Identification and characterization of a pheromone 2 specific binding protein of Euplotes octocarinatus

Europ. J. Protistol. 37, 391–403 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp

Identification and characterization of a pheromone 2 specific binding protein of Euplotes octocarinatus Matthias Möllenbeck1,* and Klaus Heckmann2 1

University of Connecticut Health Center, Department of Biochemistry, 263 Farmington Avenue, Farmington, CT, 06030, USA; E-mail: [email protected] 2 Institut für Allgemeine Zoologie und Genetik, Universität Münster, Schlossplatz 5, D - 48149 Münster, Germany Received: 3 October 2000; 27 June 2001; 30 July 2001. Accepted: 30 July 2001

Ciliates show two forms of reproduction: asexual (vegetative) and sexual reproduction (conjugation). Conjugation in Euplotes octocarinatus is induced by pheromones secreted by cells of one mating type binding to corresponding receptors on cells of a different mating type. In this study, we used heterologously-expressed pheromone 2 (Phr2) in affinity chromatography to isolate pheromone 2 binding proteins in Euplotes octocarinatus syngen 1. Using this approach, we isolated a 42-kDa protein (Phr2B) that bound Phr2. A tryptic fragment of Phr2B was used to construct degenerate oligonucleotides for PCR amplification of the macronuclear gene. The gene is predicted to encode a 58 amino acid protein. The N-terminus of the deduced protein shows significant similarity to membrane bound protein kinases of such diverse organisms as Xenopus laevis, Mus musculus and Homo sapiens, while the C-terminus contains a region which is similar to an extracellular region within chemosensory receptors of Caenorhabditis elegans. Western blotting analysis demonstrated that Phr2B specifically recognizes Phr2 and preliminary results indicate that antibodies raised against a C-terminal peptide of Phr2B inhibit cell mating. Further studies should be undertaken to investigate the precise role that Phr2B plays in preconjugative intercellular communication in E. octocarinatus. Key words: Euplotes octocarinatus; Gamone-receptor interaction; Heterologous expression; Intercellular communication; Preconjugant interaction.

Introduction Cells continually exchange information with their environment and intercellular communication is a basic feature of living organisms. In ciliates, intercellular communication is required for sexual propagation. A variety of sexual signals have been found to function in preconjugant interactions. These interactions are mediated by a class of molecules called gamones. In Blepharisma (Miyake 1968), E. octocarinatus (Heckmann and Kuhlmann 1982, 1986), E. patella (Kimball 1942), E. raikovi (Luporini et al. 1982, 1983), Dileptus *corresponding author

anser (Vinnikova and Tavrovskaja 1973), and several other species (Miyake 1996) gamones are excreted, and are therefore referred to as pheromones. Gamones of Paramecium (Kitamura 1988), E. crassus (Heckmann 1964), E. minuta (Nobili 1966) and E. vannus (Heckmann 1963) are not excreted, and in these species the gamones are most likely bound on the cell surface (Miyake 1996). Gamones are thought to function by binding to receptors. In Blepharisma and E. octocarinatus study of preconjugant interaction began with the “gamone-receptor hypothesis” (Miyake 1981a, b, 1996), which is based on the assumption that a gamone of one mating type binds to a specific recep0932-4739/02/37/04-391 $ 15.00/0

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tor on cells of the complementary mating type. This gamone-receptor binding induces formation of conjugant pairs (Miyake 1981b; Heckmann 1992, 1995; Miyake 1996; Görtz et al. 1999). On the other hand, preconjugant interaction in E. raikovi has been explained by the “self-recognition hypothesis” (Luporini and Miceli 1986), which posits that a gamone of one mating type binds to a receptor on the cells of the same mating type. This gamone-receptor binding inhibits the formation of conjugant pairs. Gamones of other mating types also bind to the same receptor, which reduces the inhibition, resulting in the formation of conjugant pairs (Luporini et al. 1996). Recently it was shown for this species that the pheromone membranebound forms represent the cell binding sites and potential signaling receptors of soluble pheromones (Ortenzi et al. 2000). The results of recent studies of E. octocarinatus showed that this morphological species is composed of reproductively isolated mating groups or syngens (Brünen-Nieweler et al. 1998; Möllenbeck 1999; Möllenbeck and Heckmann 1999). Nine pheromone encoding genes, representing the five known mating type alleles, have been characterized from different syngens of E. octocarinatus (Brünen-Nieweler et al. 1991, Meyer et al. 1991, 1992; Teckentrup et al. 1996; Brünen-Nieweler et al. 1998; Möllenbeck and Heckmann 1999). In syngen 1 of E. octocarinatus 10 mating types are distinguished. The mating types belonging to this syngen are determined by four codominant alleles (mt1–mt4); six mating types by the heterozygous combination of the alleles, and four mating types by their homozygous combination (Heckmann and Kuhlmann 1982, 1986). Each of these alleles governs the expression of a specific pheromone (Phr1–4). These pheromones attract potential mates and induce cells of other mating types to prepare for conjugation (Plümper et al. 1995; Kuhlmann et al. 1997). All four pheromones have been isolated (Weischer et al. 1985; Schulze Dieckhoff et al. 1987). While the pheromones of E. octocarinatus are well studied on the molecular level, little is known about the corresponding receptors. The purpose of this study was to identify pheromone 2 binding proteins in different cell lines of E. octocarinatus syngen 1. Further experiments should answer the question of whether these proteins also bind other pheromones or whether they exhibit specificity to pheromone 2 and therefore represent potential Phr2-receptors.

