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Identification and partial characterization of plasma membrane polypeptides of Crithidia guilhermei, crithidia deanei and Crithidia oncopelti
Comp. Biochem. Physiol. Vol. 88B, No. 4, pp. 1091-1096, 1987 Printed in Great Britain
0305-0491/87 $3.00+ 0.00 © 1987PergamonJournals Ltd
IDENTIFICATION A N D PARTIAL CHARACTERIZATION OF PLASMA MEMBRANE POLYPEPTIDES OF CRITHIDIA GUILHERMEI, CRITHIDIA D E A N E I AND CRITHIDIA ONCOPELTI S. GIOVANNI DE SIMONE,* M. J. SOARES,t~ W. DE SOUZAt and S. GOLDENBERC~ *Departamento de Imunologia, Fundaggo Oswaldo Cruz, Av. Brazil 4365, Rio de Janeiro, RJ, Brazil and Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Niter6i, RJ, Brazil; ~'Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, ILl, Brazil; ~:Departamentode Ultra-estrutura e BiologiaCelular, Fundagfio Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; §Departamento de Bioquimica e Biologia Celular, Fundagfio Oswaldo Cruz, Rio de Janeiro, RJ, Brazil (Received 12 February 1987)
Abstract--1. Components of the cell surface of Crithidia guilhermei, Crithidia deanei and Crithidia oncopelti were radioiodinated by the iodogen technique. The distribution of proteins in the detergent-poor (DPP) and detergent-enriched phase (DRP) were studied using a phase separation technique in Triton X-114 and one- and two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulphate (1D and 2D SDS-PAGE). 2. Significant differences were noted in the proteins present in the DRP when the three species were compared. 3. Two major bands with mol. wt 28,000 and 56,000 and motility in the pH gradient of 7.4 and 6.3, respectively, were observed in C. guilhermei, but not discernible in C. deanei and C. oncopelti. 4. One polypeptide with mol. wt 50,000 and pI 4.9 was identified in the DRP of C. deanei. 5. A broad band with mol. wt 68,000-140,000 and pI 4.7-5.5 was clearly observed in the DRP of C. deanei and one or two polypeptides only present in the DPP were observed in the three Crithidia species analyzed. 6. Our observations show that C. guilhermei has characteristic surface polypeptides not found in C. deanei and C. oncopelti. 7. Our results, in association with those reported by others, show that the phase separation using Triton X-114 offers a simple approach to the separation and further analysis of a select group of proteins from the bulk of the cellular proteins.
INTRODUCTION The proteins present in the plasma membrane of eukaryotic cells play an important structural role and are involved in different processes such as absorption and transportation of nutrients, cell recognition, etc. (Singer, 1974; Nicolson, 1976; Jacobson, 1983). In the case of parasites, many of the surface proteins represent important antigens which elicit both humoral and cell-mediated immune responses when they enter in contact with the vertebrate host. In recent years, a large amount of work has been done aiming at the identification of surface proteins in members of the Trypanosomatidae family, especially in those which belong to the genera Trypanosoma and Leishmania (Mancini et al., 1982; Andrews et al., 1984; Gardiner and Dwyer, 1983; Lanar and Manning, 1984). Few data are available on surface proteins of members of the genus Crithidia (Camargo et al., 1982; McLaughlin and Cain, 1985). Most of the existing data are on the surface proteins of trypanosomatids and do not discriminate between hydrophilic and amphiphilic integral membrane proteins. In this paper we analyzed the surface proteins of Crithidia guilhermei, a new species of Crithidia recently characterized in our laboratory (Soares et al., 1986), by surface labeling with 125I followed by
solubilization with Triton X-114 thus separating the proteins into an aqueous and a detergent phase (Bordier, 1981) and characterizing them by one- and two-dimensional polyacrilamide gel electrophoresis. Crithidia guilhermei is a parasite which presents some characteristic morphological features typical of the endosymbiont-bearingtrypanosomatids, although no endosymbionts were found. In this paper, we compare it with Crithidia deanei and Crithidia oncopelti, the only other two species of Crithidia which harbour an endosymbiont (Freymuller and Camargo, 1981). The results obtained are described in this paper.
MATERIALS AND METHODS Parasites Crithidia deanei (ATCC 30255) and C. oncopelti (ATCC 30264) were obtained from Dr Isaac Roitman (University of Brasilia, Brazil). Crithidia guilhermei which was isolated from the hindgut of the fly Phaenicia cuprina and characterized previously (Soarcs et al., 1986) was obtained from Dr Reginaldo Brazil (Department of Parasitology, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil). All strains were kept in Liver Infusion Tryptose medium (Camargo, 1964) at 28°C, with serial passages at 2-day intervals.
1091 C.B.P. $S/4B---E
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GIOVANNI DE SIMON'Eet al.
