Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans

Biochimica et Biophysica Acta 1672 (2004) 214 – 223 www.bba-direct.com

Overexpression, purification and characterization of the acidic ribosomal P-proteins from Candida albicans Dariusz Abramczyk, Marek Tcho´rzewski, Dawid Krokowski, Aleksandra Boguszewska, Nikodem Grankowski * Department of Molecular Biology, Maria Curie-Sklodowska University, Institute of Microbiology and Biotechnology, Akademicka Street 19, 20-033 Lublin, Poland Received 21 October 2003; received in revised form 13 April 2004; accepted 14 April 2004 Available online 10 May 2004

Abstract In all eukaryotic cells, acidic ribosomal P-proteins form a lateral protuberance on the 60S ribosomal subunit—the so-called stalk— structure that plays an important role during protein synthesis. In this work, we report for the first time a full-length cloning of four genes encoding the P-proteins from Candida albicans, their expression in Escherichia coli, purification and characterization of the recombinant proteins. Considerable amino acid sequence similarity was found between the cloned proteins and other known fungal ribosomal Pproteins. On the basis of their phylogenetic relationship and amino acid similarity to their yeast counterparts, the C. albicans P-proteins were named P1A, P1B, P2A and P2B. Using three different approaches, namely: chemical cross-linking method, gel filtration and twohybrid system, we analyzed mutual interactions among the C. albicans P-proteins. The obtained data showed all the four P-proteins able to form homo-oligomeric complexes. However, the ones found between P1B – P2A and P1A – P2B were dominant forms among the C. albicans P-proteins. Moreover, the strength of interactions between particular proteins was different in these two complexes; the strongest interactions were observed between P1B and P2A proteins, and a significantly weaker one between P1A and P2B proteins. D 2004 Elsevier B.V. All rights reserved. Keywords: Acidic ribosomal protein; Candida albicans; Expression; P-protein interaction

1. Introduction The presence of strongly acidic proteins on the surface of the ribosome is a common feature of all organisms [1,2]. Throughout the ribosome evolution, the primary amino acid structure of this group of proteins has been highly conserved [3]. In contrast to other ribosomal proteins, the acidic ones are also found in the cell cytoplasm as a free protein pool, which is able to exchange with their ribosome-bound counterparts [5]. The P-proteins form a characteristic flexible lateral protuberance on the large ribosomal subunit—the so-called stalk—that plays an important role in the interaction of translation factors with the ribosome. In Prokaryotae such

* Corresponding author. Tel.: +48-81-537-5950; fax: +48-81-5375959. E-mail address: [email protected] (N. Grankowski). 0304-4165/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2004.04.005

as Escherichia coli, the stalk of the 50S ribosomal subunit forms a pentameric complex composed of two L7/L12 protein dimers (L7 differs from L12 by an acetylated N-terminus), attached to L10 protein through their N-terminal domain. This protein complex takes part in stimulation of the elongation factor-dependent GTP hydrolysis [4]. The eukaryotic stalk of the 60S ribosomal subunit is functionally equivalent to the bacterial one [1,2]. In contrast to the prokaryotic acidic proteins, the eukaryotic ones can be phosphorylated in vitro, as well as in vivo, and for this reason they are called P-proteins [5]. According to the primary structure, the eukaryotic P-proteins can be classified into two groups, P1 and P2. Additionally, the P3 protein group has been described in plants [7]. The number of individual P-proteins in each group is not the same in all organisms; they vary from one P-protein in mammals [8] up to two in lower eukaryotes (e.g. yeast Saccharomyces cerevisiae has P1A, P1B, P2A and P2B) [9].

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Yeast P-proteins are able to form a P1 – P2 heterocomplex structure [10], attached to the ribosomal one through P0 protein [11]. Terminal parts of polypeptide chains of both Pproteins are functionally distinct: the N-termini—specific for each of P-protein group—are responsible for mutual interactions between the P-proteins [2], while the C-terminal part of the polypeptide contains a flexible hinge and a highly conserved domain with a phosphorylated serine residue [12,13]. This protein modification is catalyzed by two different protein kinases: protein kinase CKII [14] and a family of protein kinases RAP [6]. Phosphorylation of Pproteins may be implicated in their proteolytical degradation [15] and in the interaction with eEF2 [16]. Biological function of P-proteins is not limited to ribosome activity. They are also interesting from a medical point of view. Anti-P-protein antibodies are associated with autoimmune diseases, such as systemic lupus erythematosus (SLE) [17]. They also play an important role in infections caused by intercellular protozoan parasites: antibodies against the ribosomal P-proteins from Trypanosoma cruzi were detected in the serum of patients with chronic cardiac pathology of Chagas’ disease [18]. Such antibodies were also observed during the infection of Leishmania species [19]. In all cases, anti-P-proteins antibodies recognized the immunodominant epitope, located in the conservative Cterminal end of the polypeptide. Furthermore, P-proteins are recognized as one of the allergens in allergies caused by Alternaria alternata, Cladosporium herbarum and Aspergillus fumigatus, which are regarded as major worldwide causes of fungal allergy [20]. Candida albicans is a member of the microflora in most healthy people. However, especially in immunocompromised patients, C. albicans undergoes a transition from a harmless commensal to an opportunistic pathogen. Studies of the host – Candida relationship have emphasized the importance of a natural as well as of an adaptive immunity in anti-Candida protection, but very few well-defined Candida immunogenes have been identified and characterized so far [21]. Characterization of additional potential immunogenes would be of great value for understanding the mechanism of the anti-Candida immune response and would assist in the development of a new diagnosis. We report here the cloning and characterization of four genes encoding the P-proteins of C. albicans. The gene products were overexpressed, purified to homogeneity and the mutual P-protein interactions were analyzed as preliminary steps toward understanding of the structure and function of the ribosomal stalk of C. albicans.

