Proteome Analysis of Pitcher Fluid of the Carnivorous Plant

Trudel , J.; Grenier , J.; Potvin , C.; Asselin , A. Several thaumatin-like proteins bind to β-1,3-glucans Plant Physiol. 1998 118 1431 1438. [Crossr...
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Proteome Analysis of Pitcher Fluid of the Carnivorous Plant Nepenthes alata Naoya Hatano†,‡ and Tatsuro Hamada*,§ Harima Institute at Spring-8, RIKEN, 1-1-1 Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan, and Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, 1-308 Suematsu, Nonoichi-machi, Ishikawa-gun, Ishikawa 921-8836, Japan Received August 29, 2007

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of β-D-xylosidase, β-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated “pathogenesisrelated proteins”. These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth. Keywords: Carnivorous plants • Nepenthes • proteome • de novo sequencing • PR protein

Introduction To support their growth, higher plants usually absorb nutrients (nitrogen, phosphorus, potassium, and other minerals) from the soil via their roots. However, carnivorous plants growing in nutrient-poor soils have special organs to capture insects, to digest them, and to absorb their nutrients, as reported by Darwin in Insectivorous Plants.1 There are four types of traps in carnivorous plants: adhesive traps in Drosera, Drosophyllum, and Pinguicula; suction traps in Utricularia and Genlisea; snap traps in Dionaea and Aldrovanda; and pitfall traps in Nepenthes, Sarracenia, Heliamphora, Darlingtonia, and Cephalotus. Nepenthes, which contains tropical pitcher plants, includes more than 100 species, with the greatest diversity in Borneo and Sumatra. Scattered populations are also found in India, Sri Lanka, Australia, New Caledonia, Madagascar, the Seychelles, and some other places. The “pitcher” bud forms at the tip of the leaf and gradually expands to form a pot-shaped trap. The rim of the trap, called the peristome, is slippery and often quite colorful to attract insects. The inside wall is also slippery and waxy to prevent escape. The bottom of the trap contains a fluid, which has long been thought to include digestive enzymes. * To whom correspondence should be addressed: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichimachi, Ishikawa 921-8836, Japan. Telephone: +81-76-227-7507. Fax: +8176-227-7557. E-mail: [email protected]. † Harima Institute at Spring-8, RIKEN. ‡ Present address: Rare Sugar Research Center, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa 761-0793, Japan. § Ishikawa Prefectural University. 10.1021/pr700566d CCC: $40.75

 2008 American Chemical Society

The history of the study of these enzymes has been reviewed in detail by Frazier.2 Briefly, an enzyme was first purified by Steckelberg et al. using Ecteola cellulose chromatography; its highest proteolytic activity was observed at pH 2.2.3 Nakayama and Amagase also purified an enzyme using gel filtration and DEAE-Sephadex column chromatography.4 They showed that the enzyme activity is stable up to 60 °C and appears to cleave peptides preferentially at the carboxyl side of the aspartic acid residue.4 They proposed the name “nepenthesin” for the protease. Recently, Athauda et al. purified the enzymes nepenthesins I and II from Nepenthes distillatoria and determined their partial internal sequences.5 They first cloned the cDNAs and deduced the complete amino acid sequences of the enzymes from Nepenthes gracilis.5 There is no doubt that these proteases are major constituents of the digestive enzymes of Nepenthes. However, it has been reported that the pitcher fluid contains other enzyme activities. Tökés et al. showed that the secretions of the unopened pitcher of Nepenthes macfarlanei have lipase activity.6 Higashi et al. found strong activities of both acid and alkaline phosphatases, phosphoamidase, esterase C4, and esterase C8 in the pitcher juice of Nepenthes hybrida.7 Gel electrophoresis analyses also indicated that the pitcher fluid potentially contains several other proteins. Amagase used polyacrylamide gel electrophoresis (PAGE) to resolve the proteins of the pitcher fluid, identifying four separate bands in the fluid of the open pitcher.8 The identities of the protein bands remain unknown. Eilenberg et al. resolved the pitcher fluid protein by sodium dodecyl sulfate (SDS)–PAGE and visualized the proteins with silver staining.9 They showed that one of the major protein bands had chitinase activity.9 These The Journal of Proteome Research 2008, 7, 809–816 809 Published on Web 01/10/2008

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studies indicate the existence of other proteins in pitcher fluid. To identify all the proteins contained in pitcher fluid, we performed proteomic analysis using mass spectrometry. Complete sequence data are not available for Nepenthes. However, the growing protein sequence databases and advanced bioinformatics tools allow the identification of proteins from organisms whose genomes have not been completely sequenced. PEAKS is a de novo sequencing software package that deduces amino acid sequences from tandem mass spectrometry (MS/MS) spectra.10 Using these sequences, fasts searches of a protein database can identify homologous proteins.11,12 We tried to identify all the key proteins to answer a century-old question.