Surface iodination
Two-dimension PAGE (2D-PAGE)
Cells were collected by centrifugation (1.5 min at 9650g, using a Microfuge B miniature centrifuge, Beckman Instruments, California, USA) and washed in cold 0.15M phosphate-buffered saline, pH 7.2 (PBS). Surface proteins were radiolabeled in accordance with Markwell and Fox (1978) by incubating 1-2 x 108 washed viable parasites with 300-500/zCi of iodine (NaI, Amersham, UK) and 125/~g of 1,3,4,6-tetrachloro-3,6,diphenilglycoluril (Iodo-Gen, Pierce Chemical Co., USA) for 10 min at 0°C. Labelled parasites were washed four times in cold PBS as described above and maintained in an ice bath until extraction with detergent. Phase contrast microscopic observations of the radiolabeled cells showed that they remained motile.
Two dimension PAGE consisted in isotacophoresis (first dimension) followed by electrophoresis in a uniform 13% SDS-polyacrylamide gel (second dimension, O'Farrel et al., 1978). Ampholines in different pH ranges were combined in order to obtain a pH gradient ranging from 4.4 to 8.7. Proteins labeled with ~4C, with mol. wts ranging from 18,000 to 200,000, were run in each gel.
Detergent solubilization and phase separation in Triton X-114 The iodinated cells were solubilized in 2% Triton X-114 (Sigma Chemical Company, St Louis, USA) pre-condensed twice as described by Bordier (1981) in Tris-saline (TS) buffer (10mM Tris, 150mM NaCI, pH7.4) containing 1 mM phenilmethylsulfonic fluoride (PMSF) for 30--40 min at 0-4°C and centrifuged at 20,000 g for 30 min at 0-4°C. Supernatants, collected with the tubes on ice using Pasteur pipettes, were mixed with bromophenol blue (one drop of a 0.1% stock solution) to aid the visualization of the phase separation (Alcaraz et al., 1984) and applied onto a 6% (w/v) sucrose cushion (1:1.5 v/v). After incubation at 37°C for 10 min, samples were centrifuged at room temperature for 15min at 1000g. The upper, detergent-poor phase (DPP) was recovered above the cushion and the blue pellet (detergent-enrich phase; DRP) below it. The DRP was redissolved in TS to the original volume by incubating for 10 min at 0-4°C. To the DPP was added more Triton X-114 (10% stock) and bromophenol blue to yield a final concentration of 2% and the phase separation step was proceeded twice, as described above. The pellet was discarded and then washed DPP and DRP were prepared for SDS-PAGE.
Isolation of the detergent from the proteins Zinc chloride was added to the samples to make a final concentration of 0.05 M and the proteins were precipitated by the addition of 5 volumes of cold acetone. The pellet was collected by centrifugation and the supernatant solution containing the Triton X-114 was discarded. The proteins were resuspended in water and precipitated again with 5 volumes of cold acetone. After centrifugation (9650g, 5 min), the pellet was washed with 50% cold acetone, dried at room temperature and stored at -70°C until use.
SDS-gel electrophoresis Samples of the proteins (DPP and DRP) precipitated with acetone were solubilized in the same volume of hot sample buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1.0% SDS and 0.5% fl-mercaptoethanol) and further boiled for 3 min. Proteins were separated by sodium dodeeyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on gels containing a 7.0-15% linear gradient of polyacrylamide (Laemmli, 1970). After the electrophoresis the gels were stained with Commassie Brilliant Blue 250R, dried and autoradiographed using Sakura Medical Imaging films (Sakura,Japan)and Kodak-X-O-Matic regular intensifying screens (Eastman Kodak Co., Rochester, N.Y., USA). Apparent protein M r was interpolated from the migration of ferritin (220,000), phosphorylase b (94,000), bovine serum albumine (67,000), catalase (60,000), ovalbumin (43,000), lactate dehydrogenase (36,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and lactalbumine (14,400) (Calibration Kit Proteins, Pharmacia Fine Chemicals, Uppsala, Sweden).
