Chapter CHAPTER 2: IDENTIFICATION OF Curvularia lunata ALLERGENS 43 Chapter 2.1 INTRODUCTION Amongst the various fungal aero allergens found in the Singapore environment, the genus Curvularia (as explained earlier) was found to be a fungus of great importance. Several studies carried out previously on Curvularia suggest it to be an important allergenic fungus of medical importance (Gupta et al., 1999; Gupta et al., 2000; Chew et al., 2000; Asero and Botazzi, 2001; Schroeder et al., 2002; Bisht et al., 2002; Green et al., 2003; Calhoun, 2004). Although much literature has described Curvularia to be an important fungus, very few reports (two studies) have actually tried to isolate and characterize individually, the underlying allergenic components of Curvularia in detail. The first study describes the amino terminal sequence (GLTQKSAPWGLGADTIVAVELDSY) of a glycoprotein allergen (Cur l 1) showing similarity in sequence and activity with serine proteases (Gupta et al., 2004). The latest study on Curvularia lunata describes cloning, expression and characterization of a 48 kDa recombinant enolase allergen, named as Cur l (Sharma et al., 2006). The first and foremost step for generating a total recombinant allergen repertoire from C.lunata, high throughput identification of allergens is required. Various genomics and proteomics methods for rapid and high throughput identification of proteins can be utilized for this purpose. On the genomics side, methods such as whole genome shortgun sequencing (Venter et al., 1992), genome microarrays (Liu et al., 2006) and Expressed Sequence Tagging (EST) (Adams et al., 1991) are commonly used. On the proteomics side, protein microarrays (Petrik, 2006), high performance liquid chromatography (HPLC), Surface-Enhanced Laser Desorption Ionization - Time Of Flight (SELDI-TOF) (Elek and Lapis, 2006), Two Dimensional Sodium Do-decyl 44 Chapter Sulfate Poly Acrylamide Gel Electrophoresis (2D SDS PAGE) followed by mass spectrometry (Lee, 2001), Isotope-Coded Affinity Tags (ICAT) (Allison et al., 2006), methods are being used. A combinatorial method exploiting more than one of the above mentioned techniques can prove more useful for better allergen identification and isolation. 2.1.1 Expressed Sequence Tagging for rapid allergen transcript identification The sequence tagging approach is one of the most effective approaches towards large scale expressed proteome profiling. In this technology, a library of directionally cloned partial DNA sequences from randomly selected cDNA clones (termed as ESTs) is generated. These clones are then sequenced and the generated sequences are aligned with the available known nucleotide and protein databases for putative matches. Single pass sequencing of these clones creates high throughput expressed proteome sequences for a particular organism spanning various regions of the proteome (Adams et al., 1991; 1993). This approach has been successfully used in discovering novel expressed genes in many cell/tissue/organ types (Gong et al., 1994; Gross et al., 2001; Jia et al., 2001; Escribano and Coca-Prados, 2002). It also provides the profile and abundance of the expressed genes in the source cDNA library (Adams et al., 1995). The cDNA library being representative of the expressed genes, ESTs provide a powerful technique for indirect genome identification. The deduced amino acid sequences from the cDNAs corresponding majority of the mRNAs help in elucidating the primary structure of the expressed proteins (Yamamoto and Sasaki, 1997). Moreover, identification of differentially expressed genes between two states (e.g. normal and 45 Chapter diseased, early and late, juvenile or adult) is possible by using this technique (Schmitt et al., 1999). To date, there are over 250 reported publications which used ESTs in identification of fungal genes. The majority of them concentrate on identification of differentially expressed genes in pathogenic fungi in order to find out possible pathogenesis related genes (Mammadov et al., 2005; Sexton et al., 2006); identification of novel enzymes or other biochemicals for various biotechnological interests (Morrita et al., 2006; Shibuya et al., 2006). Recently, ESTs were used for identification of fungal allergens from Beauveria bassiana (Westwood et al., 2006). EST approach by itself does not provide information about specific proteins and hence, other techniques in combination with EST are required. 