Experimental Procedures Sample Preparation. Nepenthes alata plants were grown in a Wardian case under available light at 20–25 °C. The trap liquid was collected from closed pitchers or newly opened pitchers (within 24 h of lid opening). Approximately 5–10 mL of fluid could be collected from a single pitcher. The pitcher fluid samples were filtered through a 0.22 µm polyvinylidene difluoride (PVDF) membrane and stored at 4 °C until use. To concentrate the proteins present in these solutions, the samples were individually subjected to ultrafiltration [Centricon YM10, molecular weight cutoff (MWCO) of 10000; Millipore, Eschborn, Germany]. They were mixed with 1/5 volume of 5× SDS loading buffer and boiled for 5 min. The samples were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) or MS-compatible silver stain (ProteoSilver; SigmaAldrich Co., St. Louis, MO), according to the instruction manual. In-Gel Digestion. In-gel digestion with trypsin was performed basically according to a published method.13,14 The protein bands were excised from the gel and diced into small pieces. The gel pieces were destained by rinsing them twice in 30% acetonitrile containing 25 mM NH4HCO3 for 30 min each. After they were shrunk by dehydration in 100% acetonitrile, which was then removed, the gel pieces were dried naturally at room temperature for 30 min. The disulfide bonds of any cysteines were reduced by incubation with 10 mM dithiothreitol in 25 mM NH4HCO3 at 56 °C for 1 h and alkylated with 55 mM iodoacetamide in 25 mM NH4HCO3 at room temperature for 45 min in the dark. After the gel pieces had been washed with 25 mM NH4HCO3, they were dehydrated twice with 50% acetonitrile containing 25 mM NH4HCO3 for 30 min each and with 100% acetonitrile once for 5 min. They were then dried naturally at room temperature for 30 min and rehydrated with 20 µL of trypsin solution (10 ng/µL in 25 mM NH4HCO3; Promega Corp., Madison, WI) on ice for 30 min. After the excess solution had been removed, digestion was performed at 37 °C overnight. The resulting peptides were extracted with 50% acetonitrile containing 0.1% trifluoroacetic acid. The extracts were dried in a vacuum concentrator and dissolved in 6 µL of 0.1% formic acid for mass spectrometric analysis. Mass Spectrometric Analysis. The digested peptides were subjected to liquid chromatography (LC)-MS/MS analysis. LC-MS/MS analysis was performed on a Q-Tof2 quadrupole/ time-of-flight (TOF) hybrid mass spectrometer (Micromass, Manchester, U.K.) interfaced with a capillary reversed-phase liquid chromatography system (Micromass CapLC system).15 A 90 min linear gradient from 5 to 45% acetonitrile in 0.1% formic acid was produced and was split in a 1:20 ratio. The gradient solution was then injected into a nano LC column 810

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Figure 1. Silver-stained SDS-PAGE of Nepenthes pitcher fluid. Pitcher fluid samples were collected from closed pitchers (lane C) or newly opened pitchers (lane O), and proteins concentrated from equal volumes of samples (130 µL) were subjected to SDS-PAGE. The seven protein bands selected for protein identification are indicated by arrows with numbers.