RESULTS
Solubilization and phase-separation o f Crithidia proteins To identify and compare the major proteins exposed at the parasite surface, we iodinated coanoamastigotes of C. guilhermei as well as of the two endosymbiont-bearing species C. deanei and C. oncopelti. Total cellular proteins of surface-iodinated parasites were separated by S D S - P A G E and the labeled proteins were revealed by autoradiography and Commassie Blue staining. M i n o r apparent qualitative differences in the pattern of proteins were observed (data not shown). A b o u t 12-15 radiolabeled proteins and several bands stained by Commassie Blue were identified on the surface of the three species of Crithidia analyzed. The two phases ( D R P and DPP) obtained from Triton X-114 extracts of radioiodinated Crithidias were analyzed by S D S - P A G E after staining with Commassie Blue and autoradiography (Fig. 1). A large number of bands were observed in gels stained with Commassie Blue, mainly in D P P (Fig. 1 lanes D - F ) . N o significant differences were observed in such gels among the three Crithidias analyzed. The major radiolabeled membrane proteins were recovered in the D R P of C. deanei (Fig. 1 lane G) and C. guilhermei (Fig. 1 lane I). In the case of C. deanei there was a major broad band from 68,000 to 92,000 and one band with mol. wt 50,000. Other minor bands of lower mol. wts were also seen. In the case of C. guilhermei (Fig. 1 lane I) three or four major bands with mol. wts of 90,000, 85,000, 68,000, 56,000 and 28,000 were evident. In the case of C. oncopelti (Fig. 1 lane H), poorly labeled proteins (88,000, 68,000 and 63,000) were seen. Few radiolabeled membrane proteins were recovered in the D P P (Fig. 1 lanes J-L). Two polypeptides with mol. wts of 78,000 and 70,000 were seen in C. deanei (Fig. 1 lane J). In the case of C. oncopelti (Fig. 1 lane K). and C. guilhermei (Fig. 1 lane L), one band with a mol. wt of about 76,000 was seen. To evaluate whether the differences observed in the D R P proteins of Crithidia species were due to proteins not solubilized by 2% Triton X-114 that could be contaminating the D R P , the insoluble proteins were treated with 4 % SDS and, after centrifugation, analyzed by S D S - P A G E . The autoradiograms (lanes A, B and C) and the staining of the gels with Commassie Blue (lanes D, E and F) are shown in Fig. 2. One major radiolabeled polypeptide with mol. wt 80,000 and two other polypeptides (75,000-78,000 and 130,000-140,000) were observed in the gels of C. oncopelti (lane B) and C. guilhermei (lane C), respectively. These proteins were insoluble in 2% Triton X-114 presumably because isoelectric precipitation
1093
Crithidia plasma membrane polypeptidcs
MW
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Fig. l. SDS-PAGE (7-15%) of proteins from radiolabeled parasites after phase separation and Commassie Blue staining (A-F) or autoradiography (G-L). A, D, G, J: Crithidia deanei; B, E, H, K: Crithidia oncopelti; C, F, I, L: Crithidia guilhermei. DRP, detergent-rich phase; DPP, detergent-poor phase. Small arrows in G and I indicate the position of species-specific proteins. Exposition of the gels of the DPP was twice longer than those for the DRP.
MW •, I 2 2 0
000
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Fig. 2. SDS-PAGE (7-15%) of insoluble proteins in 2% Triton X-114 but which were solubilized in 4% SDS and analyzed by autoradiography (A-C) or Commassie Blue staining (D-F). A, D: Critkidia deanei; B, E: Crithidia oncopelti; C, F: Crithidia guilhermei. Numbers to the right of the figure indicate the migration of proteins of known molecular mass (MW).
1094
GIOVANNIDE SIMONEet al.
Fig. 3. Two dimensional autoradiography of polyacrilamide gel electrophoresis of DRP proteins labelled with 12~Ifrom C. deanei (A), C. oncopelti (B) and C. guilhermei (C). Each gel was loaded with 105cpm and the position of 14C-mol. wt markers run together in the gel is shown in the left side of the figure. The number at the horizontal figure axis refers to the pH gradient.
was unaffected by the nonionic detergent under the pH and ionic strength used (Hallan and Wrigglesworth, 1976). However, the reel. wts of these proteins were different from those observed with D R P proteins.
Two dimension electrophoresis of partially purified amphiphilic proteins When surface radioiodinated Crithidias were phase-separated and aliquots of the detergent phase were submitted to 2D-PAGE, relatively simple poly-
1095
Crithidia plasma membrane polypeptides
peptide profiles were seen by autoradiography. Four major bands, with Mr ranging from 68 to 92,000 and pI 4.7-5.5 (Fig. 3) were seen in all species examined. Two of these bands were more evident in C. deanei (Fig. 3 lane A) and C. oncopelti (Fig. 3 lane B) than in C. guilhermei (Fig. 3 lane C). One band with M r 50 and pI 4.9 was observed in C. deanei (Fig. 3 lane A). Two additional major bands, with mol. wt 28,000 and 56,000 and pI of 7.4 and 6.3, respectively (Fig. 2 lane C), were present in C. guilhermei. DISCUSSION
Most of the work on the identification of surface proteins of trypanosomatids has been done using whole radiolabeled cells solubilized in SDS. Using this approach proteins, most of which are glycoproteins, characteristic of certain species or even characteristic of developmental stages of certain species, have been identified (Camargo et al., 1982; McLaughlin and Cain, 1986; review in Chance, 1979). This approach, however, does not discriminate proteins which strongly interact with the membrane lipid bilayer (integral amphiphilic proteins) from the hydrophilic proteins. It has been shown recently (Bordier, 1981) that membrane proteins would differentially distribute in the two phases formed by the non-ionic detergent Triton X-114 at moderate temperature. Alcaraz and collaborators (1984) envisioned that those proteins capable of binding detergent would segregate to the lower hydrophobic phase, whereas those that did not bind detergent would remain