EXPERIMENTAL
PARASlTo1.oC.Y
14, 121-133 (1992)
Trypanosoma cruzi: Stage Expression Calmodulin-Binding Proteins
of
GEORGE A. ORR, HERBERT B. TANOWITZ, AND MURRAY WITTNER Departments
of Molecular Pharmacology, Pathology, and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, U.S.A.
ORR, G. A., TANOWITZ, H. B., AND WITTNER, M. 1992. Trypanosomi cruzi: Stage expression of calmodulin-binding proteins. Experimental Parasitology 74, 127-133. The subcellular distribution of calmodulin-binding proteins in three life stages of Trypanosoma cruzi was analyzed by a [‘ZsI]calmodulin gel overlay procedure under conditions where proteolysis was kept to a minimum. It was found that T. cruzi contains a complex profile of calcium-dependent calmodulin-binding proteins and that several of these polypeptides were differentially expressed at specific stages of development. The majority of these stagespecific polypeptides was found in the particulate fractions of the replicative stages of the parasite, i.e., epimastigote and amastigote. These studies suggest that calcium and calmodulin may play an important central role in the growth and differentiation of this parasite. We have also assessed the calmodulin content of the various Me stages by immunoblot analysis. These studies identified a 1CkDa immunoreactive peptide present at equivalent levels in epi-, trypo-, and amastigote stages (extracelhdar). 0 1992 Academic RUSS, 1~. INDEX DESCRIPTORS AND ABBREVIATIONS: Hemoflagellates: ‘ftypnnosoma cruzi; calmodulin; kDa, kilodalton; EGTA, ethylene bis(oxyethylenenitrilo)tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Trypanosoma cruzi is a hemoflagellate and the etiologic agent of Chagas’ disease, the most important cause of heart disease in many areas of Latin America. This organism undergoes a complex series of morphological transformations within the insect vector and the mammalian host (Brener 1973). The molecular mechanisms that result in these developmental changes remain poorly understood. To explore these control mechanisms we have begun to investigate the signal transduction pathways of this parasite which are homologous to those in higher organisms known to be involved in the regulation of cell growth and differentiation. Calmodulin is a ubiquitous regulatory protein involved in a variety of calciumdependent cellular processes including cell cycle control, protein secretion, cytoskeletal organization, and cell motility (Means 1988). Calmodulin has been shown to be
present in Ttypanosoma but little is known of the cellular targets (Ruben et al. 1983, 1990; Ruben and Patton 1987). Ruben et al. (1990) have suggested that calmodulin may serve only limited specialized functions in Trypanosoma. This hypothesis was based on a variety of observations including the identification of a single calmodulin-binding protein by affinity chromatography in T. brucei homogenates, the virtual absence of calmodulin-binding proteins in both T. cruzi and T. brucei cytosolic extracts by a [1251]calmodulin gel overlay procedure, and the absence of detectable calmodulin stimulation of trypanosomal enzymes normally regulated by calmodulin in higher eukaryotes. These enzymes included CAMP phosphodiesterase (Walter and Opperdoes 1982), calcium ATPase (McLaughlin 1985), and adenylate cyclase (Voorheis and Martin 1981). A major problem with many of these early studies was that cell fractionation was performed in the virtual absence of any protease inhibitors. It is now well 127 0014-4894192$3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
128
ORR,TANOWITZ,AND
established that many, if not all, calmodulin-binding proteins are exquisitely sensitive to proteolysis with the concomitant loss of all calmodulin-dependent binding activity (Kincaid and Vaughan 1986). With enzymes the problem is further compounded by the fact that after loss of calmodulin-binding activity, enzymatic activity is stimulated to that observed in the presence of calmodulin. In fact Telez-Inon et al. (1985) have reported the presence of a CAMP phosphodiesterase activity regulated by calcium/calmodulin in T. cruzi. We decided, therefore, to utilize a general approach, namely the the [‘251]calmodulinoverlay procedure (Slaughter and Means 1987) to determine the calcium-dependent calmodulin-binding proteins in the various cellular compartments of T. cruzi under conditions where proteolysis was kept to a minimum. In addition, we were also interested in whether there were differences in the calmodulin-binding proteins in the various life cycle stages of the organism, i.e., epimastigote, trypomastigote, and amastigote (extracellular). MATERIALS
ANDMETHODS
Parasite. The Tulahuen strain of T. cruzi was maintained in A/J mice (Jackson Laboratories, Bar Harbor, ME) by syringe passage (Trischmann et a/. 1978)and trypomastigotes were harvested from infected L,E, myoblasts as previously described (Rowin ef al. 1983). Trypomastigotes were separated from host cell debris and purified by passage through a Whatman No. 1 filter. This procedure removed all microscopically viable host debris as determined by light microscopy (1000x) and low-power transmission electron microscopy (3200x). Epimastigotes were maintained in liver infusion tryptose broth supplemented with 10% fetal calf serum (GIBCO). Extracellular amastigotes were prepared according to the method of Andrews et al. (1988a). We recognize that the extracellular amastigotes used in these studies may not have all of the attributes of intracellular amastigotes. However, they have been found to have unique stage-specific antigens (Andrews et al. 1988b). Therefore, at the present time these extracellular amastigotes may be the only reliable model of amastigotes available. Cell fractionation procedures. Organisms (2.5 x lO’/ml) were suspended in Buffer A (10 mM phos-
WITTNER
