). Analogous experiments using monoclonal antibodies demonstrated that these are able to clearly distinguish between different hemoflagel-

late genera. In contrast to the polyclonal antibody, which is able to recognize a p60 homologue in all hemoflagellates tested, the monoclonals are only able to detect the trypanosomal, but not the leishmanial or crithidial protein (Fig. 4). Not surprisingly, the monoclonal antibodies also do not react with non-trypanosomal protozoa. These results indicate that p60, though present in all trypanosomatidae tested, apparently is not very highly conserved within this family.

Anti p60 antibodies can discriminate between different trypanosomes. The anti p60 antibodies were further tested for their ability to discriminate between different trypanosomal subgenera and species. Not surprisingly, all species of the subgenus Trypanozoon (T.b. brucei, T.b. rhodesiense and T.b. gambiense) react equally well with the polyclonal antiserum (Fig. 5). In addition, the three representatives of the subgenus Nannomonas (T. congolense stocks K44 and 1841, T. simiae) react strongly with this antibody. In contrast to these salivarian trypanosome species, two representa-

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Fig. 3. Presence of p60 in various trypanosomatids. Immunostaining of hemoflagellatecell lysateswith the polyclonalanti p60 antibody. Lanes 1 and 7: T. brucei (controls); lane 2: C. fasciculata; lanes 3 and 4: L. major; lanes 5 and 6: L. donovani. All lanes contain similar amounts of total cellular protein.

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Fig. 4. Monoclonal anti-p60 antibodies are specific for trypanosomes. Western blots of total cellular lysates were immunostained with the monoclonal antibody 2D G3. All lanes contain similar amounts of total cellular proteins. Lanes 1 and 13: T.b. brucei (controls); lane 2: C. fasciculata; lanes 3 and 4: L. major; lanes 5 and 6: L. donovani; lane 7: E. acervulina (oocysts); lane 8: E. acervulina (sporozoites); lane 9: T. pyriformis; lane 10: D. discoideum; lane 11: C. reinhardtii; lane 12: G. lamblia.

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bound ligand, p60 is clearly different from trypanosomal calmodulin, which has been characterized earlier [32]. In contrast to calmodulin, and also to other mammalian phenothiazine-binding proteins which have been described earlier [29], the interaction of p60 with phenothiazine is not dependent on Ca 2÷ ions, and a quantitative elution is only achieved in the presence of chlorpromazine in the elution buffer. The detailed mode of interaction of p60 with phenothiazines is currently being studied. Mono- and polyclonal antibodies have been raised against p60. These antibodies, which are highly specific for p60, have served to investigate the possible presence of p60 analogues in other organisms. No such crossreacting protein has been detected outside the family Trypanosomatidae. It is particularly interesting to note that p60 has not been found in Eirneria, an organism which also

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Fig. 5. Immunostaining of different trypanosomalspecieswith the polyclonal anti p60 antibody. Lanes 1 and 9: T.b. brucei (controls); lane 2: T.b. rhodesiense; lane 3: T.b. gambiense; lane 4: T. congolense (stockK44); lane 5: T. congolense (stock 1841); lane 6: T. simiae; lane 7: T. cruzi; lane 8: T. rangeli. tives of stercorarian trypansomes, T. cruzi (Schizotrypanum) and T. rangeli (Herpetosoma) exhibit no reactivity towards this polyclonal p60 antiserum (Fig. 5). A survey of all available monoclonal antibodies resulted in two distinct staining patterns. One group of antibodies reacted to similar extents with T. brucei, T. congolense and T. sirniae, as exemplified in Fig. 6. The other group reacted strongly with all members of the brucei group, but did not react, or only very faintly so, with T. congolense and T. simiae (see Fig. 7). Both groups show no reactivity towards T. cruzi and T. rangeli (see Figs. 6 and 7).

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Discussion

Phenothiazine affinity chromatography of detergent-solubilized proteins of T. brucei revealed the presence of a single major high-molecularweight protein (p60), which is retained by the

Fig. 6. Immunostaining of different trypanosomal species with the monoclonal antibody 2D B10-1. Lanes 1 and 9: T.b. brucei (controls); lane 2: T.b. rhodesiense; lane 3: T.b. gambiense; lane 4: T. congolense (stock K44); lane 5: T. congolense (stock 1841); lane 6: T. simiae; lane 7: T. cruzi; lane 8: T. rangeli.