in the upper, detergentdepleted phase. In the present study, we have first solubilized the proteins of three Crithidia species using Triton X-114 and then separated the membrane proteins depending on their ability to be included or excluded from the detergent during phase separation. A comparison of proteins which remain in the detergent-containing phase by 2D-PAGE showed a certain degree of homology among the parasites examined. However, amphiphilic polypeptides specific for species could be demonstrated, as is the case of polypeptides of mol. wt of 28,000 and 56,000 found in C. guilhermei and the polypeptide of 50,000 found in C. deanei. These polypeptides were also seen in 2-dimensional PAGE. It is important to point out that the radiolabeled polypeptides detected by 2-D-PAGE in C. deanei and C. oncopelti are acidic, with pI of 4.7 to 5.5. Only in C. guilherrnei polypeptides with higher pI were detected. Some proteins found in the detergent-enriched phase were not completely solubilized in 2% Triton X-114 but could be visualized when 4% SDS was used. It is possible that these polypeptides represent transmembrane proteins which interact with cytoskeletal components, as previously shown in other systems (Gienney et al., 1982; Glenney and Glenney, 1984; Tiruppathi et al., 1986). It is important to point out that radiolabeled polypeptides were seen in the detergent-poor phase of the three Crithidias analysed. In the case of C. oncopelti the labeling was more evident than that observed in the DRP. These observations suggest that peripheral proteins are exposed on the surface of trypanosomatids,
Our observations, in association with others reported previously (Bouvier et al., 1985; Etges et al., 1985; Kumar, 1985), indicate that Triton X-I14 is very useful for the analysis' of plasma membrane polypeptides. It is interesting to point out that in the trypanosomatids analyzed up till now, the proteins have been detected mainly in the detergent-rich phase (Bordier et al., 1986; Etges et aL, 1985). In the case of Plasmodiurn gallinaceum, a polypeptide was seen in the detergent-poor phase (Kumar, 1985). It is also important to point out that the pattern of labeled surface polypeptides seen in one- and twodimensional P A G E of C. guilhermei was different from the other two strains of Crithidia analyzed. This observation reinforces the suggestion, made based on morphological observations and analysis of the electrophoretic mobility of six enzymes, that C. guilhermei is a new species of Crithidia (Soares et al., 1986). REFERENCES Alcaraz G., Kinet J. P., Kumar N. and Metzzwer H. (1984)
Phase separation of the receptor for immunogiobulin E and its subunits in Triton X-II4. J. biol. Chem. 259, 14922-14927. Andrews N. W., Katzin A. M. and Colli W. (1984) Mapping of surface glycoproteins of Trypanosoma cruzi by two dimensional electrophoresis. Eur. J. Biochem. 140, 599-604. Bordier C. (1981) Phase separation of integral membrane proteins in Triton X-I14 solution. J. biol. Chem. 256, 1604--1607. Bordier C., Etges R. J., Ward J., Turner M. J. and Cardoso de Almeida M. L. (1986) Leishmania and Trypanosoma surface glycoproteins have a common giycophospholipid membrane anchor. Proc. natn. Acad. Sci. USA 83, 5988-5991. Bouvier J., Etges R. J. and Bordier C. P. (1985) Identification and purification of membrane and soluble forms of the major surface protein of Leishmania promastigotes. .L biol. Chem. 260, 15504-15509. Camargo E. P. (1964) Growth and differentiation of Trypanosoma cruzi--I. Origin of metacyclic trypanosomes in liquid media. Rev. Inst. Med. Trop. Sao Paulo 6, 93-100. Camargo E. P., Mattei D. M., Barbieri C. L. and Morel C. M. (1982) Electrophoretic analysis of endonucleasegenerated fragments of K-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids. J. Protozool. 29, 251-258. Chance M. (1979) The identification of Leishmania. In Problems in the Identification of Parasites, and their Vectors (Edited by Taylor A. C. and Muller R.), pp.
55-74. BlackweU Scientific Publications, Oxford. Etges R. J., Bouvier J., Hoffman R. and Bordier C. (1985) Evidence that the major surface proteins of three Leishmania species are structurally related. Molec. Biochem. Parasit. 14, 141-149. Freymuller E. and Camargo E. P. (1981) Ultrastructural differences between species of trypanosomatids with and without endosymbionts. J. Protozool. 28, 175-181. Gardiner P. R. and Dwyer D. M. (1983) Radioiodination and identification of externally disposed membrane components of Leishmania tropica. Molec. Biochem. Parasit. 8, 283-295. Glenney J. R. and Glenney P. (1984) The microvillus 110K cytoskeletal protein is an integral membrane protein. Cell 37, 743-751. Glenney J. R. Jr., Osborn M. and Weber K. (1982) The intracellular localization of the microvillus 110K protein, a component considered to be involved in side-on mem-
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brane attachment of F-actin. Exp. Cell. Res. 138, 199-205. HaUam C. and Wrigglesworth J. M. (1976) Hydrogen--Ion titration studies on erythrocyte membranes. Biochem. J. 156, 159-165. Kumar N. (1985) Phase separation in Triton X-I14 of antigens of transmission blocking immunity in Plasmodium gallinaceum. Molec. Biochem. Parasitol. 17, 343-358. Jacobsen B. S. (1983) Interaction of the plasma membrane with the cytoskeleton: an overview. Tissue and Cell 15, 829-852. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. Lanar D. E. and Manning J. E. (1974) Major surface proteins and antigens on the different in vivo and in vitro forms of Trypanosoma cruzi. Molec. Biochem. Parasit. 11, 119-131. Mancini P. E., Strickler J. E. and Patton C. L. (1982) Identification and partial characterization of plasma membrane polypeptides of Trypanosoma brucei. Biochim. biophys. Acta 688, 399-410. Markwell M. A. K. and Fox C. F. (1978) Surface-specific iodination of membrane proteins of viruses and eucaryotic cells using 1,3,4,6-tetrachloro-3, 6-diphenylglycoluril. Biochemistry 17, 4807-4817.
