1 Department of Microbiology and Immunology, School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand
2 Horticulture and Food Research Institute of New Zealand, Palmerston North, New Zealand
Correspondence
Vernon K. Ward
vernon.ward{at}stonebow.otago.ac.nz
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ABSTRACT |
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INTRODUCTION |
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The tertiary structure of the family 18 glycohydrolase Serratia marcescens chitinase A (SmchiA) has been solved (Perrakis et al., 1994). The structure consists of an
/
barrel, indicative of the family 18 glycohydrolases, and an immunoglobulin-like fold containing conserved tryptophan residues, involved in binding native chitin (Uchiyama et al., 2001
). Other conserved aromatic residues in the catalytic domain are aligned with the tryptophan residues present in the immunoglobulin-like fold (PKD domain) and lead into the catalytic pocket. These aromatic residues may aid in feeding the insoluble chitin chain into the catalytic pocket and thus to the active site. Conserved aromatic residues in the catalytic site are proposed to form hydrophobic interactions with the hydrophobic faces of the alternating glucosamine units of chitin, thus producing the binding subsites 5, 3, 1, +1 and +2 (Fig. 1
). The chitin chain is fed into the binding pocket and cleavage occurs between the 1 and +1 subsites to remove dimers from the reducing end of the chitin chain (Nagy et al., 1998
; Uchiyama et al., 2001
). Endochitinases possess a more open cleft, allowing random binding of a substrate and hence the generation of random-sized products from a long-chain substrate.
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We have characterized a putative baculovirus chitinase encoded by Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) orf110 and confirmed that it encodes an active chitinase. We confirmed that it is a canonical baculovirus chitinase with a range of features similar to those of other baculovirus chitinases; however, the activity of this enzyme is dependent upon the substrate used. We showed that short-chain substrates can act as inhibitors of the enzyme and have proposed that short-chain fluorescent substrates, indicating both endo- and exochitinase activity, show activity through misloading into the active site. In contrast, this enzyme is solely an exochitinase when assayed against a long-chain chitin substrate.
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METHODS |
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Purification of native EppoNPV chitinase.
Fourth-instar E. postvittana larvae were inoculated with EppoNPV and extracted 10 days later. Approximately 30 insects were homogenized in 1 ml 50 mM Tris/HCl (pH 8·0), 1 mM EDTA, 1 mM dithiothreitol (DTT), 1 mM PMSF. The homogenate was clarified in a benchtop microfuge at 13 000 r.p.m. for 10 min. The supernatant was applied to a Mono Q HR5/5 column equilibrated with 50 mM Tris/HCl (pH 8·0), 1 mM EDTA, 1 mM DTT, 0·02 % Tween 20, and eluted with a gradient of 0400 mM NaCl over 15 ml in the same buffer. Fractions with chitinase activity were pooled and then separated in five 0·2 ml injections onto a Superose-12 HR10/30 column that was equilibrated and eluted with 50 mM Tris/HCl (pH 8·0), 1 mM EDTA, 1 mM DTT, 0·02 % Tween 20.
N-terminal sequencing of EppoNPV chitinase.
Purified EppoNPV chitinase was concentrated tenfold by using a 30 kDa MWCO Ultrafree centrifugal filter (Millipore), separated by SDS-PAGE (10 % gel) and transferred onto a PVDF membrane by using Towbin transfer buffer. The membrane was stained with modified Coomassie blue G250 stain (0·0125 g Coomassie blue, 20 ml methanol, 30 ml H2O), destained with 50 % methanol and rinsed in H2O. The band of correct size was excised and submitted to the Protein Microchemistry Facility, Department of Biochemistry, University of Otago, New Zealand, for N-terminal sequencing.
Cellular localization of EppoNPV.
