Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity

Analysis of major intracellular proteins of Aspergillus fumigatus by MALDI mass spectrometry: Identification and characterisation of an elongation factor 1B protein with glutathione transferase activity q Stephen Carberry 1 , Claire M. Neville 1 , Kevin A. Kavanagh, Sean Doyle * National Institute for Cellular Biotechnology, Department of Biology, National University of Ireland Maynooth, Co. Kildare, Ireland Received 13 January 2006 Abstract Aspergillus fumigatus is a recognised human pathogen, especially in immunocompromised individuals. The availability of the annotated A. fumigatus genome sequence will significantly accelerate our understanding of this organism. However, limited information is available with respect to the A. fumigatus proteome. Here, both a direct proteomic approach (2D-PAGE and MALDI-MS) and a sub-proteomic strategy involving initial glutathione affinity chromatography have been deployed to identify 54 proteins from A. fumigatus primarily involved in energy metabolism and protein biosynthesis. Furthermore, two novel eukaryotic elongation factor proteins (eEF1Bc), termed ElfA and B have been identified and phylogenetically confirmed to belong to the eEF1Bcclass of GST-like proteins. One of these proteins (ElfA) has been purified to homogeneity, identified as a monomeric enzyme (molecular mass = 20 kDa; pI= 5.9 and 6.5), and found to exhibit glutathione transferase activity specific activities (mean ± standard deviation, n= 3) of 3.13 ± 0.27 and 3.43 ± 1.0 lmol/min/mg, using CDNB and ethacrynic acid, respectively. Overall, these data highlight the importance of new approaches to dissect the proteome of, and elucidate novel functions within, A. fumigatus. 2006 Elsevier Inc. All rights reserved. Keywords: Aspergillosis; Proteomics; 2D-PAGE; Oxidative stress; Protein synthesis; GST; Glutathione The field of fungal proteomics is gathering pace as a result of the simultaneous occurrence of fungal genome sequence availability and advances in high sensitivity protein mass spectrometry. Indeed, the recent publication of a two-dimensional protein reference map for, and the analysis of stress-induced changes in the proteome of, Schizosaccharomyces pombe clearly illustrates this point [1,2]. Proteomic analysis of the filamentous fungus, Trichoderma harzianum, has also yielded insights into the mitochondrial proteome of this mycoparasitic organism [3]. Many Aspergillus species have merited attention from a biotechnological standpoint, as a consequence of a secreted proteome rich in industrially useful enzymes [4]. Until recently, however, little information was available with respect to either the technical requirements for protein extraction or the proteome content of most Aspergillus species. However, dissection of the secreted proteome (secretome) of Aspergillus flavus has demonstrated the ability of this organism to adapt to altered conditions such as the presence of various carbon sources (e.g., rutin) [5]. With respect to the human pathogenic fungus, A. fumigatus, Bruneau et al. adopted a proteomic approach to identify glycosylphosphatidylinositol-anchored proteins which are involved in the organisation of the fungal cell wall [6]. More recently, we have undertaken the identification of nonribosomal peptide synthetases in A. fumigatus using a combined proteomic and molecular approach [7]. Along with Candida albicans,A. fumigatus is a significant opportunistic pathogen of immunocompromised individuals, 0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.01.078 q Abbreviations: CDNB, 1-chloro-2,4-dinitrobenzene; 2D-PAGE, two-dimensional electrophoresis. * Corresponding author. Fax: +353 1 7083845. E-mail address: [email protected] (S. Doyle). 1 These authors contributed equally to this work. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 BBRC ARTICLE IN PRESS indeed approximately 5% of all hospital based nosocomial deaths are due to Aspergillus infections [8]. The recent completion of the sequencing of the A. fumigatus genome and the availability of the in silico annotated genome at http://www.cadre.man.ac.uk [9] should enable detailed molecular dissection of the biology of this organism. Moreover, both unannotated and annotated genome data can be interrogated to further our knowledge of the A. fumigatus proteome and the organismal response to altered environmental conditions. For instance, a greater understanding of the role played by glutathione transferases (GSTs) in mediating resistance to oxidative stress and antifungal drugs or xenobiotics is required and we have recently identified, characterised, and cloned three GST genes from A. fumigatus (gstA-C) [10]. However, GST-like domains have also been detected in distinct proteins such as Ure2p, Mak16, and EF1Bc(a subunit of the EF1B complex) [11]. Furthermore, although GST activity has recently been ascribed to the elongation factor protein complex EF1B isolated from rice and trypanothione S-transferase activity detected in EF1B complex from Leishmania major, no detection of GST activity in native EF1Bchas been possible [12]. In an attempt to purify the GST proteins (GstA-C) from A. fumigatus, glutathione (GSH)–Sepharose affinity chromatography was employed. Although GstA, B or C were undetectable, a number of other proteins were detected, one of which was identified as a eukaryotic elongation factor 1Bc. This manuscript represents one of the first detailed descriptions of a proteomic approach to identify intracellular proteins in A. fumigatus and also the use of a sub-proteomic approach, involving affinity chromatography and MALDI mass spectrometry to identify new biochemical functions in fungal species. Materials and methods Protein extraction. Aspergillus fumigatus (ATCC 26933) was cultured in Sabouraud’s media (500 ml cultures), at 37 C with shaking at 200 rpm. Mycelia were harvested, filtered under pressure, and washed with PBS and resuspended in lysis buffer (100 mM Tris–HCl, 50 mM NaCl, 20 mM EDTA, 10% (v/v) glycerol, 30 mM DTT, 1 mM PMSF, and 1 lg/ml pepstatin A, pH 7.5; 5 ml of lysis buffer per gram of mycelia). Lysis was accomplished by grinding in liquid N 2 followed by brief sonication. Mycelial lysates were centrifuged (10,000g; 30 min) to remove cell debris and the subsequent supernatant analysed by 2D-PAGE following TCA/ acetone precipitation [13]. Two-dimensional electrophoresis. Protein separation by 2D-PAGE was as follows: extracts containing 250 lg of protein were resuspended in 8 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (v/v) Triton X-100, 10 mM Tris–HC1, 65 mM dithiothreitol (DTT), and 0.8% pH 4–7 carrier ampholytes, and loaded onto Immobiline Dry strips (IPG strip; Amersham) in the pH range 4–7. Gels underwent active rehydration at 50 V for 10 h, followed by a further 10 h focusing with a total of 26250 V applied. Following IEF, gels were equilibrated in reducing buffer (50 mM Tris–HCl, 6 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, and 2% (w/v) DTT, pH 6.8) for 20 min followed by equilibration in alkylation buffer (50 mM Tris– HCl, 6 M urea, 2% (w/v) SDS, 30% (v/v) glycerol, and 2.5% (w/v) iodoacetamide, pH 6.8) for a further 20 min. The IPG strips were placed on homogeneous 12% SDS–PAGE gels and electrophoresed for 20 h at 100 V using the ProteanXi-II Cell (Bio-Rad Laboratories). Resulting gels were stained with Coomassie brilliant blue R and scanned using a Typhoon Trio Variable Mode Imager (Amersham Biosciences (Europe) GmbH, Freiburg, Germany). Chromatographic procedures. Aspergillus fumigatus cell lysates were centrifuged as above and the supernatant collected while the pellet was discarded. Ammonium sulphate solution (100% (w/v)) was then added to the supernatant to yield 65% ammonium sulphate (65% solution) which was stirred at 4 C overnight. This suspension was then centrifuged, the supernatant removed, and the pellet resuspended in a minimum volume of lysis buffer. Resuspended material was extensively dialysed against 2 ·50 volumes of 25 mM Tris–HCl, 50 mM NaCl, pH 7.4 (buffer A), and the dialysate was centrifuged at 10,000gfor 10 min and then filtered (0.45 lM) prior to chromatography. A GSH–Sepharose column (3 ml volume) was equilibrated with buffer A and following dialysate application and washing, was eluted with 50 mM Tris–HCl, 15 mM GSH, pH 9.2 (buffer B). Fractions (0.5 ml) were collected. SDS–PAGE analysis of neat or TCA precipitated fractions was carried out as previously described [13]. GSH– Sepharose column fractions were subsequently pooled (350 lg/10 ml), applied to a Q-Sepharose resin (1.5 ·8 cm; flow rate: 1 ml/min), and 0.5 ml fractions collected following elution with a 25 ml linear gradient of 0–0.5 M NaCl in 25 mM Tris–HCl, pH 