2.1.2 Identification of allergens by 1D and 2D SDS PAGE followed by tandem mass spectrometry Proteomic analysis has been one of the most powerful methods for identification of novel proteins as well as in studying protein expression in organisms under different environmental conditions (Elinbaum et al., 2002). Along with the transcriptome analysis, it reveals post-translational regulation and modifications of extracellular proteins (Oda et al., 2006). In this approach, a 1D or 2D SDS PAGE of the total protein is used to generate a proteomic profile of the organism. These bands/spots as generated by the protein gels are cut, trypsinized and sent for tandem mass spectrometric identifications by Matrix Assisted Laser Desorption/Ionization – Time Of Flight (MALDI-TOF) and Mass Spectrometry (MS-MS). Mass spectrometry generates peptide mass fingerprints and peptide fragment ion data which are then used 46 Chapter to search for protein candidates in the NCBI database as well as other locally available or generated databases. Proteomic identification of the fungal proteins is been used extensively for various purposes (Brosson et al., 2006; Carberry et al., 2006; Kalari et al, 2006; Oda et al., 2006). Proteomics method of 2D SDS PAGE followed by mass spectrometry was used for identification of fungal allergens by a group of researchers from Taipei Veterans General Hospital, Taiwan. Not so long ago, a serine protease allergen (Rho m 2) from Rhodotorula mucilaginosa (Chou et al., 2005), enolase from Penicillium citrinum and Aspergillus fumigatus (Lai et al., 2002) and a 33kDa heatlabile alkaline serine protease-like allergen from P. citrinum (Shen et al., 1997) were identified using this method. 2.1.3 Identification of allergens by western blotting Although, the two methods mentioned earlier are robust and are well known for high throughput identification of proteins, they identify the underlying genes or proteins by homology alignments (BLASTX in case of ESTs and BLASTP in case of Proteomics). Homology alignment may give a clue about the identity of the protein but cannot be used as confirmative to prove a protein to be an allergen. Hence, immunochemistry is commonly combined with 1D or 2D SDS PAGE. As the allergens are IgE binding proteins, they are detected by using patients` sera (containing IgEs) which specifically bind to the respective allergenic proteins separated on a 1D of 2D protein gel. The bands/spots are then cut, digested with trypsin to generate random peptides and are sent for mass spectrometric identification. Western blotting has been commonly used for identification of molecular weights of the allergenic proteins. Recently, Barbieri et al. (2005) used this technique to identify the allergenic components from the fungus 47 Chapter Metarhizium anisopliae. Although this technique can identify the putatively allergenic components in total protein extract, protein identification is not possible. Hence, in the present study, we combined all the three techniques: Western blotting, Proteomics and ESTs were combined to obtain the confirmed identity of the allergenic components. Firstly, putative allergens were obtained by generating ESTs. Total protein extracts from Curvularia were run on a 1D SDS PAGE and the components were separated by their molecular weights. These were then transferred to a nitrocellulose membrane and western blotting (using allergic patients` sera) was carried out in order to identify the allergenic components. Simultaneously, 2D SDS PAGE was run to separate these proteins by molecular weights as well as isoelectric point (PI). Then, these bands/spots (from the corresponding 1D and 2D gels) were cut and sent for mass spectrometric identification. The generated peptides were then compared with the in-house generated ESTs as well as with the global protein databases in order to establish the allergen protein identity as well as to know the amino acid/nucleotide sequence. 