McLaughlin G. L. and Cain (3. D. (1985) Coil surface protein of symbiotic and aposymbiotic strains of Crithidia oncopelti and Blastocrithidia culicis. Comp. Biochem. Physiol. 828, 469-477. Nicolson G. L. (1976) Transmembrane control of the receptors on normal and tumor cells--I. Cytoplasmic influence over cell surface components. Biochem. biophys. Acta 457, 57-108. O'Farrel P. Z., Goodman H. M. and O'Farrel P. H. (1978) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1140. Singer S. J. (1974) The molecular organization of membranes. Ann. Rev. Biochem. 43, 805-833. Soares M. J., Brazil R. P., Tanuri A. and De Souza W. (1986) Some ultrastructural asp~ts of Crithidia guilhermei n.sp. isolated from Phaenicia cuprina (Diptera: Calliphoddae). Can. J. Zool. 64, 2837-2842. Swanstrom R. and Shauk P. R. (1978) X-ray intensifying screens greatly enhance the detection by autoradiography of the radioactive isotopes a2p and 125I. Analyt. Biochem 86, 184-192. Tiruppathi C., Alpers D. H. and Seetharam B. (1986) Phase separation of rat intestinal brush border membrane proteins using Triton X-114. Analyt. Biochem. 153, 330-335.
0305-0491/87 $3.00+ 0.00 © 1987PergamonJournals Ltd
IDENTIFICATION A N D PARTIAL CHARACTERIZATION OF PLASMA MEMBRANE POLYPEPTIDES OF CRITHIDIA GUILHERMEI, CRITHIDIA D E A N E I AND CRITHIDIA ONCOPELTI S. GIOVANNI DE SIMONE,* M. J. SOARES,t~ W. DE SOUZAt and S. GOLDENBERC~ *Departamento de Imunologia, Fundaggo Oswaldo Cruz, Av. Brazil 4365, Rio de Janeiro, RJ, Brazil and Departamento de Biologia Celular e Molecular, Universidade Federal Fluminense, Niter6i, RJ, Brazil; ~'Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, ILl, Brazil; ~:Departamentode Ultra-estrutura e BiologiaCelular, Fundagfio Oswaldo Cruz, Rio de Janeiro, RJ, Brazil; §Departamento de Bioquimica e Biologia Celular, Fundagfio Oswaldo Cruz, Rio de Janeiro, RJ, Brazil (Received 12 February 1987)
Abstract--1. Components of the cell surface of Crithidia guilhermei, Crithidia deanei and Crithidia oncopelti were radioiodinated by the iodogen technique. The distribution of proteins in the detergent-poor (DPP) and detergent-enriched phase (DRP) were studied using a phase separation technique in Triton X-114 and one- and two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulphate (1D and 2D SDS-PAGE). 2. Significant differences were noted in the proteins present in the DRP when the three species were compared. 3. Two major bands with mol. wt 28,000 and 56,000 and motility in the pH gradient of 7.4 and 6.3, respectively, were observed in C. guilhermei, but not discernible in C. deanei and C. oncopelti. 4. One polypeptide with mol. wt 50,000 and pI 4.9 was identified in the DRP of C. deanei. 5. A broad band with mol. wt 68,000-140,000 and pI 4.7-5.5 was clearly observed in the DRP of C. deanei and one or two polypeptides only present in the DPP were observed in the three Crithidia species analyzed. 6. Our observations show that C. guilhermei has characteristic surface polypeptides not found in C. deanei and C. oncopelti. 7. Our results, in association with those reported by others, show that the phase separation using Triton X-114 offers a simple approach to the separation and further analysis of a select group of proteins from the bulk of the cellular proteins.
INTRODUCTION The proteins present in the plasma membrane of eukaryotic cells play an important structural role and are involved in different processes such as absorption and transportation of nutrients, cell recognition, etc. (Singer, 1974; Nicolson, 1976; Jacobson, 1983). In the case of parasites, many of the surface proteins represent important antigens which elicit both humoral and cell-mediated immune responses when they enter in contact with the vertebrate host. In recent years, a large amount of work has been done aiming at the identification of surface proteins in members of the Trypanosomatidae family, especially in those which belong to the genera Trypanosoma and Leishmania (Mancini et al., 1982; Andrews et al., 1984; Gardiner and Dwyer, 1983; Lanar and Manning, 1984). Few data are available on surface proteins of members of the genus Crithidia (Camargo et al., 1982; McLaughlin and Cain, 1985). Most of the existing data are on the surface proteins of trypanosomatids and do not discriminate between hydrophilic and amphiphilic integral membrane proteins. In this paper we analyzed the surface proteins of Crithidia guilhermei, a new species of Crithidia recently characterized in our laboratory (Soares et al., 1986), by surface labeling with 125I followed by
solubilization with Triton X-114 thus separating the proteins into an aqueous and a detergent phase (Bordier, 1981) and characterizing them by one- and two-dimensional polyacrilamide gel electrophoresis. Crithidia guilhermei is a parasite which presents some characteristic morphological features typical of the endosymbiont-bearingtrypanosomatids, although no endosymbionts were found. In this paper, we compare it with Crithidia deanei and Crithidia oncopelti, the only other two species of Crithidia which harbour an endosymbiont (Freymuller and Camargo, 1981). The results obtained are described in this paper.