Material and methods Cells and culture conditions: The following cell lines of E. octocarinatus syngen 1 were employed: 69(1)VII (mt1mt1), 1(14)-VIII (mt2mt2), and 3(58)-IX (mt3mt3). The Euplotes cells were grown in SME (synthetic medium for Euplotes) at room temperature either in 1.5 l Fernbach flasks (after Freiburg 1993) or as mass cultures in 15 l carboys (Kusch and Heckmann 1988), using the photosynthetic flagellate Chlorogonium elongatum, which was grown as described elsewhere (Schulze Dieckhoff et al. 1987), as food source. Escherichia coli were grown in Luria-Bertani (LB) medium. E. coli strain DH5α (Promega, Heidelberg, Germany) was used for heterologous expression of pheromone 2 as well as for plasmid propagation. Heterologous expression and labeling of pheromone 2: For the heterologous expression of pheromone 2, the pMAL-p2 vector from New England Biolabs (Schwalbach, Germany) was used. The sequence of the native pheromone 2 was expressed as a fusion protein with the maltose binding protein (MBP). Cells of the E. coli strain DH5α were transformed with this vector construct, grown in LB medium (+150 µg/ml ampicillin, 0.2% glucose) to an absorption A600 of 0.5 and finally induced with IPTG (isopropyl-β-D-thiogalactopyranoside) for 2 h. The periplasmic proteins were isolated by cold osmotic shock (Neu and Heppel 1965). The MBP domain of this fusion protein binds tightly to amylose, therefore allowing affinity purification. After elution with 10 mM maltose, labeling of the resulting fusion protein MBP::Phr2 was performed with the “DIG (digoxigenin) Protein Labeling and Detection” Kit (Boehringer Mannheim, Mannheim, Germany). According to the manufacturer’s manual, the labeling reaction took place for 2 h at room temperature with a 70 x molar surplus of DIG-NHS (digoxigenin-3-0-methyl-carbonylε-amino-caproicacid-N-hydroxy-succinimide-ester). Therefore an additional purification of the labeled fusion protein MBP::Phr2 with gel filtration chromatography was necessary. A superdex 75 HR 10/30 column (Pharmacia) and a FPLC apparatus, provided by the same manufacturer, were used. The detection of the labeling reagent was done by enzyme-catalyzed chemilumescence using CSPD (disodium 3-(4-methoxyspiro{1,2-dioxyetane-3,2tricyclo-[3.3.1.13,7]decan}-4-yl)phenylphosphate) as a substrate (Engler-Blum et al. 1993). Each of the described steps of expression, labeling and purification of the fusion protein was documented with SDS-PAGEs. Biological activity: In order to determine the biological activity of the fusion protein MBP::Phr2, Euplotes cells of different mating types were concentrated by gentle centrifugation (300 × g; 30 s) two days after the last feeding, and then suspended in new medium at a density of 4 × 103 cells per ml. Different concentrations of protein (MBP::Phr2 or native Phr1, respectively) in SME were added to 125 µl of these test cells. Mating tests were carried out with digoxigenin labeled as well as