2.2 MATERIALS AND METHODS 2.2.1 Expressed Sequence Tagging of C. lunata for allergen identification 2.2.1.1 Fungal culture and raw material A pure culture of Curvularia lunata (obtained in-house previously) was cultured in Erlenmeyer flasks (1L) containing 200 ml of 3% Sabouraud`s liquid medium (Oxoid) at 28˚C for 12-15 days until sufficient sporulation occurred. This method was preferred as it was known to yield a highly potent and allergenic extract (Gupta et al., 1999). At 48 Chapter the end of the incubation period, the spore-mycelial mass (fungal mat) was collected in a 50 ml (Falcon) tubes. The mat was then washed thoroughly with distilled water to remove spent medium and was lyophilized overnight. 2.2.1.2 Bacterial strains The following bacterial strains (E.coli) were used for the preparation of C.lunata cDNA library and ESTs: XL1-Blue [N1] ∆(mcrA) 183 ∆(mcrCB-hsdSMR-mrr)173 end A1 supE44 thi-1 recA1gyr 1A96 relA1 lac[F’proAB lacIqZ ∆M15Tn10(Tetr)] SOLRTM me14-(McrA-) ∆(mcrCB-hsdSMR-mrr)171 sbcC recB recJ uvrC umuC::Tn5(Kanr)lac gyrA96 relA1 thi-1 endA1 λR [F’ proAB lacIqZ ∆m15)c Su- BL-21 (DE3) F-ompThsdSB(r-Bm-B)galdcm(DE3)pLysS ExAsist ® interference-resistant helper phage (~1.0 x 1010 pfu/ml). Single-strand size is 7.3kb [co-migrates with ~5kb of double-strand linear DNA on 1% (w/v) agar]. 2.2.1.3 Curvularia lunata mRNA extraction One gm of the dried fungal mat was powdered with liquid nitrogen. RNA extraction was performed using RNeasy mini kit (QIAGEN) as per manufacturer’s protocol. The eluted total RNA was used for further isolation of mRNA using Poly (A) Quick mRNA isolation kit (Stratagene) as per manufacturer’s protocol. 2.2.1.4 Curvularia lunata λZAPII cDNA library The cDNA library of the extracted C.lunata mRNA (as mentioned above) was prepared (with the help of Ms. Wong Fei Ling) using uni-ZAP (Stratagene) XR vector system (Figure 2.1) as per manufacturer’s protocols. A primary library of 105 phage 49 Chapter was amplified to generate a higher titer of 109 pfu. Inserts of lengths between 0.5 and 2.5 kb were found on preliminary survey. 2.2.1.5 Curvularia lunata EST clones Exassist helper phage was used for pBluescript phagemid excision (Figure 2.2) from λZAP using the host E.coli, XL1-MRF strain. The single-stranded phagemid was converted to the double-stranded one using SOLR E.coli strain. Isolated individual colonies with the phagemid with (cloned cDNA) insert were subcultured onto plates containing 2% Luria Bertani (LB)-agar (DIFCO) and allowed to grow. A total of 3,000 colonies were picked from the plates and kept as glycerol stock (15% glycerol) at -80˚C till further use. These colonies were then inoculated in 5ml of 2% LB liquid medium and cultured for 16-20 hours at 37˚C. Plasmid extraction was performed using QIAprep kit (QIAGEN). These plasmids were then stored at -20˚C till use. The inserts from the extracted plasmids were then sequenced from the 5` end. 2.2.1.6 Sequencing of the inserts Sequencing of the inserts was carried out using ABI PrismTM dye terminator cycle sequencing ready reaction kit (Applied Biosystems). Each 20µl PCR reaction involved a mixture containing 4µl of BigDyeTM, 2.5X sequencing buffer (Applied Biosystems), 250-500ng template DNA, 3.2pmol T3 primer and sterile double distilled water to make up the volume. Thermal cycling steps (30 cycles) were as follows: denaturation – 96˚C for 30s, annealing - 50˚C for 15s and extension – 60˚C for min. Sequencing was carried out using PTC-100TM thermal Controller (MJ Research). 50 Chapter Figure 2.1: Map of Uni-ZAP XR insertion vector Figure 2.2: Map of pBluescript SK (+/-) phagemid 51 Chapter Precipitation of the PCR product after sequencing was carried out using 2µl of 3M sodium acetate, pH 4.6, 50µl of 95% ethanol, 2µl of 125mM EDTA and 10µl of sterile distilled water. The mixture was centrifuged at 13,000g for 20min after incubation at 20˚C for 30 min. The pellet was washed with 500µl of 70% ethanol and air dried before loading it on a sequencer. Purified products were subjected to ABI Prism (ABI 3100) automated DNA sequencer (Applied Biosystems). The sequencing services were provided by DNA Sequencing Laboratory (DSL), Department of Biological Sciences, National University of Singapore. 