(PepMap C18, 75 µm × 150 mm; LC Packings, Sunnyvale, CA) at 100 nL/min. The eluted peptides were sprayed directly into the mass spectrometer. The MS/MS data were acquired with MassLynx (version 4.0; Micromass) using automatic switching between MS and MS/MS modes and converted to a single text file (containing the observed m/z values of the precursor peptide, the fragment ion m/z values, and intensity values) with ProteinLynx (version 2.0; Micromass). The files were analyzed with Mascot MS/MS Ions Search (version 2.1.6; Matrix Science Ltd., London, U.K.) to assign the obtained peptides to the NCBI nonredundant database (NCBInr 20060718; 3784285 sequences). One missed cleavage site was allowed. For de novo sequencing, the files were analyzed with PEAKS Studio (version 3.0; Bioinformatics Solutions Inc., Waterloo, ON). Using these software programs, we set the parameters as follows: parent mass error tolerance, (0.1 Da; fragment mass error tolerance, (0.1 Da; enzyme, trypsin; post-translational modifications, oxidation (Met), carbamidomethylation (Cys), and propionamidation (Cys). For protein identification, the criteria were as follows: (1) Mascot scores above the statistically significant threshold (P < 0.05) and (2) at least one top-ranked unique peptide matching the identified protein. cDNA Cloning of N. alata Nepenthesin I, Thaumatin-like Protein, and Chitinase. Total RNAs were isolated from closed whole pitchers (various sizes) and from a whole pitcher opened for 1 day, as described by Kim and Hamada.16 They were then purified with the RNeasy Mini Kit (Qiagen GmbH, Hiden, Germany) and RNase-free DNase (Qiagen GmbH) to remove any contaminating genomic DNA. Poly(A)-rich RNA was purified from the total RNAs using the PolyATtract mRNA Isolation System (Promega Corp.). First-strand cDNAs for 3′- and 5′RACE PCR were synthesized from the poly(A)-rich RNA, using the SMART RACE cDNA Amplification Kit (Clontech Laboratories, Palo Alto, CA). To clone the N. alata nepenthesin I (NaNEP1) and thaumatin-like protein (NaTLP1) cDNAs, degenerate primers for 3′-RACE PCR were designed on the basis of the amino acid sequence of N. gracilis nepenthesin I (protein ID BAD07474.1) and the amino acid sequences of the TOF-MS fragments, respectively (Figure 2). The primer sequences were 5′-CA(A/G)TG(T/C)CA(A/G)CCITG(T/C)ACICA(A/G)TG(T/C)TT(T/C)AA(T/C)CA-3′ and 5′-GGIGGITG(T/C)AA(T/C)AA(T/ C)CCITG(T/C)ACIGTITT(T/C)AA(A/G)AC-3′ to amplify the 3′ends of the NaNEP1 and NaTLP1 cDNAs, respectively. The 3′-

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Figure 2. Amino acid sequences of N. alata nepenthesin I (NaNEP1) (A) and thaumatin-like protein (NaTLP1a) (B) deduced from cloned cDNA sequences (GenBank accession numbers AB266803 and AB267384, respectively). Peptides matched to these sequences (ions score of >25) by Mascot search are shown in bold. The regions corresponding to the degenerate primers used for 3′-RACE PCR are boxed.

untranslated region (UTR) sequences of the 3′-RACE PCR clones were used to amplify the full coding sequences of the NaNEP1 and NaTLP1 cDNAs by 5′-RACE PCR. To amplify the partial sequence of the N. alata chitinase (NaCHIT1) cDNA, degenerate primers for RT-PCR were designed on the basis of the conserved amino acid sequences of several plant class IV chitinases (Figure 4). The primer sequences were 5′-CTCA(A/ G)AA(T/C)TG(T/C)GG(T/G)TG(T/C)GC(T/C)GC(A/G)AA(T/ C)CT(C/G/A)TGTTG-3′ (forward primer) and 5′-CA(A/G)AA(A/ G)TGTCC(A/G)GT(T/C)TC(A/G)TG(A/T)GTGA(A/C)(A/ G)TGAGC(A/G)AA-3′ (reverse primer). The sequence of the PCR clone was used to design an NaCHIT1-specific primer for 5′RACE PCR. The 5′-UTR sequence of the 5′-RACE PCR clone was used to amplify the full coding sequence of the NaCHIT1 cDNA by 3′-RACE PCR. Amplification was performed as described in the SMART RACE cDNA Amplification Kit instructions (Clontech Laboratories). The fragments derived from RT-PCR and 3′- and 5′RACE PCR were subcloned into the pGEM-T Easy vector (Promega Corp.). Nucleotide sequences were determined with an ABI PRISM 3100 DNA Analyzer (Applied Biosystems, Foster City, CA).