8.0. Peak fractions containing were identified by SDS–PAGE and enzymatic activity analysis (glutathione conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and ethacrynic acid) [14]. Native molecular mass determination was undertaken by size exclusion chromatography using an A ¨KTA Purifier 100 system (Amersham Biosciences, UK) whereby a Superose 12 column (10 ·300 mm) was equilibrated in PBS at a flow rate of 0.4 ml/min. The concentrated material from Q-Sepharose was loaded on the column and 0.5 ml fractions collected. MALDI mass spectrometry. Mass spectrometry was carried out using an Ettan MALDI-ToF mass spectrometer (Amersham Biosciences (Europe) GmbH, Freiburg, Germany). Protein samples for peptide mass determination were excised from 2D-PAGE gels using an automated spot cutter (Amersham Biosciences (Europe) GmbH, Freiburg, Germany), digested with trypsin and deposited (1 ll) with 1 lla-cyano4-hydroxycinnaminic acid (4-HCCA; 5 mg/200 ll of 50% (v/v) acetonitrile in 0.1% (v/v) aqueous trifluoroacetic acid) onto mass spectrometry slides, and allowed to dry prior to delayed extraction, reflectron ToF analysis at 20 kV [10]. Internal calibrants, Angiotensin III (Sigma–Aldrich) and ACTH fragment 18–39 (Sigma–Aldrich), were used to calibrate all spectra. Protein identification was carried out either by m/zdata interrogation of (i) the NCBI nr database available as part of the mass spectrometer Evaluation Software Version 2.01 or (ii) a local FASTA version of the annotated A. fumigatus genome available at http://www.cadre.man.ac.uk. The annotation system employed on this website (i.e., AfuNg where N and g define the chromosome and gene number, respectively) and described by Mabey et al. [9] has been adopted in this manuscript. Tandem mass spectrometry was performed by commercial arrangement (Taplin Biological Mass Spectrometry Facility, Harvard Medical School, MA, USA) using an LCQ DECAXP Plus ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA). Enzyme activity determination. GST assays were carried out as described by Habdous et al. [14]. Briefly, assays were carried out in a total volume of 200 ll including 20 ll of protein sample with CDNB/ethacrynic acid, GSH in 0.1 M potassium phosphate (pH 5.8) at final concentrations of 0.5/0.2 and 2.5 mM, respectively. Reactions were initiated by the addition of the protein sample and a blank was created by the addition of 20 ll of 0.1 M potassium phosphate (pH 5.8) instead of the protein samples. The increase in absorbance was measured at 340 nm (corresponding to the enzymatic conjugation of CDNB/ethacrynic acid to GSH by GST). Phylogenetic analysis. Sequence alignments and bootstrap neighbour joining phylogenetic trees were generated using the Clustal X 1.81 program available at ftp://ftp-igbmc.u-strasbg.fr/pub/ClustalX/, using the program default parameters. A bootstrapping value of 1000 was used, with bootstrapping values noted at tree branch points. Trees were visualised using Treeview (http://taxonomy.zoology.gla.ac.uk/rod/treeview.html). S. Carberry et al. / Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 1097 ARTICLE IN PRESS Results Aspergillus fumigatus proteome analysis A total of 180 spots were excised from 2D-PAGE gels (Fig. 1A) which resulted in the identification of 50 distinct proteins (in 65 individual spots), by MALDI-MS (Table 1). Significant expectation values were not achieved for the remaining 115 spots to give positive identification. The percentage sequence coverage obtained ranged from 9.4% (peroxisome biogenesis factor) to 50.5% (enolase) and observed molecular masses ranged from 75 kDa to less than 25 kDa. Several proteins exhibited different observed molecular mass to the theoretical mass suggesting the occurrence of post-translational modification. The majority of proteins identified appear to be involved in energy production, with proteins characteristic of the glycolytic pathway, citric acid pathway, pentose phosphate pathway, fatty acid metabolism, and oxidative phosphorylation clearly evident (Fig. 1B and Table 1). Other classes of proteins identified include structural proteins, signalling proteins, heat shock and heat shock chaperone proteins, transcription factors, and two hypothetical proteins. A number of proteins (i.e., catalase 1, HSP 70 chaperone HscA, alanine aminotransferase, thiamine biosynthesis protein Nmt1, 14-3-3 family