2. Materials and methods 2.1. Materials and strains All restriction endonucleases, DNA-modifying enzymes, Taq DNA polymerase, isopropyl-h-D-thiogalactopyranoside

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(IPTG) were purchased from MBI Fermentas. The pLM1 vector, in which the promoter region was replaced by introducing the T7 promoter, was derived from pKEN vector. The DEAE-cellulose (DE-52) was from Whatman Biosystem Ltd. Phenylmethylsulfonyl fluoride (PMSF), ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) and all chemicals used for yeast handling and the h-galactosidase assay, were obtained from Sigma-Aldrich. [g-32P]ATP (3000 Ci/pmol) was from Amersham. The QIAquick PCR purification kit, and the DNA extraction kit were from QIAGEN. C. albicans strain CCM 8215 (ATCC10321), obtained from the Czech Collection of Microorganisms, was cultivated in YPD medium (2% pepton, 1% yeast extract, 2% glucose) up to the exponential growth phase at 26 jC. E. coli DH5a and BL21 (DE3) were used for general cloning and protein expression, respectively. Bacterial cells were cultivated in Luria – Bertami (LB) medium (1% pepton, 0.5% yeast extract, 1% NaCl). 2.2. DNA manipulations and construction of a genomic library of C. albicans Manipulation of DNA, Southern blotting and conditions of hybridization were as described by Sambrook and Russell [22]. Isolation of genomic DNA was done according to Moller et al. [23]. The genomic library of C. albicans was constructed by ligation of total EcoRI-digested genomic DNA in similarly digested pUC19 vector. Transformation of E. coli was done by the TSS method [24]. DNA sequencing was carried out using the ABI PRISM 310 Genetic Analyzer and Big Dyek Terminator Cycle Sequencing Kit (PE Biosystem). 2.3. Cloning and mutagenesis of the C. albicans P-protein genes The genomic library of C. albicans was screened with a degenerated DEG probe recognizing the conservative 3Vend fragment of eukaryotic genes encoding ribosomal Pproteins. Based on the nucleotide sequence alignment of seventeen genes for P-proteins a degenerated probe ‘‘DEG’’ was prepared (Fig. 1). Colony hybridization was performed according to Sambrook and Russell [22]. Bacterial colonies harboring the genomic DNA library were transferred and fixed to the nitrocellulose membranes by baking at 80 jC for 1 h. Prehybridization was done at 60 jC for 1 h in the solution: 6  SSC; 5  Denhardt’s solution; 0.5% SDS and 100 Ag/ml denatured/fragmented salmon sperm DNA. Hybridization was performed for 6 h at 60 jC with the same solution containing [g-32P]radiolabeled DEG probe. After incubation, the membrane washing was carried out twice for 15 min at room temperature with 2  SSC (0.3 M NaCl, 0.03 M trisodium citrate) and 0.1% SDS and once for 15 min at 60 jC with 0.2  SSC and 0.1% SDS. Identification of positive clones

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Fig. 1. Oligonucleotide degenerated DEG probe used for screening of the genomic DNA library of C. albicans. (A) Symbols describe genes for P-proteins with GenBank accession numbers: P2ALT (AAU87806)—A. alternata; P1CLA (X85180), P2CLA (X78223)—C. herbarum; P1ASC (M26504), P1BSC (M26507), P2ASC (M26503), P2BSC (M26505)—S. cerevisiae; P1SCP (M33137), P2SCP (M33138)—Schizosaccharomyces pombe; P1DIC (X56193), P2DIC (X56192)—Dictyostelium discoideum; P1HUM (NM001003), P2HUM (NM001004)—human; P1RAT (X15097), P2RAT (X15098)—rat. Non-identical nucleotides are shown in light color. Identical amino acids are indicated by hyphens. The DEG probe is shown in bold. Letter X indicates the degenerated nucleotide. (B) 17-mer degenerated DEG probe with specific description for every degenerated X nucleotide site.