Results and Discussion SDS-PAGE of Nepenthes Pitcher Fluid. The genus Nepenthes is comprised of carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. To analyze all the secreted enzymes in the pitcher, we performed a proteomic analysis of the pitcher fluid. Pitcher fluids were collected from closed pitchers or newly opened pitchers to prevent contamination with the structural proteins of insects. Both solutions were strongly acidic (pH 4). To concentrate the proteins, we tested a standard TCA precipitation procedure and compared it with the use of MWCO (10000) filters. The MWCO

filter was selected because the standard TCA precipitation procedure resulted in poor recovery of the proteins. The concentrated proteins from equal volumes of samples (130 µL) were subjected to SDS-PAGE followed by silver staining, and distinct protein bands were detected only from the sample collected from a newly opened pitcher (Figure 1). The experiment was repeated with similar results. This suggests that the pitcher fluid in closed pitchers contains little protein and that the protein levels increase dramatically after the pitcher has matured. To identify the proteins, 10 mL of the pitcher fluid from a newly opened pitcher was concentrated and analyzed with SDS-PAGE followed by CBB staining. The protein band pattern was almost identical to that obtained previously with silver staining, except that bands 2 and 3 seemed to be joined together, forming a thick protein band (data not shown). Protein Identification by Mascot. To identify the protein bands (bands 1–7 in Figure 1), we performed in-gel trypsin digestion followed by nano LC-electrospray ionization/MS/ MS analysis. The MS/MS spectra were used to search the NCBI nonredundant protein sequence database using Mascot, as described in Experimental Procedures. Because no genomic or protein data are available for N. alata, we expected to identify homologous proteins. The proteins identified (except human keratins, porcine trypsin, and bovine caseins, presumably from the MWCO filter) are summarized in Table 1. In this experiment, only one kind of peptide fragment matched each identified protein. N. gracilis nepenthesin I (NCBI ID 41016421), which was cloned by Athauda et al.,5 was identified in three samples (bands 1-3). The protein with the greatest number of matching peptides (band 2 at 65 kDa) was considered to correspond to N. alata nepenthesin I. The number of matching peptides reflects the amount of protein, because the same peptide was not selected for MS/MS analysis within a period of 200 s. Although N. alata nepenthesin I was expected to be The Journal of Proteome Research • Vol. 7, No. 2, 2008 811

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Figure 3 812

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Figure 3. Best results of amino acid sequence homology searches using fasts3 for protein band 1 (A), band 5 (B), and band 6 (C). Multiple short peptide sequences obtained by de novo sequencing were subjected to the fasts3 homology search against the UniProt Reference Clusters (UniRef100) protein database. Closely matched peptides were aligned with the amino acid sequences of Medicago varia β-xylosidase (UniProt accession number A5JTQ3) (A), Vitis riparia β-1,3-glucanase (UniProt accession number Q69D51) (B), and A. thaliana endochitinase (UniProt accession number O24658) (C). Amino acid identities are indicated with a colon and similarities with a period. Expectation (E) values provide a very accurate estimate of how often a peptide alignment score would occur by chance.

Figure 4. Comparison of the amino acid sequences of N. alata chitinase (NaCHIT1, GenBank accession number AB289807) and A. thaliana chitinase (AtCHIT, UniProt accession number O24658). Amino acid identities are indicated with asterisks. Short peptide sequences obtained by de novo sequencing are shown in bold. The regions corresponding to the degenerate primers used for cloning are boxed.

an abundant protein, only one kind of peptide sequence matched it. This can be attributed to the fact that the basic amino acids arginine and lysine, which are the cleavage sites of trypsin, are unevenly distributed in nepenthesin I (data not shown). Because the MS spectra were acquired for mass-tocharge ratios (m/z) of 400–1700, only four kinds of tryptic peptides were subjected to analysis by tandem MS/MS in this experiment. Actinidia deliciosa thaumatin-like protein (NCBI ID 71057064) was identified with two samples (bands 2 and 7). Because of the larger number of matching peptides, this protein (band 7 at 21 kDa) was deemed to correspond to N. alata thaumatin-like protein. Thaumatin and thaumatin-like proteins are pathogenesis-related (PR) proteins, the synthesis of which is normally stimulated upon the infection of plants