proteins, and uridine diphosphate-glucose-4 epimerase) (Table 1) were individually present in more than one spot, at identical molecular mass, which indicates subtle charge differences possibly due to side-chain deamination. Most proteins exhibited molecular mass and pIvalues close to the predicted values, except for NAD-dependent formate dehydrogenase pIobserved = 7.7; pI-predicted = 8.7. Moreover, protein Afu5g14680 (unknown function) presented at pI4.7, 4.9, and 5.2 with molecular masses all below the theoretical 25 kDa value. Analysis of this sequence by BLAST search indicated hypothetical proteins in Aspergillus nidulansXM_656639 and Gibberella zeae XM_390618 with similarity to Afu5g14680. The theoretical and actual mass of hypothetical protein Afu2g10030 differed by 7 kDa and BLAST analysis showed similarity to hypothetical proteins in A. nidulans XM_658516 and Botrytis cinerea 47 62 100 32.5 25 kDapH47 44% 8% 6% 18% 3% 6% 15% Energy HSP Hypothetical ProteinBiosynthesis Structural Transcription Other A B Fig. 1. (A) 2D-PAGE separation of whole protein extract from A. fumigatus. Whole protein extracts (250 lg) were subjected to isoelectric focussing on Immobiline Dry strips (pH range 4–7) followed by SDS–PAGE and Coomassie brilliant blue staining. (B) Functional classification of all A. fumigatus proteins identified by MALDI-MS whereby >60% of proteins were involved in either energy generation or protein biosynthesis. HSP, heat shock protein. 1098 S. Carberry et al. / Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 ARTICLE IN PRESS Table 1 A. fumigatus proteins (n= 50) present after culture in Sabouraud media following identification by 2D-PAGE and MALDI-MS (Fig. 1) CADRE I.D. Spot No. % Coverage M r (kDa) pIProposed function Afu1g07480 1C7 26.8 50.16 6.9 Coproporphyrinogen III oxidase Afu1g10130 D6 24.7 48.47 5.8 Adenosylhomocysteinase Afu1g10350 F12 35.3 44.74 6.5 Phosphoglycerate kinase Afu1g10630 1A7 44.4 42.18 5.7 S-Adenosylmethionine synthetase Afu1g12170 1F7 21.8 48.27 6.7 Translation elongation factor EF-Tu Afu1g13500 E5 23.5 74.81 6.1 Transketolase Afu1g13490 2D4 29.7 33.43 5.3 Spermidine synthetase Afu2g00720 B7 14.4 51.92 5.6 Aldehyde dehydrogenase Afu2g03290 2A4 25.7 29.08 4.8 14-3-3 Family protein ArtA Afu2g04230 1F1 10.9 46.80 6.0 Fumarylacetoacetate hydrolase fahA Afu2g07420 B5 22.4 72.46 5.8 Fimbrin Afu2g09960 G8 30.9 74.45 5.4 Mitochondrial HSP70 chaperone Ssc Afu2g10030 1E11 22.3 28.25 6.2 Hypothetical protein Afu2g11150 1H3 24.6 52.26 5.3 Secretory pathway gdp dissociation inhibitor Afu2g13240 A7 24.5 56.4 5.7 V-type ATPase subunit B Afu3g00590 2B6 41.0 15.18 5.8 Asp-hemolysin Afu3g01110 E6 17.2 61.41 5.8 GMP synthase Afu3g02270 A3 27.1 79.9 5.6 Catalase 1 Afu3g02270 H2 27.1 79.9 5.7 Catalase 1 Afu3g02270 F4 23.4 79.89 5.5 Catalase 1 Afu3g05450 H9 36.0 53.0 5.5 Glutamate carboxypeptidase Afu3g07630 2H3 26.8 19.61 4.7 Dihydropteroate synthase Afu3g08160 1A6 15.0 45.76 5.0 Eukaryotic translation initiation factor eIF4A Afu3g09290 G7 20.4 57.44 5.6 Phosphoglycerate mutase 2,3-biphosphoglycerate-independant Afu3g11070 A6 31.3 62.98 6.1 Pyruvate decarboxylase PdcA Afu3g11690 1D10 43.6 39.77 5.5 Fructose-biphosphate aldolase class Afu3g11690 1E10 33.1 39.77 5.8 Fructose-biphosphate aldolase class II Afu4g06620 1H1 43.9 49.35 5.8 Glutamate/leucine/phenylalanine/valine dehydrogenase Afu4g07690 G5 13.3 65.01 6.8 Phosphoribosylaminoimidazole carboxamideformyltransferase/IMP hydrocyclase Afu4g10200 B8 13.2 71.68 5.4 Transcription factor RfeF Afu4g11730 1C11 16.3 36.8 6.0 Glycerol dehydrogenase GldB Afu4g13170 1F11 45.9 34.96 6.4 Guanine nucleotide binding protein b-subunit Afu4g13170 1G11 36.4 34.96 6.4 Guanine nucleotide binding protein b-subunit Afu5g01030 IH8 43.4 36.12 6.5 Glyceraldehyde-3-phosphate dehydrogenase Ccg-7 Afu5g01030 1E8 39.2 36.12 6.2 Glyceraldehyde-3-phosphate dehydrogenase Ccg-7 Afu5g02470 1A11 29.5 38.3 6.0 Thiamine biosynthesis protein Nmt1 Afu5g02470 1B11 29.5 38.3 6.0 Thiamine biosynthesis protein Nmt1 Afu5g02470 1C10 19.9 38.3 5.7 Thiamine biosynthesis protein Nmt1 Afu5g04170 G1 15.4 80.6 4.9 Molecular chaperone HSP1 Afu5g09230 1D11 40.1 35.43 6.0 Transaldolase Afu5g10550 1A5 41.4 55.6 4.8 ATPase synthase F1 subunit b Afu5g10550 1B5 41.4 55.6 4.8 ATPase synthase F1 subunit b Afu5g10780 1B8 39.9 40.59 6.1 UDP-glucose 4-epimerase Afu5g10780 1C8 27.8 40.59 5.9 UDP-glucose 