was done by autoradiography with X-ray film (X-Omat AR, Kodak) at 80 jC with an intensifying screen for 1– 2 days. The positive colonies that hybridized specifically with the probe were recovered from the master plate. Based on the DNA sequence analysis of positive clones, we designed the following specific primers to amplify Pproteins genes from the genomic DNA library: CARP1A gene: forward 5V-GGGAATTCAGGAGTATTACAATGTCTACTGAATCTGCATTATC-3V (43-mer), Tm = 53.9 jC, reverse 5V-CCGGATCCCCCAAAATTCTTAATAATACAAGTACCCAG-3V (38-mer), Tm = 58.4 jC; CARP1B g e n e : f o r w a r d 5 V- C G G A AT T C AGGAG A ATA A CAATGTCTACTGAAGCCTCCG-3V (39-mer), Tm = 64.7 jC, reverse 5V-CGGGATCCGAAGTAGGAATGTAAATGATTCGTTAGAACG-3V (39-mer), T m = 63.7 jC; CARP2A gene: forward 5V-GGGAATTCAGGAGGAATATA ATG A A ATA C T TA G C T G C T TA C - 3 V ( 4 1- m e r) , Tm = 61.8 jC, reverse 5V-CCGGATCCCCTTATGCTGTGATCTTGAGATGCC-3V (33-mer), Tm = 65.4 jC; CARP2B gene: forward 5V-GGGAATTCAGGAG GGAAAAAATGAAATACTTAGCTGCTTAC-3V (41-mer), Tm = 62.6 jC, reverse 5 V-GGGGATCCTGCAAACAGTGCCATATAGAAGCC-3V (32-mer), Tm = 64.2 jC. EcoRI and BamHI restriction sites were inserted in all primers to facilitate subcloning. Additionally, the ribosomal binding site (RBS) was introduced in the 5V-end upstream region of the cloned gene [25], and the GTG start codon of the CARP2A was changed from GTG to ATG [26]. This point mutation was implemented in order to optimize the conditions of the P2A protein expression. The PCR products were purified and cloned in EcoRI – BamHI sites of the expression vector

pLM1, yielding series of pL-CAP plasmids. All DNA constructs were confirmed by sequencing. Then the resulting expression plasmids were used for the transformation of E. coli host strain BL21(DE3). 2.4. Heterologous expression and preparation of C. albicans P-proteins E. coli BL21(DE3) cells transformed with pL-CAP plasmids were grown in LB medium supplemented with ampicillin (250 Ag/ml). The cells were cultivated at 37 jC, until the OD at 600 nm reached value around 1.0. Then, IPTG was added to a final concentration of 0.5 mM, to induce the T7 RNA polymerase. After 60 min of induction, the culture was supplemented with rifampicin (200 Ag/ml) to inactivate E. coli RNA polymerase. The cells were maintained under agitation at 30 jC for the next 4 h and collected by centrifugation. Subsequently, cell pellets were re-suspended in disintegration buffer: 100 mM Tris –HCl, pH 7.5, 5 mM 2-mercaptoethanol, 0.5 mM EDTA, 1 mM PMSF, using approximately 5 ml of buffer per gram of wet cells. Cells were disrupted by sonication on ice for 20 min, with 10-s intervals. Cell debris was removed by centrifugation at 10,000  g for 20 min at 4 jC. The obtained supernatant was ultracentrifuged at 100,000  g for 2 h and used as fraction S-100 for isolation of the recombinant Pproteins by ethanol extraction as described previously [28]. Subsequently, the recombinant P-proteins were purified by two steps of ion-exchange chromatography. The first one was performed on DE-52-cellulose columns as described earlier [28]. The second chromatography step was per-