Pheromone 2 binding protein of Euplotes octocarinatus

unlabeled fusion protein. These mixtures were incubated at 26 ºC for 6–24 h. The selfing rates were determined in negative controls (without adding protein). The same experiments were performed with native, digoxigeninlabeled pheromone 1, which was used to study the specificity of the isolated pheromone binding protein. After production of an antibody in rabbits (Biotrend, Köln, Germany) against the potential extracellular region of the newly isolated pheromone binding protein, the mating tests were repeated, in the presence of different amounts of antibody-serum. As a control, different amounts of preimmune-serum were used to ensure that the observed effects were due to the antibody and not to the serum itself. In each case the mating competence of the cells was examined. Therefore, cells of different mating types (about 500 of each mating type) were mixed and the mating rate was determined. Affinity chromatography: 1 mg of anti-digoxigenin magnetic particles (Boehringer Mannheim, Mannheim, Germany) were prepared according to the manufacturer’s manual. The particles were incubated with about 3–5 µg digoxigenin labeled fusion protein for 1 h at 4 ºC. Cilia and cirri (isolated after Freiburg 1993) of about 1 million Euplotes cells were lysed in 100 µl SDS sample buffer including proteases inhibitors (50 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycin, 1 mM Pepstatin, 3 mM E64). The lysed cilia and cirri were incubated with magnetic particles loaded with digoxigenin labeled fusion protein for 1h at 4 ºC. To remove all components that had not bound the sample was washed three times with 20 mM KPO4 buffer (pH 7.5) and 0.15 M NaCl. Elution was performed with 0.1 M Glycin-HCl, pH 2.5. The proteins of the eluates as well as of the supernatants, resulting from the washes, were precipitated with trichloracetate acid (0.5M) and analyzed either by SDS-PAGEs or – after “semi-dry blotting” (Kyhse-Anderson 1984) – by Western blots. Polymerase chain reaction: DNA was amplified enzymatically based on the method of Saiki et al. (1988) with thermostable Taq DNA polymerase in a DNA thermal cycler (Perkin Elmer, Überlingen, Germany). Amplifications were performed using 50 ng of genomic DNA prepared from Euplotes cells as described previously (Godiska et al. 1987; Meyer et al. 1992). PCRs were carried out in a total volume of 100 µl containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5–3 mM MgCl2, 100 µM of each dNTP, 0.25 µM of two gene specific primers or one gene specific primer in combination with a telomere specific oligonucleotide and 2.5 U of Taq DNA polymerase. For amplification the reaction mixtures were denatured at 94 °C for 4 min, followed by 30 cycles of 1 min at 94 °C, 1 min at 55°C, 2 min at 72 °C, and a final incubation for 5 min at 72 °C. PCR products were analyzed by agarose gel electrophoresis. The following primers were used for amplification of the corresponding macronuclear chromosomes: R1: 5′-TTCATGATAATATTCGCTCC-3′ T C C T A T T

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R6: 5′-GCTTATTAGTATGCCCTCA-3′ R7: 5′-TGATGGGCATACTAATAAGC-3′ Te: 5′-CCCCAAAACCCCAAAACCCCAAAAC-3′ Synthesis of cDNA by reverse transcriptase (RT)PCR: For RT-PCR the 3′-RACE system for Rapid Amplification of cDNA Ends (BRL, Eggenstein, Germany) was used. One µg of poly(A)+-RNA was reverse transcribed using an oligo dT-primer and reverse transcriptase as suggested by the manufacturer. Two µl of this first-strand cDNA were subjected to PCR using gene specific primers. Water instead of first-strand DNA was used for the control reaction. For the isolation of the mRNA, either the “mRNA Isolation systems” (BRL) or the “Dynabead mRNA Direct”-Kit (Dynal, Hamburg, Germany) was used according to the manuals. For amplification of cDNA products the following primers were used (Fig. 3): R8: 5′-ATGGGCATCCTAGTTTATTC-3′ R15: 5′-TTATTTATATTGGTGTTGCCG-3′ oligo dT: 5′-CCAAGCTTGGATCCGAATTC[T]17-3′ UAP (= universal amplification primer): 5′-CCAAGC TTGGATCCGAATTC-3′ Subcloning and sequencing: All PCR products were cloned into the double-stranded plasmid-vector pGEM-T 5Zf(+) (Promega, Heidelberg, Germany). Preparation of the plasmid DNA was carried out with a Qiagen (Düsseldorf, Germany) plasmid kit. Positive clones were sequenced by the dideoxy chain-termination method (Sanger et al. 1977). Southern hybridization: About 8 µg of total DNA of Euplotes cells 69(1)-VII (mt1mt1), 1(14)-VIII (mt2mt2), and 3(58)-IX (mt3mt3) was separated on 0.8% (w/v) native agarose gels and blotted onto nylon membranes after Southern (1975). The pheromone specific probe was labeled with digoxigenin (DIG)-dUTP (DNA Labeling and Detection Kit Nonradioactive, Boehringer Mannheim) and detection was again performed by enzyme-catalyzed chemiluminescence using CSPD as a substrate Engler-Blum et al. 1993. Materials: Restriction enzymes were from Boehringer Mannheim, BRL and Promega. The sequenase version 2.0 DNA sequencing kit was purchased from USB (Bad Homburg, Germany), E. coli strain DH5α was also obtained from Promega. All chemicals used were of analytical grade and were supplied by Boehringer Mannheim, Pharmacia, LKB (Freiburg, Germany) and Sigma (Deisenhofen, Germany).