MATERIALS AND METHODS Parasites Crithidia deanei (ATCC 30255) and C. oncopelti (ATCC 30264) were obtained from Dr Isaac Roitman (University of Brasilia, Brazil). Crithidia guilhermei which was isolated from the hindgut of the fly Phaenicia cuprina and characterized previously (Soarcs et al., 1986) was obtained from Dr Reginaldo Brazil (Department of Parasitology, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil). All strains were kept in Liver Infusion Tryptose medium (Camargo, 1964) at 28°C, with serial passages at 2-day intervals.
1091 C.B.P. $S/4B---E
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GIOVANNI DE SIMON'Eet al.
Surface iodination
Two-dimension PAGE (2D-PAGE)
Cells were collected by centrifugation (1.5 min at 9650g, using a Microfuge B miniature centrifuge, Beckman Instruments, California, USA) and washed in cold 0.15M phosphate-buffered saline, pH 7.2 (PBS). Surface proteins were radiolabeled in accordance with Markwell and Fox (1978) by incubating 1-2 x 108 washed viable parasites with 300-500/zCi of iodine (NaI, Amersham, UK) and 125/~g of 1,3,4,6-tetrachloro-3,6,diphenilglycoluril (Iodo-Gen, Pierce Chemical Co., USA) for 10 min at 0°C. Labelled parasites were washed four times in cold PBS as described above and maintained in an ice bath until extraction with detergent. Phase contrast microscopic observations of the radiolabeled cells showed that they remained motile.
Two dimension PAGE consisted in isotacophoresis (first dimension) followed by electrophoresis in a uniform 13% SDS-polyacrylamide gel (second dimension, O'Farrel et al., 1978). Ampholines in different pH ranges were combined in order to obtain a pH gradient ranging from 4.4 to 8.7. Proteins labeled with ~4C, with mol. wts ranging from 18,000 to 200,000, were run in each gel.
Detergent solubilization and phase separation in Triton X-114 The iodinated cells were solubilized in 2% Triton X-114 (Sigma Chemical Company, St Louis, USA) pre-condensed twice as described by Bordier (1981) in Tris-saline (TS) buffer (10mM Tris, 150mM NaCI, pH7.4) containing 1 mM phenilmethylsulfonic fluoride (PMSF) for 30--40 min at 0-4°C and centrifuged at 20,000 g for 30 min at 0-4°C. Supernatants, collected with the tubes on ice using Pasteur pipettes, were mixed with bromophenol blue (one drop of a 0.1% stock solution) to aid the visualization of the phase separation (Alcaraz et al., 1984) and applied onto a 6% (w/v) sucrose cushion (1:1.5 v/v). After incubation at 37°C for 10 min, samples were centrifuged at room temperature for 15min at 1000g. The upper, detergent-poor phase (DPP) was recovered above the cushion and the blue pellet (detergent-enrich phase; DRP) below it. The DRP was redissolved in TS to the original volume by incubating for 10 min at 0-4°C. To the DPP was added more Triton X-114 (10% stock) and bromophenol blue to yield a final concentration of 2% and the phase separation step was proceeded twice, as described above. The pellet was discarded and then washed DPP and DRP were prepared for SDS-PAGE.
Isolation of the detergent from the proteins Zinc chloride was added to the samples to make a final concentration of 0.05 M and the proteins were precipitated by the addition of 5 volumes of cold acetone. The pellet was collected by centrifugation and the supernatant solution containing the Triton X-114 was discarded. The proteins were resuspended in water and precipitated again with 5 volumes of cold acetone. After centrifugation (9650g, 5 min), the pellet was washed with 50% cold acetone, dried at room temperature and stored at -70°C until use.
SDS-gel electrophoresis Samples of the proteins (DPP and DRP) precipitated with acetone were solubilized in the same volume of hot sample buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1.0% SDS and 0.5% fl-mercaptoethanol) and further boiled for 3 min. Proteins were separated by sodium dodeeyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on gels containing a 7.0-15% linear gradient of polyacrylamide (Laemmli, 1970). After the electrophoresis the gels were stained with Commassie Brilliant Blue 250R, dried and autoradiographed using Sakura Medical Imaging films (Sakura,Japan)and Kodak-X-O-Matic regular intensifying screens (Eastman Kodak Co., Rochester, N.Y., USA). Apparent protein M r was interpolated from the migration of ferritin (220,000), phosphorylase b (94,000), bovine serum albumine (67,000), catalase (60,000), ovalbumin (43,000), lactate dehydrogenase (36,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,100) and lactalbumine (14,400) (Calibration Kit Proteins, Pharmacia Fine Chemicals, Uppsala, Sweden).