phate, pH 7.5, containing 0.1 mM EGTA, 25 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 pg/ml each of aprotinin, leupeptin, and pepstatin) and subjected to four cycles of rapid freeze/ thawing. Cytosol was removed by centrifugation at 14,000 rpm in an Eppendorf centrifuge (Model 5415) for 10 min. The pellet was washed with Buffer A and then extracted for 30 min at 4°C with Buffer A containing 1% Triton X-100 (w/v). Detergent-soluble components were isolated by centrifugation as described above. The pellet was then extracted with Buffer A containing 1% SDS and 1 mdithiothreitol at 90°C for 5 min followed by centrifugation. SDS-PAGEI[‘2’IJcalmodulin overlay. Samples were subjected to SDS-PAGE (10%) analysis as described by Laemmli (1970). Detection of calmodulin-binding proteins by the direct gel overlay was performed according to a modification of the procedure of Glenny and Weber (1983). In this study the blocking buffer contained 5% BSA (w/v) and 0.1% gelatin (w/v). Bovine testes calmodulin was purified and radioiodinated as described by Wasco et al. (1989). Although it has been shown that trypanosome and bovine brain calmodulin share many common sequences we recognize that some differences may exist. SDS-PAGEiimmunoblofting. Trypanosomal proteins were resolved on a SDS-PAGE gel (15%) and transferred to nitrocellulose as described by Wasco et al. (1989). After blocking in 5% BSA in Tris-saline, pH 7.4, blots were incubated with rabbit anti-T.b. rhodesiense-calmodulin (clone Y’AT1.2) (1: 1000 dilution) (Schleck et al. 1991; Schleck and Patton 1991)for 2 hr at room temperature and washed, and the immune complexes were visualized by 1251-proteinA (2 x 10’ cpmiml) followed by autoradiography at - 70°C in the presence of an intensifying screen. RESULTS
Epimastigotes, trypomastigotes, and amastigotes were fractionated into cytosolic, membrane, and particulate extracts, as described above, and equivalent amounts (extracts from 4 X IO6 organisms) subjected to 10% SDS-PAGE (Fig. 1). It is clear that calmodulin bound in a calciumdependent manner to a number of discrete proteins in both the cytosolic and particulate fractions from the various developmental stages. These binding proteins ranged in subunit molecular weight from greater than 200 kDa to less than 20 kDa. Specificity was established by showing that the inclusion of EGTA or an excess of nonradioactive cal-
STAGES OF CALMODULIN-BINDING part. E T A
PROTEINS
129
Certain of these calcium-dependent, calmodulin-binding proteins (designated by * . - 200 and l for amastigote and epimastigote97 . z-97 specific, respectively) are differentially ex66 * -66 . pressed at specific stages of development. 4b* -44 The vast majority of these stage-specific calmodulin-binding proteins are particulate and are found only in the replicative stages, i.e., epimastigote and amastigote. In the cytosol from all three life forms the same spectrum of calmodulin-binding proteins, -97 -66 albeit in different amounts, was found sug-44 gesting that these proteins were performing general “housekeeping” functions. The -26 membrane fraction from all life stages and the particulate fraction from trypomastiFIG. 1. [‘251]Calmodulin gel overlay of cytosolic gotes (nonreplicative) contained a paucity membrane and particulate extracts from three life of calmodulin-binding proteins. stages. The gel overlay procedure was performed as The gel overlay procedure was also perdescribed in text. (Top) [‘*‘I]Calmodulin binding in the formed on epimastigote and trypomastigote presence of 0.5 mM calcium. (Bottom) [‘251]Calmodulin binding in the presence of 0.5 mM EGTA. (E, epi- extracts which had been stored at -70°C mastigote; T, trypomastigote; A, extracellular amasti- for 4 days. It is apparent from Fig. 2 that gote) * and l indicate calmodulin-binding proteins although the calcium-dependent binding specific to A and E, respectively. Values in the right profile is qualitatively similar to the earlier margin indicate position of molecular mass marker experiment, enhanced non-calciumproteins in kDa. dependent binding is now observed in both the cytosolic and particulate extracts. This modulin (data not shown) abolished the ma- is a phenomenon which we consistently objority of the calmodulin-dependent binding. served after prolonged storage of mammaThere are a few low-molecular-weight pro- lian sperm and is presumably due to proteteins present in the cytosolic and particu- olysis (unpublished work). For this reason late extracts of amastigotes which bind cal- it is not possible at the present time to demodulin in a calcium-independent manner. termine whether the small number of calSeveral studies have shown intense non- cium-independent calmodulin-binding procalcium-dependent binding to low-molec- teins observed in Fig. 1 are genuine or the ular-weight proteins in particulate extracts result of the procedure. from various cells and tissues (Slaughter To determine whether calmodulin itself is and Means 1987; Eldik and Burgess 1983). differentially expressed at specific stages of In fact, it has been shown that [‘251]cal- development the various fractions were modulin will bind to histones in the pres- subjected to immunoblot analysis using anence of EGTA but not in the presence of tisera raised against T. brucei calmodulin calcium (Eldik and Burgess 1983). In this (Schleck er al. 1991; Schleck and Patton study we have performed the EGTA con- 1991). Their data confirm that the protein in trol by first incubating with [‘251]calmodulin the present study examined by this antiin the presence of calcium, removing ex- body is a T. cruzi calmodulin. Moreover, cess calmodulin, and then washing rapidly these proteins undergo calcium-dependent with EGTA to remove calmodulin bound in mobility shifts and comigrate with calmodulin (Schleck et al. 1991; Schleck and Pata calcium-dependent manner (Fig. 1). CCl2’ 200 2
E
T
A
memb. ETA
l
130
ORR, TANOWITZ,
Cytosol JET
memb.