4-epimerase Afu5g14680 2D7 39.6 25.42 4.7 Hypothetical protein Afu5g14680 2G7 34.8 25.42 4.9 Hypothetical protein Afu5g14680 2H7 34.8 25.42 5.2 Hypothetical protein Afu6g04740 1E6 26.7 43.88 5.9 Actin Afu6g04920 1B9 33.0 45.73 7.7 NAD-dependant formate dehydrogenase Afu6g06750 2D3 28.0 30.05 4.7 14-3-3 Family protein Afu6g06750 2C3 28.0 30.05 4.8 14-3-3 Family protein Afu6g06770 1H2 50.5 47.29 5.6 Enolase Afu6g07720 E7 15.8 66.8 5.8 Phosphoenolpyruvate carboxykinase Afu6g07770 F3 24.6 55.12 6.2 Alanine aminotransferase Afu6g07770 C11 18.2 55.12 6.3 Alanine aminotransferase Afu6g07770 H10 33.1 55.12 5.9 Alanine aminotransferase Afu6g08050 1C2 31.4 55.78 5.9 6-Phosphogluconate dehydrogenase decarboxylating Afu6g12930 F2 16.9 85.5 6.4 Aconitate hydratase Afu7g01860 F3 28.7 65 5.5 Heat shock protein Sti1 Afu7g05070 G10 22.5 51.25 5.7 FAD-dependent oxidoreductase Afu7g05720 1G6 9.6 39.1 5.9 Peroxisome biogenesis factor (continued on next page) S. Carberry et al. / Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 1099 ARTICLE IN PRESS CNS01C5R. Conserved Domain analysis [15] of Afu2g10030 indicted that it may possess an RNA Recognition Motif (RRM). The phytanol-CoA dioxygenase family protein, (Afu8g00230) which is a peroxisomal enzyme catalysing the first step of phytanic acid a-oxidation, has not been previously identified in A. fumigatus, however it exhibited similarity with hypothetical protein AN9227.2 in A. nidulans. A Conserved Domain search identified both Afu8g00230 and AN9227.2 as containing a phytanol-CoA dioxygenase domain as well as a COG5285 domain. In Burkholderia fungorum LB400, a protein containing a COG5285 domain is involved in biosynthesis of mitomycin antibiotics and the polyketide, fumonisin. Dihydropteroate synthase (Afu3g07630) has been associated with drug resistance in Saccharomyces cerevisiae and has been shown to be derived from a polycistronic gene also responsible for the production of two other functional proteins [16]. Catalase 1 (Afu3g02270) involved in oxidative stress and identified here as three separate protein spots has been implicated in A. fumigatus pathogenicity [17]. Almost 44% (22/50) of all proteins identified were derived from intron-containing genes. For instance, 3 peptides of m/z2027.99, 2567.11, and 2630.31 were detected in pyruvate decarboxylase (Afu3g11070) which derive from three distinct splicing events. In addition, two peptides derived from exon–exon splicing were detected in each of seven proteins (catalase, glutamate carboxypeptidase, vacuolar synthase subunit B, enolase, secretory pathway dissociation inhibitor, F1-ATPase (b-subunit), and translation elongation factor EF-Tu) (Table 1). A single peptide, in each of the 14 remaining proteins, was identified which resulted from exon splicing (data not shown). Aspergillus fumigatus sub-proteome analysis GSH–Sepharose affinity chromatography was undertaken in order to selectively detect and purify glutathione binding proteins in A. fumigatus. Affinity column elution with 5 mM GSH (in 50 mM Tris–HCl, pH 9.2) did not result in the displacement of any bound proteins (data not shown) and all resultant GSH-binding proteins were displaced from the column in the presence of 15 mM GSH. Overall, the total protein yield was 7.4 mg/g mycelia in cell lysate supernatant and the yield of GSH-binding proteins was 5.3 lg/g mycelia which indicates that these proteins represent 0.0007% of total soluble protein in A. fumigatus. The presence of three proteins was detected by SDS–PAGE analysis following GSH–Sepharose chromatography and a peptide (SVDVVEEYLQDR) was identified by Tandem-MS from the protein present in band 3 (Figs. 2A and B). Database interrogation http://www.tigr.org;http://www.cadre.man.ac.uk with this sequence identified a putative translation elongation factor 1Bctype protein (Afu8g00580; ElfB) with 10% sequence coverage (Fig. 2B). Further analysis of GSH-binding proteins by 2D-PAGE (Fig. 2C) demonstrated that at least 10 proteins (molecular mass range: 18–40 kDa) were present following GSH–Sepharose affinity chromatography. All GSH-binding proteins were excised from 2D-PAGE gels and analysed by MALDI-MS. Four proteins were identified based on comparison to both the NCBI nr database and a FASTA version of the in silico-annotated A. fumigatus genome available at http://www.cadre.man.ac.uk (Table 2). Specifically, the most