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Fig. 7. Immunostainingof differenttrypanosomalspecieswith the monoclonalantibody2D G3. Lanes 1 and 9: T.b. brucei (controls); lane 2: T.b. rhodesiense; lane 3: T.b. gambiense; lane 4: T. congolense (stockK44); lane 5: T. congolense(stock 1841); lane 6: T. simiae; lane 7: T. cruzi; lane 8: T. rangeli. features a well structured cortex of membraneassociated microtubules, though of a somewhat different architecture than the pellicle of the hemoflagellates [33]. Immunoblot analyses of different members of the family of the Trypanosomatidae have demonstrated that this protein is not strongly conserved. Furthermore, the degree of conservation of immunoreactivity of the p60 homologues apparently does not follow the established systematic groups [34]. When using the polyclonal antiserum, weak, but distinct crossreactivity is observed with representatives of the genus Leishmania, and a strong reaction is found with Crithidia. On the other hand, the polyclonal antiserum which is able to recognize different genera, does not react with some subgenera within the genus Trypanosoma, in that it recognizes representatives of the salivarian, but not of the stercorarian trypanosomes. The monoclonal antibodies are far more re-

strictive in that none of them reacts with organisms outside the genus Trypanosoma. Even within this genus, no reaction is detectable with the stercorarian species T. cruzi and T. rangeli. On the other hand, all monoclonal antibodies tested react similarly with T.b. brucei, T.b. gambiense and T.b. rhodesiense. A more complex pattern of reactivity is observed in the subgenus Nannomonas. One set of monoclonal antibodies (represented by the prototype 2D B 10-1) reacts strongly both with T. congolense and T. simiae, while the other set (represented by the prototype 2D G3) does not, or only faintly so. However, either set of monoclonals, as well as the polyclonal serum, exhibit a much weaker reaction with T. congolense stock 1841 than with either T. congolense K44 or with T. simiae. This differential reactivity of our monoclonal antibodies towards the different stocks of T. congolense is particularly interesting with regard to the widely held suspicion that the species T. congolense is indeed a collection of diverse organisms. This view has lately gained strong experimental support from chromosome analysis of different T. congolense isolates [35]. Interestingly, the pattern of species specificity of the monoclonal antibodies is not correlated with their epitope specificity as determined by the staining pattern of peptide maps of p60. Hence, both groups, as defined by their reactivity towards T. congolense and T. simiae, do contain individual antibodies of either epitope specificity. These observations clearly indicate that the monoclonal anti-p60 antibodies may be of wider practical use for the identification of, or distinction between trypanosomes, and most notably T. congolense isolates, of uncertain origin. Similarly species-specific antibodies, though directed towards a different protein, have recently been obtained by immunization with total T. congolense [36]. Further work is clearly required to evaluate the potential usefulness of p60 antibodies as diagnostic tools. On the other hand, the availability of p60 antibodies will now allow the investigation of the structure and the functional significance of p60. A more detailed investigation of the biochemistry of p60 may lead to a better understanding of the mechanisms of action of phenothiazine drugs

44 in the t r y p a n o s o m e cell a n d m a y e v e n t u a l l y allow the r a t i o n a l d e s i g n o f m o r e specific, a n d m o r e p o t e n t t r y p a n o c i d a l drugs.

Acknowledgements W e w o u l d like to t h a n k U r s u l a K u r a t h for h e r skillful t e c h n i c a l assistance, B. S t a d l e r for his continuous help and advice, E. Schweingruber for thoughtfully reading the manuscript a n d R. B r a u n for his c o n t i n u o u s i n t e r e s t a n d m a n y h e l p f u l discussions. W e a r e i n d e b t e d to t h e following coll e a g u e s for p r o v i d i n g cell cultures, extracts o r

blots: R. P e c k , D e p t . of P r o t o z o o l o g y , U n i v e r sity of G e n e v a ; R. Parish, D e p t . of P l a n t B i o l o g y a n d B. G o t t s t e i n , D e p t . o f P a r a s i t o l o g y , U n i v e r sity o f Z u r i c h ; W . H a e u s e r m a n n , C I B A - G E I G Y A G , A u b o n n e ; K. B l a s e r , D e p t . of Clinical I m munology, A. Boschetti, Dept. of Biochemistry a n d T. W y l e r , D e p t . o f Z o o l o g y , all at the U n i versity of B e r n ; R. E t g e s , D e p t . o f B i o c h e m i s t r y , U n i v e r s i t y o f L a u s a n n e . This s t u d y was supp o r t e d b y grants f r o m t h e U N D P / W o r l d B a n k / W H O Special P r o g r a m m e for R e s e a r c h and T r a i n i n g in T r o p i c a l D i s e a s e s a n d f r o m t h e Swiss N a t i o n a l Science F o u n d a t i o n .

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