part
ET
FT
AND
WITTNER
contained EGTA it is not surprising that the majority of the calmodulin is found in the cytosolic extracts. There was minor labeling observed in both the detergent and particulate extracts from all three life stages. Currently, it is not possible to distinguish between a minor population of calmodulin tightly bound to subcellular structures in a calcium-independent manner and calmodulin nonspecifically trapped during cell fractionation. Densiometric scanning indicates that the relative content of calmodulin in epimastigotes, trypomastigotes, and extracellular amastigotes is 1:0.85:0.89. DISCUSSION
FIG. 2. [‘251]Calmodulin gel overlay of cytosolic membrane and particulate extracts of epimastigotes and trypomastigotes after storage at - 70°C for 4 days. (Top) [‘2SI]Calmodulin binding in the presence of 0.5 mA4 calcium. (Bottom) [‘251]Calmodulin binding in the presence of 0.5 mM EGTA.
ton; data not shown). Recent studies have shown that the calmodulin of T. cruzi and that of T. brucei are almost identical differing in one conservative amino acid replacement (Chung and Swindle 1990). Figure 3 shows that the three life stages studied contained a single immunoreactive polypeptide of 14 kDa. Since all fractionation buffers Epi. %-iii
TWP. P”-C M P”
Amast. C
M
P’
kDa 14FIG. 3. Anti-calmodulin immunoblot analysis of cytosolic, membrane, and particulate extracts from three life stages. See text for details (C, cytosol; M, membrane; P, particulate).
The gel overlay procedure has proved to be a convenient method for determining the absence or presence of calmodulin-binding proteins in a variety of organisms (Glenny and Weber 1983; Slaughter and Means 1987). The argument has been made that this procedure may underestimate the number of calmodulin-binding proteins since some proteins may not renature sufficiently following SDS-PAGE and the various washings necessary to remove the SDS. However, to date, all calmodulin-binding proteins identified by other independent criteria, i.e., affinity chromatography and chemical or photochemical crosslinking, have been shown to interact with [lz51]calmodulin using the SDS-PAGE/gel overlay procedure (Slaughter and Means 1987). This is the first report demonstrating that T. cruzi possess a complex profile of calcium-dependent calmodulin-binding proteins. Analysis of three of the life stages of the organism indicates that several of these binding proteins are differentially expressed at specific stages of development. The majority of these stage-specific proteins were found in the particulate fractions of the replicative stages of T. cruzi, i.e., epimastigote and amastigote. At the present time the function of any of these
STAGES OF CALMODULIN-BINDING
calmodulin-binding proteins, either constitutively or differentially expressed, is not known. However, the complexity of their composition suggests that calcium/ calmodulin signaling pathways occupies a central role in the growth and development of the parasite, as is the case in higher eukaryotic organisms. It is of interest that those life stage forms that replicate, i.e., epimastigotes and amastigotes, have the largest numbers of calmodulin-binding proteins, whereas the trypomastigote has a paucity of these proteins. The implications of these observations in the development of the parasite as well as the signal transduction mechanisms in each form are currently a subject of investigation. Our data indicating the presence of significant numbers of calcium-dependent calmodulin-binding proteins in T. cruzi are at variance with those of Ruben et al. (1990). These workers concluded that calmodulin must have a limited and specialized function in trypanosomes due to the relatively few calmodulin-binding proteins found in their trypanosomal preparations. We believe that the lack of calmodulin-binding proteins in their study may have been due to proteolysis. In fact, the major labeling observed by Ruben et al. (1990) was calcium-independent and consisted primarily of low-molecular-weight polypeptides. We have also assessed the calmodulin content of the various T. cruzi life stages by immunoblot analysis using T. brucei calmodulin antisera. Our studies have identified a 1CkDa immunoreactive polypeptide present in equivalent amounts in the three life stages suggesting that the amount of calmodulin does not significantly change during the life cycle of the parasite. In recent years there has been increasing interest in investigating the signal transduction pathways in T. cruzi and its potential role in morphogenesis. For example, the addition of CAMP analogs or inhibitors of CAMP phosphodiesterases has been shown
PROTEINS
131