Results and discussion Isolation, labeling and determination of biological activity of recombinant Phr2 Heterologous expression The sequence encoding pheromone 2 of E. octocarinatus exhibits three in-frame UGA stop

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codons, encoding cysteine (Meyer et al. 1991). In order to express the pheromone 2 encoding gene in E. coli, we used overlap extension PCR (Ho et al. 1989) to change the UGA codons to the universal cysteine codon UGC. After cloning into the expression vector pMAL-p2, Phr 2 was expressed in E. coli as a fusion protein with the maltose binding protein MBP (MBP::Phr2). The pMAL-p2 vector was chosen, because it causes the export of the synthesized fusion protein into the periplasm of E. coli. Here the formation of disulfide bonds is favored because of the oxidative conditions (Plückthun et al. 1988). For E. raikovi it was shown that the cysteines in the pheromones form disulfide bonds (Raffioni et al. 1991) and there is evidence that this is also the case in E. octocarinatus. All investigated pheromones of E. octocarinatus exhibit a high content (about 10%), an even number and

well conserved positions of cysteine residues, though the overall similarity of the proteins is low (Heckmann 1992). Brünen-Nieweler et al. (1994) have shown that PDI (protein disulfide isomerase) treatment of heterologously expressed pheromone 3 results in a higher biological activity. The formation of disulfide bonds could further explain the observed stability of the pheromones at high temperatures (Heckmann 1992). It is known that varying amounts of the same recombinant protein are produced in different E. coli strains (Riggs 1990). Using E. coli strain DH5α we could obtain about 17.5 mg of fusion protein per liter E. coli cells. Fig. 1 shows the E. coli proteins before and after induction with IPTG (lanes 1 and 2, respectively) as well as the elute of the periplasm proteins after affinity chromatography with amylose (lane 3). The fusion protein migrates with an apparent

Fig. 1. 12% SDS-PAGE under reducing conditions and stained with Coomassie brilliant blue. Lanes: m, protein markers; 1, E. coli periplasm proteins before induction with IPTG; 2, E. coli periplasm proteins after induction with IPTG; 3, fusion protein gel purified on Superdex 75 FPLC; 4, digoxigenin labeled fusion protein gel purified on Superdex 75 FPLC

Pheromone 2 binding protein of Euplotes octocarinatus

molecular mass of about 60 kDa. This is slightly higher than the calculated molecular weight of the fusion protein MBP::Phr2, which is 54 kDa (Mw MBP: 42.7 kDa; Mw Phr2: 11.3 kDa). The reason for this difference in migration is probably due to the Phr2 part of the fusion protein. It is known that the native Phr2 migrates under reducing condition in SDS-PAGE like an 18.5 kDa protein (Heckmann 1992). Next to the major band representing the fusion protein, we believe that the several faint bands migrating faster than the MBP::Phr2 fusion protein represent degradation products. Additionally, the elute exhibits another protein of about 18.5 kDa. This protein is most likely an E. coli protein. Partial sequencing of this protein revealed similarities to cytoplasmic aminoaspartate transferases of E. coli (Heek, pers. comm.). Labeling and purification In order to label the MBP::Phr2 the digoxigenin-anti-digoxigenin indicator system (Kessler et al. 1990), which can be used for labeling DNA and RNA (Kessler et al. 1990) as well as proteins, peptides and glycoconjugates (Haselbeck and Hösel 1990), was chosen because of its high sensitivity and the fact that digoxigenin has only been found in plants of the genus Digitalis. Therefore no cross-reactivity with endogenous digoxigenin in Euplotes could be observed (Kühnel, pers. comm.). After labeling the MBP::Phr2, FPLC gel chromatography was performed to remove any digoxigenin which has not bound to the ligand, and to eliminate the degradation products. Fig. 1 shows the fusion protein before labeling and after purification of the labeled fusion protein (lanes 3 and 4, respectively). Due to the labeling reaction the fusion protein is shifted. A shift is also obvious in the E. coli protein, which could not be removed by FPLC. A possible reason for this could be that it interacts with the fusion protein. In contrast to the E. coli protein, the degradation products could be removed by FPLC. Biological activity In order to investigate the influence of the maltose binding protein as well as the labeling reagent in terms of biological activity and specificity, the labeled fusion protein was examined in biotests.