RESULTS
Solubilization and phase-separation o f Crithidia proteins To identify and compare the major proteins exposed at the parasite surface, we iodinated coanoamastigotes of C. guilhermei as well as of the two endosymbiont-bearing species C. deanei and C. oncopelti. Total cellular proteins of surface-iodinated parasites were separated by S D S - P A G E and the labeled proteins were revealed by autoradiography and Commassie Blue staining. M i n o r apparent qualitative differences in the pattern of proteins were observed (data not shown). A b o u t 12-15 radiolabeled proteins and several bands stained by Commassie Blue were identified on the surface of the three species of Crithidia analyzed. The two phases ( D R P and DPP) obtained from Triton X-114 extracts of radioiodinated Crithidias were analyzed by S D S - P A G E after staining with Commassie Blue and autoradiography (Fig. 1). A large number of bands were observed in gels stained with Commassie Blue, mainly in D P P (Fig. 1 lanes D - F ) . N o significant differences were observed in such gels among the three Crithidias analyzed. The major radiolabeled membrane proteins were recovered in the D R P of C. deanei (Fig. 1 lane G) and C. guilhermei (Fig. 1 lane I). In the case of C. deanei there was a major broad band from 68,000 to 92,000 and one band with mol. wt 50,000. Other minor bands of lower mol. wts were also seen. In the case of C. guilhermei (Fig. 1 lane I) three or four major bands with mol. wts of 90,000, 85,000, 68,000, 56,000 and 28,000 were evident. In the case of C. oncopelti (Fig. 1 lane H), poorly labeled proteins (88,000, 68,000 and 63,000) were seen. Few radiolabeled membrane proteins were recovered in the D P P (Fig. 1 lanes J-L). Two polypeptides with mol. wts of 78,000 and 70,000 were seen in C. deanei (Fig. 1 lane J). In the case of C. oncopelti (Fig. 1 lane K). and C. guilhermei (Fig. 1 lane L), one band with a mol. wt of about 76,000 was seen. To evaluate whether the differences observed in the D R P proteins of Crithidia species were due to proteins not solubilized by 2% Triton X-114 that could be contaminating the D R P , the insoluble proteins were treated with 4 % SDS and, after centrifugation, analyzed by S D S - P A G E . The autoradiograms (lanes A, B and C) and the staining of the gels with Commassie Blue (lanes D, E and F) are shown in Fig. 2. One major radiolabeled polypeptide with mol. wt 80,000 and two other polypeptides (75,000-78,000 and 130,000-140,000) were observed in the gels of C. oncopelti (lane B) and C. guilhermei (lane C), respectively. These proteins were insoluble in 2% Triton X-114 presumably because isoelectric precipitation
1093
Crithidia plasma membrane polypeptidcs
MW
94 oom,..i 6 7 O00n,,.~
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ii
n
c .... | Commas
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.
slo
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r radiography
Fig. l. SDS-PAGE (7-15%) of proteins from radiolabeled parasites after phase separation and Commassie Blue staining (A-F) or autoradiography (G-L). A, D, G, J: Crithidia deanei; B, E, H, K: Crithidia oncopelti; C, F, I, L: Crithidia guilhermei. DRP, detergent-rich phase; DPP, detergent-poor phase. Small arrows in G and I indicate the position of species-specific proteins. Exposition of the gels of the DPP was twice longer than those for the DRP.
MW •, I 2 2 0
000
94000
•~
6ff 000 60 000
-,-
4 3 000
'..,
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Fig. 2. SDS-PAGE (7-15%) of insoluble proteins in 2% Triton X-114 but which were solubilized in 4% SDS and analyzed by autoradiography (A-C) or Commassie Blue staining (D-F). A, D: Critkidia deanei; B, E: Crithidia oncopelti; C, F: Crithidia guilhermei. Numbers to the right of the figure indicate the migration of proteins of known molecular mass (MW).
1094
GIOVANNIDE SIMONEet al.
Fig. 3. Two dimensional autoradiography of polyacrilamide gel electrophoresis of DRP proteins labelled with 12~Ifrom C. deanei (A), C. oncopelti (B) and C. guilhermei (C). Each gel was loaded with 105cpm and the position of 14C-mol. wt markers run together in the gel is shown in the left side of the figure. The number at the horizontal figure axis refers to the pH gradient.
was unaffected by the nonionic detergent under the pH and ionic strength used (Hallan and Wrigglesworth, 1976). However, the reel. wts of these proteins were different from those observed with D R P proteins.