to induce differentiation of epimastigotes into metacyclic trypomastigotes (GonzalesPerdome et al. 1988; Rangel-Aldao et al. 1988; Heath et al. 1990). In addition, Tellez-Inon et al. (1985) have demonstrated the presence of a calmodulin-regulated CAMP-dependent phosphodiesterase. Furthermore, there have been suggestions that the G-proteins are present in this parasite (Eisenschlos et al. 1986). Moreover, our laboratory has investigated calcium homeostasis in both trypomastigotes and epimastigotes (Morris er al. 1991)and the presence of a inositol phosphate/diacylglycerol pathway in the epimatigote form has recently been demonstrated (Docampo and Pignataro 1991). In summary, we have shown that when precautions are taken to minimize proteolysis, T. cruzi contains a complex profile of calcium-dependent calmodulin-binding proteins and that several of these polypeptides are differentially expressed at specific stages of development. The functional significance of these calmodulin-binding proteins is currently under active investigation. Although calmodulin inhibitors have been used to block the transformation from one life stage to another (GonzalezPerdomo et al. 1988); the significance of these observations is unclear since these compounds may also be cytotoxic. We are currently characterizing the genes for these calcium-dependent calmodulin-binding proteins by screening T. cruzi genomic and stage-specific cDNA expression libraries with [1251]calmodulin with the expectation that this will lead to a better understanding of calcium-dependent signal transduction in this parasite and the role that it plays in growth and development. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants AI-26368, AI-12770, AI-29747, MH 45654, and P30-CA13330. G.A.O. was a recipient of NIH RCDA Award HDO0.577and an Irma T. Hirschl,
132
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Monique Weill-Caulier Career Scientist Award. The authors thank Drs. Curtis L. Patton and Sarah Schleck, Yale University, for providing us with rabbit anti-CaM as well as helpful discussions regarding this manuscript, and Vickie Braunstein and Wanda Rivera for excellent technical assistance. REFERENCES ANDREWS, N. W., HONG, K. S., ROBBINS, E. S., AND NUSSENZWEIG,V. 1988a. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Experimental Parasitology
64, 47ti84.
ANDREWS, N. W., ROBBINS,E. S., LEY, V., HONG, K. S., AND NUSSENZWEIG,V. 1988b. Developmentally regulated, phospholipase C-mediated release of the major surface glycoprotein of amastigotes of Trypanosoma cruzi. Journal cine 167, 300-314.
of Experimental
BRENER,Z. 1973. Biology of Trypanosoma nual Review of Microbiology
Medi-
cruri. An-
27, 347-382.
CHUNG, S-H., AND SWINDLE, J. 1990. Linkage of the calmodulin and ubiquitin loci in Trypanosoma cruzi. Nucleic Acids Research 18, 4561-4569. DOCAMPO,R., AND PIGNATARO,0. P. 1991. The inositol phosphateidiacylglycerol signalling pathway in Trypanosoma
cruzi. Biochemical
Journal
275, 407-
411. EISENSCHLOS,C. D., PALADINI, A. A., MOLINA Y VEDIA, L., TORRES,H. N., AND FLAWIA, M. M. 1986. Evidence for the existence of an N,-type regulatory protein in Trypanosoma cruzi membranes. Biochemical
Journal
237, 913-917.
ELDIK, L. J. V., AND BURGESS,W. H. 1983. Analytical subcellular distribution of calmodulin and calmodulin-binding proteins in normal and virustransformed fibroblasts. Journal of Biological Chemistry
258, 4539-4547.
GLENNY, J. R., JR., AND WEBER, K. 1983. Detection of calmodulin binding polypeptides separated in SDS-polyacrylamide gels by a sensitive [‘2SI]calmodulin gel overlay assay. In “Methods in Enzymology,” Vol. 102, pp. 204-220. Academic Press, New York. GONZALES-PERDOMO,M., ROMERO,P., AND GOLDENBERG,S. 1988.Cyclic AMP and adenylate cyclase activators stimulate Trypanosoma cruzi differentiation. Experimental Parasitology 66, 205-212. HEATH, S., HIENY, S., AND SHER, A. 1990. A cyclic AMP inducible gene expressed during the development of infective stages of Trypanosoma cruzi. Molecular and Biochemical Parasitology 43, 133-142. KINCAID, R. L., AND VAUGHAN, M. V. 1986. In “Calcium and Cell Function” (W. Y. Cheung, Ed.), pp. 43-70. Academic Press, New York. LAEMMLI, U. K. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature
(London)
227, 680-685.
AND WITTNER MCLAUGHLIN, J. 1985. A high affinity Ca’+dependent ATPase in the surface membrane of the bloodstream stage of Trypanosoma rhodesiense. Molecular and Biochemical Parasitology 15, 189201. MEANS, A. R. 1988. Molecular mechanisms of action of calmodulin. Recent Progress in Hormone Research 44, 223-262.
MORRIS,S. A., TANOWITZ, H. B., BILEZIKIAN, J. P., AND WITTNER, M. 1991. Modulation of host cell metabolism by Trypanosoma cruzi. Parasitology Today 7, 82-87.