by pathogens.17 Because the MS/MS spectra of peptides from one protein (band 1, 70 kDa) corresponded to β-D-xylosidase of Oryza sativa and Arabidopsis thaliana (NCBI ID 62701894 and 62321271, respectively), the protein was predicted to be N. alata β-D-xylosidase. This enzyme hydrolyzes the β-1,4 bond between two xylose saccharides in xylan (polyxylose), which is one of the most important components of hemicellulose in plants. The MS/MS spectra of the peptides identified another protein (band 5, 32 kDa) as being homologous to O. sativa β-1,3-glucanase (NCBI ID 50917635). Thaumatin-like protein and β-1,3-glucanases are PR proteins (PR-5 and PR-2, respectively), which are important in the defense against plant diseases.18 The MS/MS spectra of band 4 (40 kDa) and band 6 (27 kDa) revealed no significant matches to sequences in the The Journal of Proteome Research • Vol. 7, No. 2, 2008 813

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Table 1. Summary of Proteins Identified with Mascot band

molecular mass (kDa)

1

70

2

65

3 4 5 6 7

63 40 32 27 21

no.

NCBI accession number

1 2 3 1 2 1

gil41016421 gil62701894 gil62321271 gil41016421 gil71057064 gil41016421

1

gil50917635

1 2

gil129320 gil71057064

matched peptidesa

Mascot score

protein name

taxonomy

aspartic proteinase nepenthesin I β-xylosidase, putative β-xylosidase aspartic proteinase nepenthesin I thaumatin-like protein aspartic proteinase nepenthesin I no significant match putative β-1,3-glucanase no significant match protein P21 thaumatin-like protein

N. gracilis O. sativa A. thaliana N. gracilis Ac. deliciosa N. gracilis

GPLSLPSQLDVTK (4) GQETPGEDPVTASR (2) TMIGNYEGTPCK (2) GPLSLPSQLDVTK (15) APGGCNNPCTVFK (3) GPLSLPSQLDVTK (8)

63 58 54 60 56 53

O. sativa

LLKSTTISK (1)

58

Glycine max Ac. deliciosa

TDQYCCNSGSCGPTDYSR (18) APGGCNNPCTVFK (20)

142 110

a Numbers in the parentheses are the numbers of matched peptides. They correlate well with protein abundance, because the same peptide was not selected for MS/MS within a period of 200 s. They also include the numbers of chemically modified forms such as carbamidomethyl cysteine and propionamide cysteine.

nonredundant NCBI database. We repeated the searches with the “no enzyme” parameter. In this search, MS/MS data from the protein corresponding to band 3 (63 kDa) matched N. gracilis nepenthesin II (NCBI ID 41016423). Thus, the results of protein identification with Mascot were incomplete. To identify these refractory proteins (especially bands 1 and 4-6), we used de novo sequencing of the tryptic peptides from the MS/MS data and conducted sequence homology searches with the fasts program. However, with these techniques, the digested peptides from highly abundant proteins often interfered with the identification of minor proteins. To remove them from the query sequences, we first cloned the cDNAs of the N. alata nepenthesin I and thaumatin-like protein genes. cDNA Cloning of N. alata Nepenthesin I and Thaumatinlike Protein. To isolate the N. alata nepenthesin I (NaNEP1) and thaumatin-like protein (NaTLP1) genes, degenerate primers were designed on the basis of the amino acid sequences of N. gracilis nepenthesin I (QCQPCTQCFNQ) and the peptide (GGCNNPCTVFKT) that matched Ac. deliciosa thaumatin-like protein identified with the Mascot search. Using these primers, 3′-RACE PCR was performed to clone the C-terminal regions of the genes. Gene-specific primers were designed on the basis of the DNA sequences of the correct clones. Using these primers, 5′-RACE PCR was performed to clone the full-length cDNA sequences of the NaNEP1 and NaTLP1 genes. Only one type of NaNEP1 cDNA clone (GenBank accession number AB266803) and three types of NaTLP1 clones [NaTLP1a, NaTLP1b, and NaTLP1c (GenBank accession numbers AB267384, AB267385, and AB267386, respectively)] were isolated. The fulllength cDNA of NaNEP1 was 1548 bp long, and it encodes a polypeptide of 437 amino acid residues (Figure 2A). The amino acid sequence of NaNEP1 was 94.5% identical to that of N. gracilis nepenthesin I. The MS/MS data for band 2 were used to search the NaNEP1 sequence with Mascot, which generated 20% sequence coverage of NaNEP1 (Figure 2A). The calculated molecular mass of NaNEP1 was 46 kDa. In contrast, the approximate molecular mass of NaNEP1 was estimated from SDS-PAGE to be 65 kDa under reducing conditions (Figure 1). This discrepancy was also seen in N. gracilis nepenthesin I.5 Athauda et al. suggested that the discrepancy was due to the presence of carbohydrate in nepenthesin I.5 The NaTLP1a, NaTLP1b, and NaTLP1c cDNAs were 749, 737, and 728 bp long, respectively. They encode polypeptides of 225 amino acid residues (Figure 2B) and have sequence differences that result in two amino acid substitutions (I9, T9, and I9 and K193, K193, and R193, respectively). The molecular masses of the NaTLP1s 814