2.2.1.7 Sequence analyses of the inserts using various softwares The electrophoreograms (.ABI files) for various sequenced ESTs were analyzed using the Phred-Phrap-Cross_Match software package program (Version 10.0) by CodonCode Corporation (USA). This software package helps in analyzing the EST electrophoreogram sequences for base calling, sequence assembly and comparisons by classifying the sequences into various contigs. Firstly, the sequences were subjected to Phred (Ewing and Green, 1998; Ewing et al., 1998; Green and Ewing © 1993-1996) for reading the DNA sequencing trace files, base calling and assigning sequence quality value to each called base. The quality value is an error probability (logtransformed) given by the formula; Q= -10 log10 (Pe), where Q is the quality value and Pe is the error probability of a particular called base. PHD2FASTA software then extracted information from the Phred (.phd) files and created input files for next program. Briefly, this software transformed all the sequences from .ABI files to .FASTA format. Further, these transformed sequences were analyzed using 52 Chapter higher sensitivities as compared to those with Coomassie stain. This could be the reason why bands corresponding to enolase were not obtained. The reason why enolase cDNA did not showed up in the ESTs could be due to the longer length of the enolase cDNA (approx. 2.5Kb) as compared to others which were less than 1.5Kb. Allergens such as thioredoxin and alcohol dehydrogenase which were previously obtained via mass spectrometry of C. lunata 1D gel were not obtained on the 2D proteome. Spots identifying MnSOD and cyclophilin were also obtained previously on the 1D proteome. Spots corresponding to hsp70 as well as calcium binding protein allergens were previously not observed on the 1D proteome. From 1D and 2D SDS PAGE), a total of different allergen types were obtained. All of these types (except enolase) were already obtained through the EST strategy. This suggests that the allergens obtained by ESTs as well as proteomics are almost the same, except that ESTs may help in identifying more types (e.g. isoforms) as compared to those obtained by proteomics. Furthermore, peptide coverage study was done to understand the percentage of peptides for the allergens that were identified by mass spectrometry. The higher the coverage, the more confirmed would be the identity of the protein. Peptide coverage for the allergens ranged from around 14% to 92%. The highest coverage was obtained for SOD allergen (92%) followed by cyclophilin (76%) coverage. Thioredoxin, calcium binding protein and alcohol dehydrogenase had peptide coverage of 44%, 27% and 26% respectively. The lowest peptide coverage was found to be that for hsp70 (14%). Higher peptide coverage of the allergens could be exploited in future for diagnosis for a particular allergen type in the total extract (Table 2.7). Hence, the combined method of 1D as well as 2D SDS PAGE together 81 Chapter Table 2.6: Tandem mass spectrometric identification of the spots as obtained on the C.lunata 2D SDS PAGE MW: Molecular weight in kilo daltons (kDa), (E): Expected, (O): Observed Score: Protein scores ≥78 (p[...]... 37kD 25 kD 15kD 10kD CL 52 71 Chapter 2 Table 2. 4: Distribution of the IgE binding frequency (%) for the detected allergenic components MW: Molecular weight 1 15 2 18 -20 3 22 4 25 7 37 8 42 9 45 10 48-50 Curvularia inequalis 0 8 8 16 83 0 8 8 8 8 Curvularia pallescens 0 50 42 75 75 25 25 42 0 33 Curvularia fallax 0 42 33 42 75 33 25 0 25 25 Curvularia brachyspora 58 75 42 75 83 33 42 58 58 58 Curvularia. .. Curvularia lunata 8 75 42 58 92 25 33 0 58 8 Cladosporium cladosporoides 0 58 58 75 92 25 42 58 50 42 Cladosporium herbarum 8 92 75 83 58 42 25 33 50 50 Penicillium notatum 16 58 42 58 42 8 8 16 16 33 IgE binding frequency (%) Components MW (kDa) 72 5 6 28 -30 33 Chapter 2 Figure 2. 