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¨ kta Purifier System (Amersham Biotech) formed with the A equipped with high-resolution anion-exchange column Resource-Q. For all chromatography steps, the following buffer was used: 50 mM Tris – HCl, pH 7.5, 5 mM 2mercaptoethanol, 0.5 mM EDTA, 1 mM PMSF (buffer B). 2.5. Preparation of the P-protein cDNAs by RT-PCR Total RNA from C. albicans was prepared using High Pure RNA Isolation Kit (Roche). mRNA reverse transcription and polymerase chain reaction to amplify the cDNAs for P-proteins were performed using 10 Ag of total RNA as a template. RT-PCR was carried out in accordance with the manufacturer’s protocol (Roche) using Titan One Tube RTPCR. The primers used to amplify the C. albicans P-proteins cDNAs were as follows: CARP1A gene: forward 5V-TAATTACAATGTCTACTGAATCTGCATTATCATACGCC-3V (37-mer), Tm = 59.4 jC and reverse 5V-CTTAATAATACAAGTACCCAGTAAAACAGATGTTGACC-3V (38mer), Tm = 59.4 jC, CARP1B gene: forward 5V-ACAAGAATAACAATGTCTACTGAAGCCTCCG-3V (31-mer), Tm = 59.1 jC and reverse 5V-GAATGAAGTAGGAATGTAATGATTCGTTAGAACG-3V, (35-mer), Tm = 58.1 jC; CARP2A gene: forward 5V-CATTAATAGCATTTTCATGTTACTAACAAG-3V (30-mer), Tm = 52.1 jC and reverse 5VCCTTATGCTGTGATCTTGAGATGCC-3V, (25-mer), Tm = 56.8 jC CARP2B gene: forward 5V-GGAAAAAATGAAATACTTAGCTGCTTACTTATTGTTAG-3V (38-mer), T m = 57.3 jC and reverse 5V-TTCTCTCAAACCAGCAAAGGCGACAAGC-3V (28-mer), Tm = 60.3 jC. All RT-PCR products were identified by DNA sequencing. 2.6. In vitro phosphorylation and dephosphorylation of ribosomes and purified recombinant P-proteins The phosphorylation reaction mixture contained: 20 mM Tris –HCl, pH 7.5, 15 mM MgCl2, 5 mM 2-mercaptoethanol, [g-32P]ATP as phosphate donor, protein kinase CK2 isolated from C. albicans [27] and protein substrates—the purified recombinant P-proteins (2 Ag). The reaction was performed at 30 jC for 20 min and protein samples were analyzed by isoelectrofocusing (IEF) [28]. The ribosomes were isolated from C. albicans cells with a standard protocol as described previously [14]. Dephosphorylation of ribosomes was done with calf intestine alkaline phosphatase (Sigma-Aldrich) according to the procedure provided by the supplier. 2.7. Chemical cross-linking of purified recombinant C. albicans P-proteins Fifteen micrograms of the purified recombinant P-proteins in 25 Al of 20 mM phosphate buffer pH 7.0 were incubated at 25 jC for 10 min in the presence of 10 mM of freshly prepared glutaraldehyde. The reaction was stopped by addition of SDS sample buffer and protein samples were analyzed by SDS-PAGE.

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2.8. Preparation of homo- and hetero-oligomers of purified recombinant C. albicans P-proteins Interaction of C. albicans P-proteins was studied by mixing individual recombinant P-proteins in equal molar amounts in 6 M guanidine hydrochloride. Then, the denaturing reagent was removed by extensive dialysis against buffer A (50 mM Tris – HCl pH 7.5, 150 mM NaCl). The dialyzed protein solutions were clarified by centrifugation (25,000  g for 30 min). Afterwards, the protein samples were analyzed ¨ kta Purifier System equipped with Superose 12 HR 10/ by A 30 FPLC gel filtration column. The column was calibrated with the protein standards (ribonuclease A 13.7 kDa, chymotrypsinogen A 25 kDa, ovalbumin 43 kDa, bovine serum albumin 67 kDa, aldolase 158 kDa) in buffer A. 2.9. Two-hybrid system—yeast transformation, colony-lift filter and liquid b-galactosidase assay Transformation of S. cerevisiae SFY526 strain (MATa, ura3-52, his3-200, ade2-101, lys2-801, trp1-901, leu2-3, 112, canr, gal4-542, gal80-538, URA3::GAL1-lacZ) was done by using yeast –E. coli shuttle vectors: pGBT9 (GAL4(1 – 147), TRP1, ampr) carrying yeast GAL4 DNA-binding domain (BD) and pGAD424 (GAL4(768 – 881), LEU2, ampr) carrying GAL4 activation domain (AD) (Clontech Laboratories Inc). Vectors with genes for individual C. albicans P-proteins were co-transformed into yeast cell according to the manufacturer’s instruction (lithium acetate method). Yeast transformants were selected on solid synthetic minimal medium lacking tryptophan (in case of transformants carrying pGBT9 plasmid) or lacking leucine (for yeast transformants carrying pGAD424 plasmid). P-protein genes from C. albicans were amplified by PCR methods using specific primers described above (see Section 2.3). The colony-lift filter and liquid h-galactosidase assays with X-gal and o-nitrophenyl h-D-galactopyranoside were performed according to the manufacturer’s instruction (Clontech Laboratories). 2.10. Analytical methods SDS-PAGE was carried out on 13.8% acrylamide slab gel according to Laemmli’s method [29]. Protein bands were visualized by staining either with silver or with Coomassie Brilliant Blue R-250. IEF gel electrophoresis was performed on 5% polyacrylamide slab gels containing 2% ampholytes (Sigma) in the 2.5 – 5.0 pH range, as previously described [30]. Protein concentration was measured by the Bradford procedure (Bio-Rad Laboratories), using bovine serum albumin as a standard. Ribosome concentration was measured according to van der Zeijst’s method [31]. Amino acids sequence analysis of P-proteins was performed by Edman degradation method after CNBr treatment, when necessary.