Two dimension electrophoresis of partially purified amphiphilic proteins When surface radioiodinated Crithidias were phase-separated and aliquots of the detergent phase were submitted to 2D-PAGE, relatively simple poly-
1095
Crithidia plasma membrane polypeptides
peptide profiles were seen by autoradiography. Four major bands, with Mr ranging from 68 to 92,000 and pI 4.7-5.5 (Fig. 3) were seen in all species examined. Two of these bands were more evident in C. deanei (Fig. 3 lane A) and C. oncopelti (Fig. 3 lane B) than in C. guilhermei (Fig. 3 lane C). One band with M r 50 and pI 4.9 was observed in C. deanei (Fig. 3 lane A). Two additional major bands, with mol. wt 28,000 and 56,000 and pI of 7.4 and 6.3, respectively (Fig. 2 lane C), were present in C. guilhermei. DISCUSSION
Most of the work on the identification of surface proteins of trypanosomatids has been done using whole radiolabeled cells solubilized in SDS. Using this approach proteins, most of which are glycoproteins, characteristic of certain species or even characteristic of developmental stages of certain species, have been identified (Camargo et al., 1982; McLaughlin and Cain, 1986; review in Chance, 1979). This approach, however, does not discriminate proteins which strongly interact with the membrane lipid bilayer (integral amphiphilic proteins) from the hydrophilic proteins. It has been shown recently (Bordier, 1981) that membrane proteins would differentially distribute in the two phases formed by the non-ionic detergent Triton X-114 at moderate temperature. Alcaraz and collaborators (1984) envisioned that those proteins capable of binding detergent would segregate to the lower hydrophobic phase, whereas those that did not bind detergent would remain in the upper, detergentdepleted phase. In the present study, we have first solubilized the proteins of three Crithidia species using Triton X-114 and then separated the membrane proteins depending on their ability to be included or excluded from the detergent during phase separation. A comparison of proteins which remain in the detergent-containing phase by 2D-PAGE showed a certain degree of homology among the parasites examined. However, amphiphilic polypeptides specific for species could be demonstrated, as is the case of polypeptides of mol. wt of 28,000 and 56,000 found in C. guilhermei and the polypeptide of 50,000 found in C. deanei. These polypeptides were also seen in 2-dimensional PAGE. It is important to point out that the radiolabeled polypeptides detected by 2-D-PAGE in C. deanei and C. oncopelti are acidic, with pI of 4.7 to 5.5. Only in C. guilherrnei polypeptides with higher pI were detected. Some proteins found in the detergent-enriched phase were not completely solubilized in 2% Triton X-114 but could be visualized when 4% SDS was used. It is possible that these polypeptides represent transmembrane proteins which interact with cytoskeletal components, as previously shown in other systems (Gienney et al., 1982; Glenney and Glenney, 1984; Tiruppathi et al., 1986). It is important to point out that radiolabeled polypeptides were seen in the detergent-poor phase of the three Crithidias analysed. In the case of C. oncopelti the labeling was more evident than that observed in the DRP. These observations suggest that peripheral proteins are exposed on the surface of trypanosomatids,
Our observations, in association with others reported previously (Bouvier et al., 1985; Etges et al., 1985; Kumar, 1985), indicate that Triton X-I14 is very useful for the analysis' of plasma membrane polypeptides. It is interesting to point out that in the trypanosomatids analyzed up till now, the proteins have been detected mainly in the detergent-rich phase (Bordier et al., 1986; Etges et aL, 1985). In the case of Plasmodiurn gallinaceum, a polypeptide was seen in the detergent-poor phase (Kumar, 1985). It is also important to point out that the pattern of labeled surface polypeptides seen in one- and twodimensional P A G E of C. guilhermei was different from the other two strains of Crithidia analyzed. This observation reinforces the suggestion, made based on morphological observations and analysis of the electrophoretic mobility of six enzymes, that C. guilhermei is a new species of Crithidia (Soares et al., 1986). REFERENCES Alcaraz G., Kinet J. P., Kumar N. and Metzzwer H. (1984)
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McLaughlin G. L. and Cain (3. D. (1985) Coil surface protein of symbiotic and aposymbiotic strains of Crithidia oncopelti and Blastocrithidia culicis. Comp. Biochem. Physiol. 828, 469-477. Nicolson G. L. (1976) Transmembrane control of the receptors on normal and tumor cells--I. Cytoplasmic influence over cell surface components. Biochem. biophys. Acta 457, 57-108. O'Farrel P. Z., Goodman H. M. and O'Farrel P. H. (1978) High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell 12, 1133-1140. Singer S. J. (1974) The molecular organization of membranes. Ann. Rev. Biochem. 43, 805-833. Soares M. J., Brazil R. P., Tanuri A. and De Souza W. (1986) Some ultrastructural asp~ts of Crithidia guilhermei n.sp. isolated from Phaenicia cuprina (Diptera: Calliphoddae). Can. J. Zool. 64, 2837-2842. Swanstrom R. and Shauk P. R. (1978) X-ray intensifying screens greatly enhance the detection by autoradiography of the radioactive isotopes a2p and 125I. Analyt. Biochem 86, 184-192. Tiruppathi C., Alpers D. H. and Seetharam B. (1986) Phase separation of rat intestinal brush border membrane proteins using Triton X-114. Analyt. Biochem. 153, 330-335.