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were estimated from the amino acid sequences to be approximately 24 kDa. The MS/MS data for band 7 were used to search the NaTLP1a (I9 and K193) sequence with Mascot, which generated 52% sequence coverage of NaTLP1a (Figure 2B). A BLASTP search with NaTLP1a showed a high level of identity (>80%) to nine thaumatin/osmotin-like proteins. These results suggest that band 2 and band 7 proteins are homologues of nepenthesin I and thaumatin-like protein, respectively. De Novo Sequencing with PEAKS and Homology Searches. To determine the partial amino acid sequences of the proteins corresponding to bands 1 and 4-6, de novo sequencing of the peptides recovered from the in-gel-digested proteins was performed. Before this analysis, the MS/MS data were reanalyzed against the protein sequences of N. alata nepenthesin I and thaumatin-like protein, with Mascot. The peptides that matched N. alata nepenthesin I and thaumatin-like protein were removed from further analysis. The amino acid sequences were obtained by interpreting the MS/MS data using the PEAKS Studio software, which provides the sequence of a peptide without reference to a protein sequence database. The deduced amino acid sequences (PEAKS scores of >80%) were used for sequence homology searches (fasts3) (Figure 3). The fasts3 program is especially designed for the analysis of short peptide data from mass spectrometric analysis of protein digests.11 The three proteins with the lowest E values are summarized in Table 2. A very low E value indicates a high degree of similarity between the query sequence and the matching sequence from the database. These results suggest that the proteins corresponding to bands 1, 5, and 6 are homologues of β-D-xylosidase, β-1,3-glucanase, and chitinase, respectively. Another protein (band 4) had no significant similarity to any sequence in the publicly available databases. This confirms the identifications of the proteins corresponding to bands 1 and 5 by Mascot search. β-1,3-Glucanase and endochitinase are PR proteins, like thaumatin-like protein.18 Plants secrete various hydrolytic enzymes to protect themselves when they are attacked by pathogens, such as bacteria and fungi, and these enzymes are called PR proteins. PR proteins are generally characterized as protease resistant.19,20 van Loon explained this as being necessary for them to function in the intercellular spaces in which proteolytic enzymes are abundant.21 Most thaumatin-like proteins have molecular masses of 22 kDa and are stabilized by eight disulfide bonds. Selitrennikoff suggested that the highly stabilized structure allows the proteins to be very resistant to protease degradation.22 Nepenthes distillatoria nepenthsein I also has a large number of cysteine residues (12 residues/

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Table 2. Summary of the Best Three Proteins in Homology Searches

Eb

band

UniProt ID

homologue

taxonomy

1

A5JTQ3 A5JTQ2 Q3V5Q1 Q69D51 O82673 P23431 O24658 Q06209 Q05K38

β-xylosidase β-xylosidase R-L-arabinofuranosidase β-1,3-glucanase β-1,3-glucanase glucan endo-1,3-β-glucosidase putative endochitinase basic endochtinase CHB precursor chitinase precursor

Medicago varia M. varia Raphanus sativus Vitis riparia Cichorium intybus × Cichorium endivia Nicotiana plumbaginifolia A. thaliana B. napus Brassica rapa

5

6

a This search was performed on July 10, 2007. occur by chance.

b

1.0 × 10-19 3.9 × 10-18 6.0 × 10-16 7.3 × 10-18 7.8 × 10-17 8.5 × 10-16 9.0 × 10-9 9.2 × 10-9 9.2 × 10-9

Expectation values (E) provide a very accurate estimate of how often a peptide alignment score would