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3. Results

Table 1 Summary of recombinant P-protein purification

3.1. Cloning and analysis of genes encoding ribosomal Pproteins from C. albicans

Purification step Protein Specific Total Purification Yield (mg) incorporation incorporation fold (%) (nmol/mg)a (nmol)a

The starting point of our studies was to use the oligonucleotide degenerated DEG probe. Briefly, to clone the required genes, we screened the C. albicans genomic library with the [g-32P]-labeled DEG probe (Fig. 1) by ‘colony hybridization’. Positive clones were isolated and analyzed by DNA sequencing. The four isolated genes encoding lowmolecular mass ribosomal P-proteins were deposited to GenBank with accession numbers: CARP1A gene— AF317659; CARP1B gene—AF317660; CARP2A gene— AF317661; CARP2B gene—AF317662. The nucleotide sequences of the cloned genes were compared to their transcripts by using RT-PCR reaction (see Section 2). The obtained results showed that all four P-protein genes were intron-less (data not shown). One of them, CARP2A, starts from the non-standard start codon [26]. The gene products were named P1A, P1B, P2A and P2B on the basis of phylogenetic relationship and amino acid similarity to their yeast counterparts (Fig. 2). Molecular masses in kDa: 10.99, 10.74, 10.86, 11.20 and theoretically calculated pI values: 3.80, 3.67, 3.71, 3.84 of P1A, P1B, P2A and P2B protein, respectively, were estimated on the basis of primary structure analysis.

(A) P1A S-100 fractionb 1000 NH4Cl/ethanol 215 DEAE-cellulose 27 Resource Q 2

4.9 14.6 26.5 70.9

4900 3139 715 141

1.0 3.0 5.4 14.5

100 64 15 3

(B) P1B S-100 fractionb 1000 NH4Cl/ethanol 221 DEAE-cellulose 42 Resource Q 14

7.8 18.6 82.8 114.1

7800 4110 3442 1560

1.0 2.4 10.6 14.2

100 53 45 20

(C) P2A S-100 fractionb 1000 356 NH4Cl/ethanol DEAE-cellulose 142 Resource Q 79

21.0 55.7 105.0 137.5

21,000 19,830 14,910 10,863

1.0 2.6 5.0 6.5

100 94 71 51

(D) P2B S-100 fractionb 1000 NH4Cl/ethanol 281 DEAE-cellulose 219 Resource Q 133

50.0 75.7 81.4 128.6

50,000 21,272 17,827 17,104

1.0 1.5 1.6 2.6

100 42 36 34

3.2. Expression and purification of recombinant P-proteins

a The reactions were performed under standard conditions as described under Section 2, except for the time of incubation which was extended to 60 min for the total phosphorylation of P-proteins. b The same amount of S-100 protein fractions as starting material inserted in the tables aimed at easier comparison of the purification steps of four recombinant P-proteins.

The P-protein genes were cloned into the pLM1 expression vector and overexpression of P-proteins in E. coli cells was obtained. The recombinant P-proteins were isolated from S-100 fraction of cell-free extract and purified as

described in Section 2.4. The detection of recombinant proteins in eluted fractions was performed by two methods: SDS-PAGE analysis and enzymatic incorporation of [g-32P]

Fig. 2. Phylogenetic relationship of C. albicans P-proteins (underlined) with other eukaryotic P-proteins based on the multiple amino acid sequence alignment. Construction of phylogenetic tree is inferred by applying ClustalW algorithm with the neighborhood joining method [46] to determine the distance between the eukaryotic P-proteins (DNASTAR 3.15). GenBank accession numbers for the sequences shown here are: A. alternata P1 (P49418) and P2 (S43109); Arabidopsis thaliana P1 (AAC73028), P2 (AAM20070) and P3 (AAM66953); Artemia salina P1 (P02402) and P2 (P02399); A. fumigatus P2 (Q9UUZ6); Ceratitis capitata P1 (CA72658) and P2 (CA70259); C. herbarum P1 (P50344) and P2 (P42039); Drosophila melanogaster P1 (P08570) and P2 (P05389); human P1 (P05386) and P2 (P05387); Leishmania infantum P2 (Q06383); Leishmania peruviani P2 (O46313); Plasmodium falciparum P2 (O00806); rat P1 (P19944) and P2 (P02401); S. cerevisiae P1A (P05318), P1B (P10622), P2A (P05319) and P2B (P02400); S. pombe A1 (P17477), A3 (CAA21791), P2a (P08094) and P2b (P17478); T. cruzi P1 (P26643) and P2 (S59918); Zea mays P1 (P52855), P2 (AAC49360) and P3 (O24413).