abundant protein (Fig. 2C-spot 3 a,b; Afu1g17120) was identified, based on 46.4% sequence coverage, as an translation elongation factor 1Bcsubunit. This protein, termed ElfA, appeared to be present as two distinct spots of identical molecular mass but different pIvalues (5.9 and 6.5, respectively) (Fig. 2C). Anion-exchange chromatography (Fig. 3A) was subsequently used to purify ElfA to homogeneity followed by assessment of protein purity by SDS–PAGE (Fig. 3B). When the GST activity of purified ElfA was analysed, both CDNB and ethacrynic acid were recognised as substrates with specific activities (mean ± standard deviation, n=3) of 3.13 ± 0.27 and 3.43 ± 1.0 lmol/min/mg, respectively. No activity was detected against DCNB and neither was any glutathione peroxidase activity detected in ElfA (data not shown). Moreover, the observed specific activities against CDNB and ethacrynic acid were much greater than those observed for recombinant A. fumigatus GstC expressed in Escherichia coli (0.01 ± 0.0009 lmol/min/mg) [10]. Size exclusion analysis identified the molecular mass of ElfA to be 20 kDa, suggesting it is present as a monomer within the cell, unlike most forms of GST which are located intracellularly in dimeric form (data not shown). These results represent the first demonstration of GST activity associated with an elongation factor 1Bcsubunit in a fungus. Moreover, it also the first demonstration of GST activity in a native elongation factor 1Bcsubunit disassociated from other components of the EF1B complex. Aspergillus fumigatus ElfA and ElfB were compared to 28 sequences of GST-like proteins from various species, previously analysed by McGoldrick et al. [11], in order to assess homology (Fig. 4). From this analysis, three distinct clades were observed namely EF1c-like, Ure2, and Table 1 (continued) CADRE I.D. Spot No. % Coverage M r (kDa) pIProposed function Afu7g05720 D8 27.0 52.01 5.4 Pyruvate dehydrogenase complex dihydrolipoamide acetyltransferase component Afu8g00230 2F4 22.3 32.65 5.6 Phytanoyl-CoA dioxygenase family protein Afu8g03930 A9 31.3 66.96 5.3 HSP70 chaperone HscA Afu8g03930 H8 22.8 66.96 5.4 HSP70 chaperone HscA CADRE I.D., A. fumigatus gene annotation nomenclature according to Mabey et al. [9] and Nierman et al. [33]. 1100 S. Carberry et al. / Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 ARTICLE IN PRESS ↓ElfA M 1 2 3 4 kDa 47.5 32.5 25 AB 0 10 20 30 40 50 60 70 80 90 0 10152025 mAU Elution volume (ml) ElfA 5 Fig. 3. (A) Purification of A. fumigatus ElfA by anion-exchange chromatography of GSH-binding proteins from A. fumigatus. GSH purified proteins were applied to a Q-Sepharose column (14 ml column volume) and eluted with an increasing salt gradient (0–0.5 M NaCl in 25 mM Tris–HCl, pH 8.0). ElfA was identified in the first peak as indicated. (B) SDS–PAGE analysis of ElfA purified by ion exchange chromatography (Q-Sepharose). Lane M, molecular mass markers; lanes 1–4, Q-Sepharose column fractions containing purified ElfA. Table 2 GSH-binding proteins of A. fumigatus separated using 2D-PAGE and identified by MALDI mass spectrometry Afu Code Spot No. % Coverage Mol. mass (kDa) pIProposed function Afu5g06060 1 14.8 18.1 4 Sulphur metabolism regulator SkpA, putative Afu1g03970 2 10 25 3.2 Mitochondrial translation initiation factor IF-2, putative Afu1g17120 3a,b 46.4 24.3 5.9 ElfA; elongation factor 1-c, putative Afu4g09130 4 25.8 24 10 Mannosyltransferase, putative 2 1 43 b pH 3 10 kDa 3 a 100 62 47.5 32.5 25 A B C Fig. 2. (A) SDS–PAGE analysis of GSH-binding proteins from A. fumigatus. (B) Tandem mass spectrometry identified peptide SVDVVEEYLQDR which was specific to the sequence of A. fumigatus ElfB (CADRE I.D: Afu8g00580) (C) 2D-PAGE analysis of GSH-binding proteins. Isoelectric focussing on Immobiline Dry strips (pH range 3–10) followed by SDS–PAGE and Coomassie brilliant blue staining. Proteins in spots 1–4 were identified by MALDIMS, one of which was ElfA (CADRE I.D: Afu1g17120; Table 2). ElfB was not detectable following 2D-analysis. S. Carberry et al. / Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 1101 ARTICLE IN PRESS MAK16. Both ElfA and ElfB appear in the EF1c-like clade of GSTs showing homology to EF1c, both clearly distinct from the Ure2 and MAK16 clades. ElfA and EAA47645 (56% similarity) appear to be orthologues, as do ElfB and Q00717 (59% similarity), confirming that