6: C lunata protein bands corresponding to IgE binding bands (from 1D western blots) excised from the 15% SDS PAGE for tandem... Total No of No Unigenes* 0100 079 020 0 1 42 181 0300 0400 22 9 0500 28 1 0600 319 350 0700 0800 408 451 0900 1000 494 1100 508 120 0 545 603 1300 1400 644 1500 679 1600 709 No of contigs 009 020 033 041 050 066 081 086 100 114 135 150 159 170 184 20 1 ESTs represented by all contigs 021 058 119 171 21 9 28 1 350 3 92 449 506 5 92 655 697 756 821 891 % Redundancy 12. 00 19.00 28 .66 32. 50 33.80 35.83 38. 42 38 .25 38.77... Hence, bands between the molecular weight range of 15-50kDa were sent for mass spectrometric analysis A total of 7 bands were cut (Figure 2. 6) Out of the 7 bands cut, 5 bands corresponded to the IgE binding bands from C lunata 1D western blots and 2 bands 70 Chapter 2 Figure 2. 5: Western blot analysis using Curvularia atopic patients` sera From left to the right of each blot - Lane 1: Marker, Lane 2: Curvularia. .. (Schizosaccharomyces pombe) 29 .5 26 5 18/35 CL1754 1.91E+03 C2 Probable ribosomal protein Rps8bp (Neurospora crassa) 21 20 2 17/35 CL1677 1 .23 E+03 75 Chapter 2 (Asp f 6) from A fumigatus while band B5 showed identity to cyclophilin allergen from D lanata The molecular weights of the bands and that of the corresponding allergens were matching, suggesting confirmed identity (Table 2. 5) The identities of the bands were further... would aid in finding the probable isoforms/variants of C lunata proteins Moreover, allergens which were previously not observed in 1D SDS PAGE could also be identified by this method The 2D proteome could also be used for standardization and quality control 2. 3 .2. 2 Study of the total proteome of C lunata using 2D SDS PAGE To study the expressed proteins of C lunata, 2D SDS PAGE was carried out using... obtained peptides and hence the excised bands/spots on 1D as well as 2D SDS PAGE 2. 3 RESULTS AND DISCUSSION 2. 3.1 Expressed Sequence Tagging of C lunata for allergen identification 2. 3.1.1 Curvularia lunata cDNA library A cDNA library represents information of the encoded mRNA giving a brief picture of the pattern of expression for the organism/state/condition under study The cDNA library of C .lunata was... identification M: Broad range protein marker (Biorad) Bands C1 and C2 were bands corresponding to non-IgE binding bands and were used to validate and confirm mass spectrometry results 50KDa B1 35KDa C1 B2 25 KDa B3 B4 C2 B5 15KDa 10KDa M C lunata extract 73 Chapter 2 were used as a negative control These bands were not IgE binding bands and were used to test the validity of the mass-spectrometry results Furthermore,... to allergens was also included) 2. 2 .2 Identification of allergens by Proteomics and Western Blots 2. 2 .2. 1 Total protein extraction of the cultured fungus Total protein extraction was carried out using trichloroacetic acid (TCA) / acetone method 1g of dried fungal mat was powdered with liquid nitrogen 10 ml of TCA extraction solution (10% TCA, 0.007% DTT) was added to the powder and incubated at -20 ˚C... Proteomics 2. 3 .2. 1 Identification of the allergens using 1D western blots The resolved protein components of various Curvularia species were immobilized onto a nitrocellulose membrane followed by incubation with a total of 12 fungal atopic patients` sera reactive to Curvularia Protein components from 2 species of Cladosporium (Cladosporium herbarum and Cladosporium cladosporioides) and from Penicillium . sequence. 2. 2 MATERIALS AND METHODS 2. 2.1 Expressed Sequence Tagging of C. lunata for allergen identification 2. 2.1.1 Fungal culture and raw material A pure culture of Curvularia lunata (obtained. 009 021 12. 00 First 020 0 020 0 1 42 020 058 19.00 First0300 0300 181 033 119 28 .66 First0400 0400 22 9 041 171 32. 50 First0500 0500 28 1 050 21 9 33.80 First0600 0600 319 066 28 1. sequences showing similarity to allergens was also included). 2. 2 .2 Identification of allergens by Proteomics and Western Blots 2. 2 .2. 1 Total protein extraction of the cultured fungus Total