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into proteins as described under Section 2. P-protein fractions purified to homogeneity were pooled and kept for further analysis. The results of P-protein purifications are summarized in Table 1 and Fig. 3. 3.3. Comparative analysis of both recombinant and ribosomal P-proteins P-proteins extracted by NH4Cl/ethanol solution from C. albicans ribosomes and the recombinant P-proteins isolated from E. coli expression cells were first analyzed by IEF gel electrophoresis in pH range 2.5 –5.0 (Fig. 4). The comparative analysis of P-proteins isolated from both sources was preceded by dephosphorylation of C. albicans ribosomes, as they are endogenously phosphorylated, contrary to the recombinant P-proteins that are not. Isoelectric point values of the recombinant and dephosphorylated ribosomal Pproteins were comparable with each other, except for P1A protein. As can be seen, the migration distance of the recombinant P1A protein on the IEF gel is shifted compared to its counterpart extracted from C. albicans ribosomes. 3.4. Analysis of interactions between ribosomal P-proteins P-proteins have a tendency to form oligomeric complexes, and it has been shown, using P-proteins from other species, that the formation of P1 – P2 hetero-dimer is a significant step for the subsequent assembly of a pentameric

Fig. 4. Isoelectrofocusing analysis of the recombinant and extracted Pproteins from C. albicans ribosomes. Lines P1A, P1B, P2A and P2B— individual recombinant P-proteins (1 Ag), line R—C. albicans P-proteins from in vitro dephosphorylated ribosomes (500 Ag).

complex, probably in the form P0 –(P1 – P2)2 [11,32– 34]. In order to study the P-protein’s tendency to form homovs. hetero-oligomers, we used three different methods, commonly known in the investigation of protein –protein interactions. The first method—cross-linking with glutaraldehyde— was used for the preliminary analysis of oligomerization properties of P-proteins. The cross-linked oligomers were analyzed by SDS-PAGE (Fig. 5). The presence of monomers (MW f 14 kDa) and homo-dimers (MW f 26 kDa) was visible on the gel for all P-proteins. Three of them—P1B, P2A, P2B—formed oligomers with apparent molecular masses around 42 kDa. Cross-linked

Fig. 3. SDS-PAGE analysis of recombinant P-proteins from each step of purification. Protein samples (30 Ag) were applied on the polyacrylamide gel. Lanes stand for, respectively: (1) S-100 protein fraction containing expressed recombinant proteins; (2) protein fractions after NH4Cl/ethanol extraction; (3) protein fraction after DEAE-cellulose chromatography; (4) chromatography on the Resource Q FPLC column.

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P2A protein produced series of oligomers showing molecular masses over 42 kDa. Moreover, a small subset of P1A and P2A proteins was able to form large homooligomers, which were unable to enter the slab gel during electrophoresis. The analysis of hetero-oligomers formation was not performed by this method because the similarity of molecular masses of all P-proteins made it difficult to distinguish homo- vs. hetero-oligomeric forms. The second method—gel filtration chromatography— was adapted for further analysis of oligomeric properties of recombinant P-proteins. We were able to distinguish several different homo-oligomeric forms (Table 2A). The dominant homo-oligomeric forms for P1A, P1B, P2A and P2B proteins were complexes with molecular masses of about 46, 64, 47 and 71 kDa, respectively. Additionally, a small part of homo-oligomers of P1A and P2A proteins was eluted with the void volume of the column, indicating the polydisperse nature of such complexes, as it had already been observed with the cross-linking experiment. Analysis of hetero-interactions was proceeded by mixing two different P-proteins according to conditions described in Section 2 and subsequently, the protein samples were analyzed by gel filtration method. Using this approach enabled us to detect the hetero-oligomers in case of P1A – P2B and P1B – P2A proteins, with unique molecular masses around 68 kDa at a yield of around 90%. In the remaining cases (i.e. P1A –P2A, P1B –P2B, P1A –P1B and P2A –P2B), we only observed the appearance of homooligomeric forms characteristic of individual P-proteins (Table 2B). The third method was the two-hybrid system. Using the colony-lift filter h-galactosidase assay, we noticed that the P1A and P1B proteins fused to GAL4 DNA binding domain (BD) in the pGBT9 vector, gave a false-positive response, thus inducing the expression of h-galactosidase without any additional protein components. Such transactivation behavior of C. albicans P-proteins resembles the

Table 2 Homo- (A) and hetero-oligomer (B) forms of P-proteins analyzed by size exclusion chromatography (A)

(B)

P-protein

Molecular mass (kDa)