Table 3. List of the Identified Proteins band

molecular mass (kDa)

protein name

putative physiological function

1 2 3 4 5 6 7

70 65 63 40 32 27 21

β-D-xylosidase nepenthesin I nepenthesin II unknown β-1,3-glucanase chitinase thaumatin-like protein

unknown (pitcher lid opening?) prey digestion prey digestion unknown antibacterial effect prey digestion/antibacterial effect antibacterial effect

molecule), which are assumed to form six disulfide bonds, as suggested by computer modeling, and are considered to contribute to the remarkable stability of the protein.5 These studies suggest that the identified proteins can remain in the pitcher fluid without digestion because of the formation of several disulfide bonds. β-1,3-Glucanase hydrolyzes β-1,3linked glucans, which are major components of the cell walls of fungal pathogens. Mauch et al. showed that they can act synergistically with chitinase to suppress the growth of the pathogen.23 Interestingly, several thaumatin-like proteins bind to β-1,3-glucans24 and have β-1,3-glucanase activity.25 The PR proteins may inhibit the proliferation of putrefying bacteria on prey undergoing digestion in the pitcher. Higashi et al. isolated bacteria in the Nepenthes pitcher.7 They proposed that the prey in the Nepenthes pitcher are also dissolved by enzymes secreted by these bacteria. The PR proteins would control the amount of symbiotic bacteria in the pitcher to ensure sufficient nutrients for plant growth. Chitinase hydrolyzes chitin, a linear polymer of β-1,4-linked N-acetylglucosamine, which is a major component of the cell walls of insects and fungi. It has been suggested that chitinase plays a double role, in both carnivory and defense against pathogenic attack. This chitinolytic activity in the pitcher fluid was demonstrated by Eilenberg et al.,9 who also cloned two types of chitinase genes from Nepenthes khasiana using PCR with degenerate oligonucleotide primers designed to the conserved amino acid sequences of plant chitinases.9 These enzymes displayed a high degree of amino acid homology to plant class I chitinases.9 However, a fasts homology search with short peptide sequences, obtained by de novo sequencing the band 6 protein, showed significant homology to A. thaliana (O24658) and Brassica napus (Q06209 and Q05K38) chitinases instead of to the N. khasiana chitinases (Table 2). Chitinases are classified into classes I, I*, II, III, and IV on the basis of structural differences.26 The A. thaliana and B. napus chitinases belong to class IV. We cloned a chitinase gene from N. alata (GenBank accession number AB289807) using degenerate primers based on the conserved amino acid sequences of several plant class IV chitinases. The deduced amino acid sequence of the cloned cDNA is shown in Figure

4. This sequence is highly homologous (57% identical, allowing for 10 gaps) to that of A. thaliana chitinase (Figure 4) and less homologous (36 and 39% identical, allowing for 40 and 60 gaps, respectively) to that of N. khasiana endochitinases (NkCHIT1 and NkCHIT2, respectively; shown in the Supporting Information). On the basis of its primary structure, our cloned chitinase belongs to class IV, which comprises a group of extracellular chitinases.26 This suggests that our newly cloned chitinase is the authentic chitinase that is secreted into the pitcher fluid. β-D-Xylosidase is an enzyme that hydrolyzes xyloglucan, xylan, arabinoxylan, and arabinan, which are components of hemicellulose in the plant cell wall. Recently, it was proposed that β-D-xylosidase has a role in cell wall metabolism and plant development.27 It is supposed that this protein is involved in the opening of the pitcher lid, because its level increases rapidly after the lid is opened. However, the precise biochemical function of this protein remains unclear.

Conclusion In this study, we detected seven proteins that exist mainly in the pitcher fluid of N. alata, using SDS-PAGE with silver staining. To characterize these proteins, we performed a proteomic analysis with mass spectrometry. We identified six proteins, three of which (β-D-xylosidase, β-1,3-glucanase, and thaumatin-like protein) are newly identified in Nepenthes pitcher fluid. They are summarized in Table 3. We also identified another Nepenthes chitinase in pitcher fluid. β-1,3Glucanase, class IV chitinase, and thaumatin-like protein are PR proteins, which presumably protect pitcher fluid containing prey from bacterial growth. β-D-Xylosidase is probably more involved in pitcher development than in its carnivory.

Supporting Information Available: Comparison of the amino acid sequences of N. alata chitinase and N. khasiana endochitinases. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Darwin, C. Insectivorous Plants; John Murray: London, 1875.

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