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The labeled fusion protein could induce mating at concentrations as low as 10–9 M, while the maximum mating rate was observed with a concentration of 10–7 M. No conjugation was induced in cells of mating type VIII (mt2 mt2). This is expected if the fusion protein displays the specificity demanded of a Phr2, because cells of mating type VIII express Phr2 itself and therefore conjugation can not be induced by this pheromone. The same mating tests were performed with digoxigenin-labeled native pheromone 1. This pheromone induced mating at concentrations as low as 10–11 M and the maximum pair formation was found using a concentration of 10–9 M. As expected, pairs of cells were not observed with mating type VII (mt1 mt1). Previously it was shown that maltose binding protein (MBP) itself does not induce pair formation (Heek, pers. comm.). The observed biological activity is therefore only due to the pheromone. Because native pheromones show biological activity up to 10–13 M (Kusch and Heckmann 1988), it must be stated that the labeling reagent as well as the maltose binding protein probably reduce the biological activity (by about 10–4), but the biological specificity remains. Possible reasons for this reduced activity could be an incorrect or less efficient formation of the right secondary structure of the fusion protein. Although protein folding and correct disulfide bond formation is favored under the oxidative conditions in the E. coli cytoplasm, secretion of eukaryotic proteins with correct disulfide bonds is less efficient than the export of native bacterial exoproteins (Stader and Silhavy 1990). Differences in the structure and oxidizing properties of prokaryotic and eukaryotic catalysts for disulfide-linked protein folding may be the reason for this (Bardwell et al. 1993). It can also not be excluded that the MBP (Mw 42.7 kDa) disturbs the interaction of the pheromone with the corresponding receptor sterically.

The pheromone 2 receptor protein Affinity chromatography Affinity chromatography has been a useful tool to isolate cell surface receptors that do not change their binding properties to the corresponding ligand when solubilized. In this study affinity chromatography with MBP::Phr2 was performed using lysed cilia and cirri of cells representing different mating types. In each case mating competent cells

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were used, because preliminary experiments indicated an up-regulation of pheromone binding molecules when cells become mating competent. In these experiments the labeled fusion protein showed interaction with cilia and cirri proteins and to a lesser degree with deciliated cell bodies. It can not be excluded that the last interaction was due to remaining cilia and cirri on the cell body. Strong signals could be obtained with cilia and cirri of mating competent cells representing mating type VII as well as mating type VIII, which expresses

Phr2 itself. MBP alone showed no interaction with any Euplotes proteins. Fig. 2a shows that the affinity chromatography with MBP::Phr2 exhibited several bands. A closer investigation of these proteins showed that some of them represent parts of the antibody used against digoxigenin. The elute of cilia and cirri of cells representing mating type VII (lane 2) exhibited two bands: a protein about 60 kDa, representing the fusion protein as well as a protein of about 42 kDa. This latter band also appears in the elute of the cilia and cirri of cells of the

Fig. 2. Affinity chromatography. a. 12% SDS-PAGE under reducing conditions and stained with Coomassie brilliant blue. Lanes: m, protein markers; 1, first elute, cilia and cirri of cells of mt VIII; 2, first elute, cilia and cirri of cells of mt VII. b. Western blots. Blot A: after incubation with labeled fusion protein MBP::Phr2. Lanes: 1, first elute, cilia and cirri of cells of mt VII; 2, second elute, cilia and cirri of cells of mt VII, 3, second elute, cilia and cirri of cells of mt VIII; 4, first elute, cilia and cirri of cells of mt VIII. Blot B: after incubation with labeled native Phr1. Lanes: 1, first elute, cilia and cirri of cells of mt VII; 2, second elute, cilia and cirri of cells of mt VII, 3, second elute, cilia and cirri of cells of mt VIII; 4, first elute, cilia and cirri of cells of mt VIII.