(%)

Mixed P-proteins

Molecular mass (kDa)

(%)

P1A

>300 46 11 64 11 >300 47 25 11 71 11

16 43 41 99 1 1 90 1 8 91 9

P1A – P2A

>300 47 25 11 68 25 68 25 >300 71 11

1 24 10 65 89 11 90 10 1 10 89

P1B P2A

P2B

P1A – P2B P1B – P2A P1B – P2B

transactivation potential of the S. cerevisiae P1A and P1B protein, showing this to be a common feature in yeast species [35]. The remaining transformants with pBD-P2A, pBD-P2B as well as pAD-P1A, pAD-P1B, pAD-P2A and pAD-P2B, did not show any transactivation potential. Therefore, the pBD-P1A and pBD-P1B constructs were omitted from the studies on mutual P-protein interactions for the sake of their false-positive character. All possible interactions between hybrid-proteins were determined by the colony-lift filter assay (Table 3). The strongest signals were observed during the interaction between constructs containing P1B – P2A proteins, while the P1A – P2B gave a weaker signal. In the case of construct P1A –P2A and P1B –P2B proteins, a blue color of yeast colonies was developed not earlier than after 24 h. No interaction was observed in the constructs carrying genes for P-proteins from the same group. The qualitative results presented in Table 3 were confirmed by the quantitative liquid h-galactosidase assay with ONGP as a substrate. As shown in Fig. 6, the yeast transformant containing pAD-P1B/pBD-P2A gave the highest activity of h-galactosidase. Much weaker, but still significant, interactions were noticed for the pADP1A/pBD-P2B hybrid proteins, and weak signals—for

Table 3 Analysis of interaction between P-proteins using GAL1 promoter system

pAD-P1A pAD-P1B pAD-P2A pAD-P2B pAD

Fig. 5. SDS-PAGE of P-proteins chemical cross-linked in glutaraldehyde. Arrows indicate molecular masses of oligomeric forms of P-proteins.

pBD-P2A

pBD-P2B

+/ ++++

++ +/

pBD

The number of plus (+) signs represents the relative rates at which the transformed yeast cells turned blue after incubation at 30 jC on the filter paper. (++++) 1 – 3 h; (++) 6 – 9 h; (+/ ) >24 h. At least 10 independent transformants were tested for each assay.

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Fig. 6. Interactions between P-proteins using liquid h-galactosidase assays. Liquid h-galactosidase assay was done with the transformants of yeast strain SFY526. Abbreviations of BD and AD stand for, pGBT9 vector expressing GAL4 DNA-binding domain and pGAD424 vector expressing GAL4 DNA activation domain, respectively. The BD and AD domains were fused with respective P-proteins and described as follows: AD1A – BD2A, AD1A – BD2B, AD1B – BD2A, AD1B – BD2B, AD1A – AD1B, AD2A – BD2B, AD2B – BD2A. Letter K means control reactions: BD2B – AD, BD2A – AD, BD – AD1A, BD – AD1B, BD – AD2A, BD – AD2B and BD – AD. The data represent average values from the two sets of independent experiments performed in triplicate. h-Galactosidase units were quantified according to the Miller method [47].

the pAD-P1A/pBD-P2A and pAD-P1B/pBD-P2B hybrid constructs.

4. Discussion Almost 30 years ago, the first eukaryotic acidic ribosomal P-proteins were isolated and characterized [36] and so far, about 90 gene sequences encoding the eukaryotic Pproteins from 57 organisms have been deposited in the GenBank (data updated in February 2003). In this work, we identified the genes encoding acidic P-proteins forming at the stalk structure on the large ribosomal subunit from C. albicans. The biological function of the eukaryotic Pproteins is mainly associated with the regulation of protein synthesis at the level of elongation step. The P-proteins are characteristic of a very highly conserved acidic-domain, located in the C-terminus [1] and this conserved feature was used to design a synthetic oligonucleotide DEG probe used for isolation of the four P-protein genes from the C. albicans genomic library. It should be noted that our gene cloning was done in parallel with C. albicans sequencing project from the Stanford Genome Technology Center [http:// www-sequence.stanford.edu/group/candida/index.html] and our results are fully compatible. Considering the analyzed genes, one of the four genes—the CARP2A encoding P2A protein—started from the non-standard AUG start codon— GUG, as already described in our recent publication [26]. For this reason, we introduced a point mutation in the start codon replacing GUG by AUG during the PCR amplification in order to optimize the conditions for the P2A protein expression [37]. P-protein overexpression approach was accomplished with the expression system based on the strong T7