these proteins contain GST-like domains (Fig. 4). Discussion Here we present data on the identification of 54 intracellular proteins from the pathogenic fungus A. fumigatus using a combined 2D-PAGE/sub-proteomic/MALDI-MS approach. In addition, conclusive evidence is presented for the first time that a eukaryotic elongation factor 1Bc protein of molecular mass 20 kDa exhibits glutathione transferase activity. Archer and Dyer [18] have noted that proteomics will play an important role in future investigations into the biology and pathogenicity of A. fumigatus. Yet, it is quite surprising that reliable methods for the extraction of proteins from filamentous fungi did not emerge until the early 2000s. Since then, we, and others, have reported essentially similar strategies for mycelial lysis involving vigorous physical disruption (bead-beating [19], grinding in liquid N 2 [7,20], sonication or the use of French press technology [21]). Despite the availability of efficient approaches to cell wall disruption, it is only recently that a strategy for the identification of intracellular proteins from A. fumigatus has been forthcoming [22]. Indeed, 28 of the 50 proteins identified in our study have not previously been reported in A. fumigatus. The percentage sequence coverage range (9.4–50.5%) obtained here for MALDI-MS identification of A. fumigatus proteins is consistent with that recently reported by Medina et al. for the identification of rutininduced secreted proteins from A. flavus (7–43% sequence coverage) and Grinyer et al. who mapped mitochondrial proteins from Trichoderma harizium (8–54% sequence coverage) [3,5]. Many of the proteins identified were shown to be present in several spots following 2D-PAGE, including Fig. 4. Phylogenetic analysis of ElfA and B from Aspergillus fumigatus along with 28 predicted GST proteins from a range of fungal species. It is clear that both ElfA and B cluster in the EF1Bc-like group of GST-like proteins. EF1Bc-like proteins comprise: EAA47645 (Magnaporthe grisea); Q00717 (Aspergillus nidulans); EAA77260 (Gibberella zeae); EAA55279 (Magnaporthe grisea); NP_011717 (Saccharomyces cerevisiae); KLLA0D11594G (Kluyveromyces lactis); KLLA0F26092G (Kluyveromyces lactis); AAA16892 (S. cerevisiae); NP_012842 (S. cerevisiae); YALI0C24420G (Yarrowia lipolytica); YALI0B12562G (Yarrowia lipolytica); DEHA0D17369G (Debaryomyces hansenii); EAA57903 (A. nidulans); XP_323127 (Neurospora crassa); EAA64302 (A. nidulans); NP_587885 (Schizsaccharomyces pombe). Ure 2 proteins comprise: KLLA0D19624G (K. lactis); NP_014170 (S. cerevisiae); CAGL0J07392G (Candida glabrata); DEHA0F08635G (D. hansenii); YALI0C03069G (Yarrowia lipolytica). MAK 16 proteins comprise: NP_009377(S. cerevisiae); NP_586198(Encephalitozoon cuniculi); CAGL0G06248G (C. glabrata); KLLA0A04037G (K. lactis); DEHA0F02112G (D. hansenii); YALI0C08052G (Yarrowia lipolytica). 1102 S. Carberry et al. / Biochemical and Biophysical Research Communications 341 (2006) 1096–1104 ARTICLE IN PRESS HSP70 chaperone HscA, alanine aminotransferase, thiamine biosynthesis protein, UDP-glucose 4 epimerase, catalase, and glyceraldehyde-3-phosphate dehydrogenase. Glyceraldehyde-3-phosphate dehydrogenase was also identified from several protein spots in Candida albicans [23]. The expression of three previously unidentified proteins in A. fumigatus (pyruvate decarboxylase, glyceraldehyde3-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) has been previously reported to be upregulated in C. albicans in response to the presence of the antifungal azole agent, ketoconazole [23]. Interestingly, pyruvate decarboxylase, along with two other proteins (HSP90 and enolase), was identified as a fungal antigen expressed during invasive aspergillosis [24]. A number of identified proteins have been shown to be located in the cell wall of other fungi, included HSP90 and HSP70 in C. albicans [25], enolase and glyceraldehyde-3-phosphate dehydrogenase [26]. Others, such as catalase, ATP synthase (subunit b), and phosphoglycerate mutase were recently shown to be secreted by A. flavus [5]. Overall, it is clear that a more systematic and focused approach is required to further elucidate the functional rationale governing the unexpected localisation, and dynamic nature, of these fungal proteins which were previously thought to be predominantly of cytoplasmic origin. In an attempt to move