RANGEL-ALDAO, R., TRIANA, F., FERNANDEZ, V., COMACH G., ABATE, T., AND MONTOREANO,R. 1988. Cyclic AMP as an inducer of the cell differentiation of Ttypanosoma cruzi. Biochemical International 17, 337-344. ROWIN, K. S., TANOWITZ, H. B., WITTNER, M., NYGUEN, H. T., AND NADAL-CINARD, B. 1983. Inhibition of muscle differentiation by Trypanosoma cruzi. Proceedings of the National Academy of Sciences (USA) 80, 63906394. RUBEN, L., EGWNAGU,C., AND PATTON, C. L. 1983.
African trypanosomes contain calmodulin which is distinct from host calmodulin. Biochimica Biophysica Acta 758, 104-l 13. RUBEN, L., HAGHIGHAT, N., AND CAMPBELL, A. 1990. Cyclical differentiation of Trypanosoma brucei involves changes in the cellular component of calmodulin-binding proteins. Experimental Parasirology 70, 144153. RUBEN, L., AND PATTON, C. L. 1987. Calmodulin from Trypanosoma brucei: Immunological analysis and genomic organization. In “Methods in Enzymology,” Vol. 139, pp. 262-276. Academic Press, New York. SCHLECK,S. A., AND PATTON, C. L. 1991. Molecular cloning and characterization of CaMcruzin, a Ca2+dependent trypanosome calmodulin-binding protein. Submitted for publication. SCHLECK,S. A., AND PATTON, C. L. 1991. Recombinant trypanosome calmodulin (TCaM) detects Ca’+-dependent calmodulin-binding proteins in Trypanosoma brucei rhodesiense. Submitted for publication. SLAUGHTER,G. R., AND MEANS. A. R. 1987. Use of the ‘2sl-labeled protein gel overlay technique to study calmodulin-binding proteins. In “Methods in Enzymology,” Vol. 139, pp. 433-444. Academic Press, New York. TELLEZ-INON, M. T., ULLOA, R. M., TORRUELA,M., AND TORRES,H. N. 1985. Calmodulin and calciumdependent cyclic AMP phosphodiesterase activity in Trypanosoma cruzi. Molecular and Biochemical Parasitology 14, 143-153. TRISCHMANN,T., TANOWITZ, H. B., WITTNER, M., AND BLOOM, B. R. 1978. The role of the immune
STAGES OF CALMODULIN-BINDING response in the natural resistance of inbred strains of mice. Experimental Parasitology 45, 160-166. VOORHEIS, H. P., AND MARTIN, B. R. 1981. Characteristics of the calcium mediated mechanism activating adenylate cyclase in Trypanosoma brucei. Euro. pean Journal of Biochemistry 116, 411-477. WALTER, R. D., AND OPPERDOES, F. R. 1982. Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phos-
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phoprotein phosphatase in Trypanosoma Molecular
and Biochemical
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brucei. 6, 287-
291. WASCO, W. M., KINCAID,
R. L., AND ORR, G. A.
1989. Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella. Journal of Biological
Chemistry
264, 5104-5111.
Received 23 July 1991; accepted with revision 3 October 1991
PARASlTo1.oC.Y
14, 121-133 (1992)
Trypanosoma cruzi: Stage Expression Calmodulin-Binding Proteins
of
GEORGE A. ORR, HERBERT B. TANOWITZ, AND MURRAY WITTNER Departments
of Molecular Pharmacology, Pathology, and Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, U.S.A.
ORR, G. A., TANOWITZ, H. B., AND WITTNER, M. 1992. Trypanosomi cruzi: Stage expression of calmodulin-binding proteins. Experimental Parasitology 74, 127-133. The subcellular distribution of calmodulin-binding proteins in three life stages of Trypanosoma cruzi was analyzed by a [‘ZsI]calmodulin gel overlay procedure under conditions where proteolysis was kept to a minimum. It was found that T. cruzi contains a complex profile of calcium-dependent calmodulin-binding proteins and that several of these polypeptides were differentially expressed at specific stages of development. The majority of these stagespecific polypeptides was found in the particulate fractions of the replicative stages of the parasite, i.e., epimastigote and amastigote. These studies suggest that calcium and calmodulin may play an important central role in the growth and differentiation of this parasite. We have also assessed the calmodulin content of the various Me stages by immunoblot analysis. These studies identified a 1CkDa immunoreactive peptide present at equivalent levels in epi-, trypo-, and amastigote stages (extracelhdar). 0 1992 Academic RUSS, 1~. INDEX DESCRIPTORS AND ABBREVIATIONS: Hemoflagellates: ‘ftypnnosoma cruzi; calmodulin; kDa, kilodalton; EGTA, ethylene bis(oxyethylenenitrilo)tetraacetic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Trypanosoma cruzi is a hemoflagellate and the etiologic agent of Chagas’ disease, the most important cause of heart disease in many areas of Latin America. This organism undergoes a complex series of morphological transformations within the insect vector and the mammalian host (Brener 1973). The molecular mechanisms that result in these developmental changes remain poorly understood. To explore these control mechanisms we have begun to investigate the signal transduction pathways of this parasite which are homologous to those in higher organisms known to be involved in the regulation of cell growth and differentiation. Calmodulin is a ubiquitous regulatory protein involved in a variety of calciumdependent cellular processes including cell cycle control, protein secretion, cytoskeletal organization, and cell motility (Means 1988). Calmodulin has been shown to be