Pheromone 2 binding protein of Euplotes octocarinatus

mating type VIII (lane 1). In this case the 60 kDa fusion protein is not visible; it remained in the supernatant (not shown). In addition a protein of about 28 kDa can be seen in both lanes. Comparison with the migration of the anti-digoxigenin antibody demonstrated that the 28 kDa protein represents the light chain of the antibody. Therefore, parallel to each affinity chromatography experiment, the employed antibody was analyzed by SDS-PAGE in order to identify parts of the antibody that did not remain in the supernatant and therefore could give false positive signals. The reason why antibody parts sometimes do not remain in the supernatant is unclear. In all experiments the 42 kDa protein could – though to a different amount – be detected as long as cilia and cirri of mating competent cells were used. This result focussed our attention on the 42 kDa protein as a possible pheromone receptor. These findings raise the general question whether it is expected to find a pheromone 2 receptor in cells of mating type VIII. These cells express Phr2 and therefore do not respond to this pheromone. It has to be stated that although these cells do not react to this pheromone, it does not generally exclude the existence of the corresponding receptor. Kuhlmann and Heckmann (1989) have shown that Euplotes cells indeed express all pheromone receptors until they reach a certain stage of adolescence. After production of a pheromone, cells lose their sensitivity for this pheromone, which may be due to internalization of the corresponding receptor after binding this newly synthesized pheromone or by desensitization of these receptors. In a mutant which shows a deletion in the pheromone 2 gene, and therefore does not produce biologically active pheromone 2, conjugation can actually be stimulated by pheromone 2. Cells of a certain mating type, which are incubated with a pheromone that normally induces conjugation in these cells, but where conjugation is inhibited by shaking, lose the sensitivity to this pheromone after some hours (Kuhlmann and Heckmann, pers. comm.). The sensitivity to the other pheromones remains. Specificity To confirm the interaction of the isolated protein with Phr2, and to answer the question of whether this protein represents a general pheromone binding protein or whether it shows

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specificity to Phr2, the affinity chromatography was performed again; this time the proteins were blotted to a PVDF membrane after electrophoresis and incubated with MBP::Phr2 (Fig. 2b blot A) and native Phr1 (Fig. 2b blot B), respectively. In blot A, the fusion protein (60 kDa), the heavy as well as the light chains of the antibody and the 18 kDa E. coli protein could be detected. Additionally, in each lane a signal at 42 kDa was obvious, which resembles the described protein. On the other hand, incubated with Phr1, the fusion protein and the heavy chain of the antibody gave a signal; in this case the light chain and the E. coli protein remained in the supernatant. No signal, and therefore no interaction, was detectable in regard to the 42 kDa protein. To exclude that a loss of biological activity is the reason for the missing interaction, the employed Phr1 was examined in mating tests, which revealed that this pheromone could induce mating in competent cells at concentrations as low as 10–11M. Therefore these blots clearly demonstrate firstly the interaction of Phr2 with the 42 kDa protein and secondly that this interaction is most likely specific to Phr2. Because of these results, the 42 kDa protein represented a potential receptor and was therefore sequenced. Sequencing of the 42 kDa protein More than 50% of all proteins are modified at the N-terminus (Lottspeich et al. 1994) and can therefore not be sequenced directly by Edman degradation (Edman 1949). Therefore the isolated protein was digested with trypsin before sequencing. The resulting 10 aa peptide sequence is indicated in Fig. 3. Based on this sequence a primer was designed under consideration of the codon usage of E. octocarinatus. This primer was used in combination with a telomere specific primer for PCRs in order to amplify the corresponding macronuclear DNA molecules of cells representing mating types VII and VIII, respectively. Two faint products appeared in each case, which were isolated and reamplified. The appearance of only faint products is not surprising, because first PCRs were carried out with a degenerate primer in combination with the telomeric specific primer, which can bind to all chromosomes ends. Sequencing of these products revealed that one of these products was β-tubulin, while the other one exhibited an unknown sequence (Fig. 3). The amplification of tubulin can be

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explained, because databank searches revealed a motif in tubulin that was very similar to the obtained peptide sequence. To exclude the possibility that the sequenced protein was tubulin, the other revealed peptide sequences, which were too short to design primers, were investigated closely. None

of these short sequences matched tubulin. Due to the small amount of the sequenced protein sequencing did not reveal very strong signals in most of the cases, but a few amino acids could be identified clearly and all of them could be found in the unknown sequence (Fig. 3).