promoter. Overproduced, recombinant P-proteins were predominantly present in the soluble fraction of the cell-free extract, and purification steps were identical for all proteins (Fig. 3). The yield of the recombinant proteins ranged from 3% to more than 50% of the total soluble P-proteins (Table 1). Biochemical characterization of recombinant proteins has shown their pI values to be similar to those of the native proteins, except for the P1A protein: the wild-type P1A displays a more basic pI than its recombinant counterpart (Fig. 4). This shift might be attributed to a specific post-translational modification of the protein, which we have not yet identified. Furthermore, amino acid analysis by Edman degradation of wild-type P1A and P1B proteins indicated the existence of an Na-blocked amino terminus. The first methionine seemed to be removed before Naacetylation of the next serine, as it has been shown for yeast S. cerevisiae P1-type proteins [38,39]. Phosphorylation of the serine residue located in the conserved C-terminal region [12,13] is the essential modification for eukaryotic acidic ribosomal proteins. Phosphate incorporation into P-proteins stimulates the interaction with elongation factor 2 [16] and/or can be involved in P-protein degradation [15]. The C. albicans P-proteins are phosphorylated in vitro (data not shown) and presumably, at the Cterminal of polypeptide chain, where the amino acid motif recognized by protein kinase CK2 is located. The highly conserved C-terminal region plays a particular role in several human diseases as an antigenic determinant recognized by human antibodies [17,18]. For instance; this particular C-terminal epitope of T. cruzi P-proteins may induce a strong humoral response in chronic Chagas’ heart disease [18]. Additionally, this highly conserved polypep-

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tide can serve as an immunogenic signal in numerous fungal opportunistic pathogens [20], and therefore could be regarded as a potential universal signal similar to other antigens/allergens from C. albicans [40]. Comparative analysis of amino acids sequences revealed the P2 proteins from C. albicans to have a sequence similarity to wellknown fungal allergens, i.e. P2 proteins from A. alternata, C. herbarum and A. fumigatus [20] (Fig. 2). Consequently, this could be a suggestion of a potential immunogenic character of the C. albicans P-proteins. Although our main concern was focused on the mutual interactions among Pproteins. In this paper, we have defined the interactions among the C. albicans acidic ribosomal P-proteins with different in vitro and in vivo methods. Gel filtration chromatography and chemical cross-linking approaches indicated all P-proteins to be able to form polydispersed homo-oligomeric structures. Therefore, the formation of homo-oligomers could be recognized as a characteristic feature, since individual P-proteins are able to exist in a state called ‘molten globule’-like structure, as already observed for S. cerevisiae P-proteins, and this particular conformation is prone to form polydispersed forms [41]. The results from gel filtration chromatography showed the presence of dominant homo-oligomer forms of individual P-proteins, with molecular masses ranging from 46 to 71 kDa. It should be noted here that these values measured by size exclusion chromatography are dependent upon the use of globular protein standards. C. albicans P-proteins might have, as their yeast counterparts, an unusual elongated shape, differing them from globular proteins and consequently, the molecular mass could be obscured by this fact, and therefore molecular masses of homo-oligomers were verified by cross-linking analysis with glutaraldehyde. The analysis of the individual P-proteins showed two major protein bands with apparent molecular masses of f 14 and f 28 kDa, indicating the presence of monomeric and dimeric forms in the solution. Additionally, the higholigomeric forms were also observed, but to a smaller extent. The existence of monomers can be explained by unfavorable distribution of the amino group of lysine residues, close to the intermolecular area, which might prevent the formation of Schiff bases by the reaction of an aldehyde group of glutaraldehyde with the amino group of basic amino acids [42]. Our results, showing the tendency of individual P-proteins to form several species of homooligomers, are compatible with the previous observation, indicating single P-proteins to be prone to form polydisperse oligomeric forms [10,41]. However, it should be emphasized here that the preference of P-proteins to form hetero-oligomers has been already observed in several other species [10,11,33,34,43,44]. Finally, following this experimental path, we were able to demonstrate the ability of P-proteins to generate hetero-oligomeric forms, between P1A –P2B and P1B – P2A proteins, using the gel filtration approach, the more so as the in vivo analysis of heterointeractions between the P-proteins using the two-hybrid

system showed the strongest preference for the formation of P1B – P2A heterocomplex. These experimental data are in contrast to the S. cerevisiae model of P-protein heterointeraction, according to which the strongest mutual relations between P1A and P2B proteins are observed [10,11,44]. As shown several times in complementation experiments, there is no full compatibility between Pproteins coming from several species [45]. Even though they had quite a high degree of similarity in primary structure and biophysical properties, they have quite a large functional diversity. Therefore, contrary to S. cerevisiae data, our result where the P1B– P2A heterocomplex has been observed as the dominant dimer, further supports the general observation that every species has its own specific P-protein pool that meets its unique requirements. Nevertheless, our results continue to support the generally accepted concept of hetero-interactions between P1 and P2 proteins undergoing in eukaryotic cells.

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