present in Ttypanosoma but little is known of the cellular targets (Ruben et al. 1983, 1990; Ruben and Patton 1987). Ruben et al. (1990) have suggested that calmodulin may serve only limited specialized functions in Trypanosoma. This hypothesis was based on a variety of observations including the identification of a single calmodulin-binding protein by affinity chromatography in T. brucei homogenates, the virtual absence of calmodulin-binding proteins in both T. cruzi and T. brucei cytosolic extracts by a [1251]calmodulin gel overlay procedure, and the absence of detectable calmodulin stimulation of trypanosomal enzymes normally regulated by calmodulin in higher eukaryotes. These enzymes included CAMP phosphodiesterase (Walter and Opperdoes 1982), calcium ATPase (McLaughlin 1985), and adenylate cyclase (Voorheis and Martin 1981). A major problem with many of these early studies was that cell fractionation was performed in the virtual absence of any protease inhibitors. It is now well 127 0014-4894192$3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
128
ORR,TANOWITZ,AND
established that many, if not all, calmodulin-binding proteins are exquisitely sensitive to proteolysis with the concomitant loss of all calmodulin-dependent binding activity (Kincaid and Vaughan 1986). With enzymes the problem is further compounded by the fact that after loss of calmodulin-binding activity, enzymatic activity is stimulated to that observed in the presence of calmodulin. In fact Telez-Inon et al. (1985) have reported the presence of a CAMP phosphodiesterase activity regulated by calcium/calmodulin in T. cruzi. We decided, therefore, to utilize a general approach, namely the the [‘251]calmodulinoverlay procedure (Slaughter and Means 1987) to determine the calcium-dependent calmodulin-binding proteins in the various cellular compartments of T. cruzi under conditions where proteolysis was kept to a minimum. In addition, we were also interested in whether there were differences in the calmodulin-binding proteins in the various life cycle stages of the organism, i.e., epimastigote, trypomastigote, and amastigote (extracellular). MATERIALS
ANDMETHODS
Parasite. The Tulahuen strain of T. cruzi was maintained in A/J mice (Jackson Laboratories, Bar Harbor, ME) by syringe passage (Trischmann et a/. 1978)and trypomastigotes were harvested from infected L,E, myoblasts as previously described (Rowin ef al. 1983). Trypomastigotes were separated from host cell debris and purified by passage through a Whatman No. 1 filter. This procedure removed all microscopically viable host debris as determined by light microscopy (1000x) and low-power transmission electron microscopy (3200x). Epimastigotes were maintained in liver infusion tryptose broth supplemented with 10% fetal calf serum (GIBCO). Extracellular amastigotes were prepared according to the method of Andrews et al. (1988a). We recognize that the extracellular amastigotes used in these studies may not have all of the attributes of intracellular amastigotes. However, they have been found to have unique stage-specific antigens (Andrews et al. 1988b). Therefore, at the present time these extracellular amastigotes may be the only reliable model of amastigotes available. Cell fractionation procedures. Organisms (2.5 x lO’/ml) were suspended in Buffer A (10 mM phos-
WITTNER
phate, pH 7.5, containing 0.1 mM EGTA, 25 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride, and 5 pg/ml each of aprotinin, leupeptin, and pepstatin) and subjected to four cycles of rapid freeze/ thawing. Cytosol was removed by centrifugation at 14,000 rpm in an Eppendorf centrifuge (Model 5415) for 10 min. The pellet was washed with Buffer A and then extracted for 30 min at 4°C with Buffer A containing 1% Triton X-100 (w/v). Detergent-soluble components were isolated by centrifugation as described above. The pellet was then extracted with Buffer A containing 1% SDS and 1 mdithiothreitol at 90°C for 5 min followed by centrifugation. SDS-PAGEI[‘2’IJcalmodulin overlay. Samples were subjected to SDS-PAGE (10%) analysis as described by Laemmli (1970). Detection of calmodulin-binding proteins by the direct gel overlay was performed according to a modification of the procedure of Glenny and Weber (1983). In this study the blocking buffer contained 5% BSA (w/v) and 0.1% gelatin (w/v). Bovine testes calmodulin was purified and radioiodinated as described by Wasco et al. (1989). Although it has been shown that trypanosome and bovine brain calmodulin share many common sequences we recognize that some differences may exist. SDS-PAGEiimmunoblofting. Trypanosomal proteins were resolved on a SDS-PAGE gel (15%) and transferred to nitrocellulose as described by Wasco et al. (1989). After blocking in 5% BSA in Tris-saline, pH 7.4, blots were incubated with rabbit anti-T.b. rhodesiense-calmodulin (clone Y’AT1.2) (1: 1000 dilution) (Schleck et al. 1991; Schleck and Patton 1991)for 2 hr at room temperature and washed, and the immune complexes were visualized by 1251-proteinA (2 x 10’ cpmiml) followed by autoradiography at - 70°C in the presence of an intensifying screen. RESULTS
Epimastigotes, trypomastigotes, and amastigotes were fractionated into cytosolic, membrane, and particulate extracts, as described above, and equivalent amounts (extracts from 4 X IO6 organisms) subjected to 10% SDS-PAGE (Fig. 1). It is clear that calmodulin bound in a calciumdependent manner to a number of discrete proteins in both the cytosolic and particulate fractions from the various developmental stages. These binding proteins ranged in subunit molecular weight from greater than 200 kDa to less than 20 kDa. Specificity was established by showing that the inclusion of EGTA or an excess of nonradioactive cal-