Fig. 3. Macronuclear chromosome sequences encoding the Phr2 binding protein of the Euplotes cell lines 69(1)-VII and 1(14)-VIII (accession numbers AF234678 and AF234679).Telomeres and non-coding sequences are shown in small letters, the coding region is shown in capital letters with the deduced amino acid sequence underneath. Primers (R8 and R15) used to amplify the transcript as well as the polyA addition site are indicated. UGA encoded cysteines are framed. The amino acid sequences obtained by peptide analysis are single underlined. A degenerate primer based on the sequence of the peptide underlined in bold was used to generate the initial PCR products. The region exhibiting similarity to membrane bound protein kinases is double underlined, the consensus sequence of phosphorylation sites for protein kinases C and/or A is underlined dotted and the putative extracellular domain is shown in italics. The only deviating base (position 418) in the sequence of cell line 1(14)-VIII is shown above the sequence.

Pheromone 2 binding protein of Euplotes octocarinatus

Investigating the unknown sequence we found in mating types VII and VIII the same sequence with only one base deviation, which is located in the non-coding sequence (Fig. 3). The length of the macronuclear DNA molecules was 598 bp, including telomeres in each case. This was surprisingly short, because with an apparent molecular weight of the isolated protein of about 42 kDa, a coding sequence of about 1.200 bp (about 400 aa) was expected. To confirm that whole macronuclear DNA molecules were amplified, and to exclude recombination in the PCR reactions, a Southern blot was performed (not shown), in which a signal of about 600 bp was detectable for all cell lines. Possible reasons for this discrepancy could be posttranslational modifications, especially glycosylations. The sequence exhibited no evidence for N-glycosylation sites with the known consensus sequence Asn X Ser/Asn X Thr. There is no consensus for O-glycosylation of serines or threonines. Further analysis of the sequence showed no evidence for other posttranslational modifications. Perhaps more likely is that the protein represents a complex of subunits, and covalently bound complexes have been found in SDS-PAGE (Meyer, pers. comm.). It is also known that different receptors form homodimers (receptor tyrosine kinases) or heterodimers (receptor serine / threonine kinases) after binding ligand. The fact that all the peptide sequences were present in the macronuclear and cDNA clones of Phr2B strongly suggests that if this protein resembles a complex, it must consist of identical subunits. A third possibility could be that this protein is a composite translated from the information in different transcription units. This phenomenon, which is called trans-splicing, is found in Trypanosoma and euglenoid protozoa (Agabian 1990). Since the macronuclear genes of Euplotes are located on gene-sized pieces, and therefore represent autonomous replication units, it is possible that only a part of the protein encoding sequence is amplified in the PCRs; another part would be located on another macronuclear DNA molecule, and therefore would not be amplified. In order to identify the coding region of these macronuclear chromosomes, RT-PCRs were performed with mRNA of cells of mating type VII and VIII. In each case PCR products could be obtained, when the mRNA was isolated from mating competent cells. This suggests that the regulation of this protein might take place at the transcription level.

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The whole transcript could be obtained by PCR (R8-UAP), but sequencing it in full length was not possible. Computer analysis showed that the full transcript can form a very stable secondary structure (not shown). This may have accounted for the premature termination of the sequencing reaction. Therefore another product (R15-UAP) was amplified and sequenced. Overlapping sequence data provided all parts of the complete sequence of the Phr2B cDNA clone (see Fig. 3). Sequencing these products revealed two in-frame UGA codons, encoding cysteine in Euplotes (Meyer et al. 1991), an UAG stop codon at the end of the transcript and no introns. Sequence analysis Fig. 4 shows the result of the databank search (BlastP, Altschul et al. 1990). The N-terminus of the isolated protein shows similarity to domains of membrane-bound protein kinases of such different organisms as C. elegans, M. musculus and H. sapiens. Furthermore a conserved consensus sequence of phosphorylation sites for protein kinases C and/or A [(ST) X (RK)] (Kishimoto et al. 1985) is present (Fig. 4 a). In addition, basic amino acids are located in front of this site, which are known to have a strong effect on this kind of phosphorylation site (Woodgett et al. 1986). Whether this potential phosphorylation site actually plays a role in the signal transduction after pheromone binding is not clear. Though there is some similarity to protein kinases, this protein probably does not function as a protein kinase, because the consensus sites for ATP binding (DFG and/or GGXG) are missing. The C-terminus exhibits similarity to the extracellular domains of the C. elegans srg family (Troemel et al. 1995). 40 receptors are included in this family, some of which are described as potential pheromone receptors due to their expression patterns in chemosensory neurons. All these receptors show low similarity among each other, only a few motifs, sometimes only single aminoacids, are conserved. It is likely that these residues are important for protein function, though their precise role is not known yet. The isolated Euplotes protein contains a region that is similar to one of these motifs (Fig. 4 b). Five out of six of highly conserved amino acids in the C. elegans chemosensory receptors are present in Phr2b, while the sixth residue, glutamine, is replaced by tyrosine. The two less highly conserved amino acids are not present in the Euplotes protein.