towards a consistent nomenclature for A. fumigatus proteins, the annotation described by Mabey et al. and employed on the CADRE website at http://www.cadre.man.ac.uk has been adopted [9]. The initial availability of the unannotated genome of the A. fumigatus [http://www.tigr.org] and subsequently that of thein silico annotated version at http://www.cadre.man. ac.uk have been critical to the success of the work presented here with respect to protein identification. Moreover, the availability of the A. nidulans genome sequence [http://www.broad.mit.edu/annotation/fungi/aspergillus/] has also been of great assistance in protein annotation and identification. However, it should be noted that proteomic data also serve to validate the predictive bioinformatic tools (e.g., Artemis software [27] and GlimmerM [28,29]) used, in part, to predict intron–exon splice sites in fungal genes. Consequently, at least 29 peptides from 22 proteins have been identified as arising from mRNA formed as a consequence of intron excision. Notably, 22 of 50 proteins identified in this work derive from intron-containing genes in A. fumigatus and represent biochemical validation of bioinformatic gene prediction data. The eukaryotic elongation factor 1B complex (eEF1B) plays a key role in the elongation step of protein synthesis and is comprised of three subunits termed a,b, and c,in order of increasing molecular mass [30]. The identification of an EF1Bcprotein (ElfA) in A. fumigatus, which exhibits GST activity, significantly extends the role of S-transferase activity beyond glutathionyl conjugation to xenobiotic compounds and advances experimental and bioinformatic evidence for the presence of GST-like domains in eEF1Bc subunits [11,30,31]. In fact, a recent study has demonstrated an S-transferase activity in the eEF1Bcsubunit of the parasite Crithidia fasciculata, capable of utilising trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine), but not glutathione, as a substrate for CDNB conjugation [32].No glutathione peroxidase activity was detectable in ElfA preparations. Interestingly, Vickers et al. [12] have shown that the intact eEF1B complex is necessary for peroxidase activity found in the eEF1Bcsubunit of Leishmania major eEF1B. Microarray analysis of A. fumigatus gene expression has revealed that ElfA (termed elongation factor-1c) expression is up-regulated twofold within 1 h of a temperature shift (30–48 C) which suggests a role for ElfA in protecting against heat shock [33]. Five clusters of GST-like proteins were previously identified, three of which included EF1c, Ure2, and MAK16 [11]. Recreating a similar phylogenetic tree revealed that both ElfA and ElfB from A. fumigatus clustered and shared homology to GST-like proteins with EF1csimilarity. These data suggest that both ElfA and ElfB are eEF1Bc proteins containing GST-like domain and that this GSTlike domain is conserved in various eukaryotes. These genes could possibly have a wide range of functions including protection from oxidative stress, controlling translation in response to oxidative stress [34] or regulating protein folding in a chaperone like manner [35]. Unlike the high affinity of ElfA for GSH-affinity media, the eEF1Bcsubunits/eEF1B complexes identified by Kobayashi et al. and Kamiie et al. did not exhibit affinity for glutathioneaffinity media, although eEF1Bcsubunits purified from rice and Bombyx mori were amenable to purification by GSH-affinity chromatography [36,37] and GST activity was present in the purified EF1B complex preparations. Identification of the complete enzymatic and functional repertoire of ElfA will have to await eEF1B complex studies in A. fumigatus. In summary, our findings represent one of the first detailed identifications of the most abundant intracellular proteins of A. fumigatus, identifies the importance of subproteomic studies in fungal biology, as exemplified by the identification and characterisation of the eEF1Bcprotein, ElfA and B, and will facilitate future studies into the dynamic proteome of A. fumigatus. Acknowledgments This work was funded by the Irish Higher Education Authority through the Programme for Research in Third Level Institutions (PRTLI) Scheme-Cycle 3. Preliminary sequence data were obtained from The Institute for Genomic Research website at http://www.tigr.org. 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