STAGES OF CALMODULIN-BINDING part. E T A
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Certain of these calcium-dependent, calmodulin-binding proteins (designated by * . - 200 and l for amastigote and epimastigote97 . z-97 specific, respectively) are differentially ex66 * -66 . pressed at specific stages of development. 4b* -44 The vast majority of these stage-specific calmodulin-binding proteins are particulate and are found only in the replicative stages, i.e., epimastigote and amastigote. In the cytosol from all three life forms the same spectrum of calmodulin-binding proteins, -97 -66 albeit in different amounts, was found sug-44 gesting that these proteins were performing general “housekeeping” functions. The -26 membrane fraction from all life stages and the particulate fraction from trypomastiFIG. 1. [‘251]Calmodulin gel overlay of cytosolic gotes (nonreplicative) contained a paucity membrane and particulate extracts from three life of calmodulin-binding proteins. stages. The gel overlay procedure was performed as The gel overlay procedure was also perdescribed in text. (Top) [‘*‘I]Calmodulin binding in the formed on epimastigote and trypomastigote presence of 0.5 mM calcium. (Bottom) [‘251]Calmodulin binding in the presence of 0.5 mM EGTA. (E, epi- extracts which had been stored at -70°C mastigote; T, trypomastigote; A, extracellular amasti- for 4 days. It is apparent from Fig. 2 that gote) * and l indicate calmodulin-binding proteins although the calcium-dependent binding specific to A and E, respectively. Values in the right profile is qualitatively similar to the earlier margin indicate position of molecular mass marker experiment, enhanced non-calciumproteins in kDa. dependent binding is now observed in both the cytosolic and particulate extracts. This modulin (data not shown) abolished the ma- is a phenomenon which we consistently objority of the calmodulin-dependent binding. served after prolonged storage of mammaThere are a few low-molecular-weight pro- lian sperm and is presumably due to proteteins present in the cytosolic and particu- olysis (unpublished work). For this reason late extracts of amastigotes which bind cal- it is not possible at the present time to demodulin in a calcium-independent manner. termine whether the small number of calSeveral studies have shown intense non- cium-independent calmodulin-binding procalcium-dependent binding to low-molec- teins observed in Fig. 1 are genuine or the ular-weight proteins in particulate extracts result of the procedure. from various cells and tissues (Slaughter To determine whether calmodulin itself is and Means 1987; Eldik and Burgess 1983). differentially expressed at specific stages of In fact, it has been shown that [‘251]cal- development the various fractions were modulin will bind to histones in the pres- subjected to immunoblot analysis using anence of EGTA but not in the presence of tisera raised against T. brucei calmodulin calcium (Eldik and Burgess 1983). In this (Schleck er al. 1991; Schleck and Patton study we have performed the EGTA con- 1991). Their data confirm that the protein in trol by first incubating with [‘251]calmodulin the present study examined by this antiin the presence of calcium, removing ex- body is a T. cruzi calmodulin. Moreover, cess calmodulin, and then washing rapidly these proteins undergo calcium-dependent with EGTA to remove calmodulin bound in mobility shifts and comigrate with calmodulin (Schleck et al. 1991; Schleck and Pata calcium-dependent manner (Fig. 1). CCl2’ 200 2
E
T
A
memb. ETA
l
130
ORR, TANOWITZ,
Cytosol JET
memb.
part
ET
FT
AND
WITTNER
contained EGTA it is not surprising that the majority of the calmodulin is found in the cytosolic extracts. There was minor labeling observed in both the detergent and particulate extracts from all three life stages. Currently, it is not possible to distinguish between a minor population of calmodulin tightly bound to subcellular structures in a calcium-independent manner and calmodulin nonspecifically trapped during cell fractionation. Densiometric scanning indicates that the relative content of calmodulin in epimastigotes, trypomastigotes, and extracellular amastigotes is 1:0.85:0.89. DISCUSSION
FIG. 2. [‘251]Calmodulin gel overlay of cytosolic membrane and particulate extracts of epimastigotes and trypomastigotes after storage at - 70°C for 4 days. (Top) [‘2SI]Calmodulin binding in the presence of 0.5 mA4 calcium. (Bottom) [‘251]Calmodulin binding in the presence of 0.5 mM EGTA.
ton; data not shown). Recent studies have shown that the calmodulin of T. cruzi and that of T. brucei are almost identical differing in one conservative amino acid replacement (Chung and Swindle 1990). Figure 3 shows that the three life stages studied contained a single immunoreactive polypeptide of 14 kDa. Since all fractionation buffers Epi. %-iii
TWP. P”-C M P”
Amast. C
M
P’
kDa 14FIG. 3. Anti-calmodulin immunoblot analysis of cytosolic, membrane, and particulate extracts from three life stages. See text for details (C, cytosol; M, membrane; P, particulate).