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Fig. 4. Similarity of the Euplotes PhrB protein to other proteins. a. Similarity of the N-terminus to membrane bound protein kinases. Conserved amino acids are shown in boldface, the consensus sequence of the phosphorylation site for protein kinase C and/or A is underlined, additionally. The numbers above the sequences indicate the position in the different proteins. b. Similarity of the C-terminus to extracellular domains of the srg family of chemosensory receptors of C. elegans. The GenBank accession numbers are as follows: X. laevis, Z17205; H. sapiens, D79997; M. musculus, X 95351 and L 76158; C. elegans srg3, 1584489, srg4, 1584490, srg6, 1584492 and srg7, 1584493.

A putative membrane spanning region can be found by hydrophobicity analysis (after Kyte and Doolittle 1982) between aa 41 and 52. This region is located between the consensus sequence of phosphorylation sites for protein kinases C and/or A and the sequence showing similarity to extracellular domains of chemosensory receptors of C. elegans. All this suggested that this protein exhibits a Ncyt Cex orientation, which is e.g. shown for the transferrin receptor (Baker et al. 1987). Therefore a peptide with the sequence resembling the putative

extracellular domain (PYICFS) of the newly isolated protein (Fig. 3) was synthesized and an antibody against it was produced.

Experiments with antibody against synthetic peptide Initial experiments demonstrated that the preimmune-serum as well as the antibody-serum had no significant effect on the mating rate in very low concentrations (diluted 1:125,000 up to 1:50,000). As expected, with increasing amounts of

Pheromone 2 binding protein of Euplotes octocarinatus

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Fig. 5. Influence of preimmune-serum and antibody-serum on mating rates. The columns show the mean values of 20 individual tests and the observed ranges of mating rates are indicated by errors bars. Results from controls performed in order to test the physiological conditions of the employed cells are shown at the left next to the yscale.

preimmune- as well as antibody-serum a general decrease of mating could be observed (Fig. 5). This inhibition is most likely due to the proteins in the serum. At high concentrations (dilutions <1:1,000) both sera caused the death of the cells. Nevertheless, in the range of dilutions from 1:4,100–1:3,000 a significant difference between the sera was obvious. Cells of mating type VII incubated with MBP::Phr2 and antibody-serum exhibited an almost complete inhibition of mating (0–1%), while samples incubated with fusion protein and preimmune-serum still showed mating rates of 25–10%. Selfing was never found in the controls. The mentioned percentages are averages of a total of 20 samples in each case. With each experiment controls were performed to determine the physiological mating conditions of the cells used. Cells of mating types VII and VIII showed an average mating rate of about 85%, mating type VII cells stimulated with Phr2 a rate of about 65%. In samples with two mating types (VII and VIII, respectively) the preimmune-serum decreased the mating rate to about 7%, the anti-serum to about 4% (dilutions <1:4,100). A complete inhibition could never be observed in these cases. Such complete inhibition

would be expected assuming a universal pheromone receptor. In the case of different pheromone specific receptors mating is still expected, because even if the antibody would block all Phr2 receptors (and the experiments described above indicate this), Phr1 of mating type VII could still induce mating. It must be stated that the results of these experiments can only be evaluated as first tendencies. Purified or monoclonal antibodies, which were not available in this study, would certainly be of advantage, because no serum proteins could disturb the mating.

Conclusions In this study a Phr2 specific binding protein, Phr2B, could be isolated by affinity chromatography. Experimental results indicate that Phr2B is involved in preconjugant interaction of E. octocarinatus. These data suggest Phr2B may be a Phr2specific receptor, but further studies are necessary to confirm this. These studies will provide further insight into intercellular communication in E. octocarinatus.

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Acknowledgements: The pheromone 2 encoding gene was mutated and cloned by Dr. Puppe and kindly provided for this study. Helpful discussions with Drs Meyer, Klobutcher and Jacobs are gratefully acknowledged. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 310, Teilprojekt C1) and the Fonds der Chemischen Industrie.

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