The gel overlay procedure has proved to be a convenient method for determining the absence or presence of calmodulin-binding proteins in a variety of organisms (Glenny and Weber 1983; Slaughter and Means 1987). The argument has been made that this procedure may underestimate the number of calmodulin-binding proteins since some proteins may not renature sufficiently following SDS-PAGE and the various washings necessary to remove the SDS. However, to date, all calmodulin-binding proteins identified by other independent criteria, i.e., affinity chromatography and chemical or photochemical crosslinking, have been shown to interact with [lz51]calmodulin using the SDS-PAGE/gel overlay procedure (Slaughter and Means 1987). This is the first report demonstrating that T. cruzi possess a complex profile of calcium-dependent calmodulin-binding proteins. Analysis of three of the life stages of the organism indicates that several of these binding proteins are differentially expressed at specific stages of development. The majority of these stage-specific proteins were found in the particulate fractions of the replicative stages of T. cruzi, i.e., epimastigote and amastigote. At the present time the function of any of these
STAGES OF CALMODULIN-BINDING
calmodulin-binding proteins, either constitutively or differentially expressed, is not known. However, the complexity of their composition suggests that calcium/ calmodulin signaling pathways occupies a central role in the growth and development of the parasite, as is the case in higher eukaryotic organisms. It is of interest that those life stage forms that replicate, i.e., epimastigotes and amastigotes, have the largest numbers of calmodulin-binding proteins, whereas the trypomastigote has a paucity of these proteins. The implications of these observations in the development of the parasite as well as the signal transduction mechanisms in each form are currently a subject of investigation. Our data indicating the presence of significant numbers of calcium-dependent calmodulin-binding proteins in T. cruzi are at variance with those of Ruben et al. (1990). These workers concluded that calmodulin must have a limited and specialized function in trypanosomes due to the relatively few calmodulin-binding proteins found in their trypanosomal preparations. We believe that the lack of calmodulin-binding proteins in their study may have been due to proteolysis. In fact, the major labeling observed by Ruben et al. (1990) was calcium-independent and consisted primarily of low-molecular-weight polypeptides. We have also assessed the calmodulin content of the various T. cruzi life stages by immunoblot analysis using T. brucei calmodulin antisera. Our studies have identified a 1CkDa immunoreactive polypeptide present in equivalent amounts in the three life stages suggesting that the amount of calmodulin does not significantly change during the life cycle of the parasite. In recent years there has been increasing interest in investigating the signal transduction pathways in T. cruzi and its potential role in morphogenesis. For example, the addition of CAMP analogs or inhibitors of CAMP phosphodiesterases has been shown
PROTEINS
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to induce differentiation of epimastigotes into metacyclic trypomastigotes (GonzalesPerdome et al. 1988; Rangel-Aldao et al. 1988; Heath et al. 1990). In addition, Tellez-Inon et al. (1985) have demonstrated the presence of a calmodulin-regulated CAMP-dependent phosphodiesterase. Furthermore, there have been suggestions that the G-proteins are present in this parasite (Eisenschlos et al. 1986). Moreover, our laboratory has investigated calcium homeostasis in both trypomastigotes and epimastigotes (Morris er al. 1991)and the presence of a inositol phosphate/diacylglycerol pathway in the epimatigote form has recently been demonstrated (Docampo and Pignataro 1991). In summary, we have shown that when precautions are taken to minimize proteolysis, T. cruzi contains a complex profile of calcium-dependent calmodulin-binding proteins and that several of these polypeptides are differentially expressed at specific stages of development. The functional significance of these calmodulin-binding proteins is currently under active investigation. Although calmodulin inhibitors have been used to block the transformation from one life stage to another (GonzalezPerdomo et al. 1988); the significance of these observations is unclear since these compounds may also be cytotoxic. We are currently characterizing the genes for these calcium-dependent calmodulin-binding proteins by screening T. cruzi genomic and stage-specific cDNA expression libraries with [1251]calmodulin with the expectation that this will lead to a better understanding of calcium-dependent signal transduction in this parasite and the role that it plays in growth and development. ACKNOWLEDGMENTS This work was supported by National Institutes of Health Grants AI-26368, AI-12770, AI-29747, MH 45654, and P30-CA13330. G.A.O. was a recipient of NIH RCDA Award HDO0.577and an Irma T. Hirschl,
132
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Monique Weill-Caulier Career Scientist Award. The authors thank Drs. Curtis L. Patton and Sarah Schleck, Yale University, for providing us with rabbit anti-CaM as well as helpful discussions regarding this manuscript, and Vickie Braunstein and Wanda Rivera for excellent technical assistance. REFERENCES ANDREWS, N. W., HONG, K. S., ROBBINS, E. S., AND NUSSENZWEIG,V. 1988a. Stage-specific surface antigens expressed during the morphogenesis of vertebrate forms of Trypanosoma cruzi. Experimental Parasitology
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Received 23 July 1991; accepted with revision 3 October 1991