PCR was used to generate three EppoNPV chitinase clones for transient expression. Each amplified product was cloned into pBluescript II SK(+), sequenced to confirm product fidelity and then subcloned into the expression vector pA1, after the constitutive AcMNPV ie1 promoter. Primers F1 (5'-GGACTCGAGAAAATGTTGCGCAATTCCATATAC-3') and R1 (5'-ATTGAATTCGCGTTTACAGTTCGTCTCTCA-3') generated the full-length EppoNPV chitinase gene as an XhoIEcoRI fragment that was ligated into pA1 to create clone pA1-ChiF. Primers F1 and R2 (5'-TTTGTCGACCGATCCCAGTTCGTCTCTCATTTTG-3') replaced the stop codon with a SalI site at the C terminus in pA1, and then oligonucleotides hisF (5'-TCGACGGGTCTCACCACCACCACCACCACTGAG-3') and hisR (5'-AATTCTCAGTGGTGGTGGTGGTGGTGAGACCCG-3') were annealed and ligated into this clone as a SalIEcoRI fragment to create pA1-ChiHIS. Primers F1 and R3 (5'-TAGCGTTTTGAATTCGCCTCACATTTTGAA-3') generated clone pA1-ChiRDEL with the C-terminal RDEL sequence removed. In addition, primers AcF (5'-ACAACTTAAAATCTCGAGAAATTAAAATGTTGTAC-3') and AcR (5'-CTAAAATGATACTGAATTCATTGCTTTTACAGTTC-3') were used to amplify the AcMNPV chitinase gene as an XhoIEcoRI fragment and generate clone pA1-AcChi. Constructs were transfected into Spodoptera frugiperda IPLB-Sf9 cells by using FuGENE 6 (Roche). After 2 days incubation, the medium and cells were harvested and analysed for chitinase activity.
Expression and purification of recombinant EppoNPV chitinase.
EppoNPV orf110 was cloned and expressed in pET32a (Novagen) as a Trx fusion protein, utilizing the 6-His tag provided by the vector between trx and the EppoNPV chitinase gene. The ORF was isolated by PCR using primers chiNcoI-F (5'-TTTCCATGGC-GCTGCCCGGCGTGCC-3') and RDEL-X (5'-TAGCGTTTTGAATTCGCCTCACATTTTGAA-3') to amplify amino acids Ala18 to Met548, thus removing the N-terminal secretion signal and predicted C-terminal ER-retention sequence (RDEL). The PCR product was cloned into pBluescript II SK(+) for sequence confirmation and then subcloned into pET32a as an in-frame NcoIEcoRI fusion with trx and expressed in the Escherichia coli expression host Origami (Novagen). A 1·2 l culture containing 0·5 M sorbitol and 100 µg ampicillin ml1 was inoculated and grown at 37 °C until the OD550 reached 0·5. The culture was induced with 0·1 mM IPTG and incubated at 16 °C for 16 h. The cells were harvested, resuspended in 5 ml lysis buffer [20 mM Tris/HCl (pH 7·5), 0·3 M NaCl, 10 mM imidazole, 0·5 mg lysozyme ml1] and sonicated. The soluble fraction of the cell lysate was applied to an NiNTA column (Qiagen). The resin was washed twice with 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole (pH 8), twice with cleavage buffer [20 mM Tris/HCl (pH 7·4), 50 mM NaCl, 2 mM CaCl2] and then incubated for 16 h at room temperature with 12·5 U enterokinase (Novagen). The released chitinase was collected and the enterokinase was removed with Ekapture agarose (Novagen).
AcMNPV chitinase preparation.
A 20 ml suspension culture of S. frugiperda IPLB-Sf9 insect cells was inoculated with AcMNPV budded virus and incubated for 5 days. The cells were harvested, resuspended in PBS (pH 6·2) and lysed by using a polytron homogenizer. The lysate was centrifuged at 12 000 g for 5 min and the supernatant was concentrated twofold on a 30 kDa MWCO Ultrafree centrifugal device (Millipore). Sf9 insect cells that had not been infected with AcMNPV were treated similarly and used as a negative control.
Determination of catalytic parameters by using fluorescent oligosaccharide substrates.
Detection of chitinase activity was performed by using a fluorescent end-point assay as described by McCreath & Gooday (1992). Reactions were measured in a Cary Eclipse multiplate fluorometer (Varian) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Chitinase activity was measured by a time-course assay using the method of McCreath & Gooday (1992)
, except that activity was measured in McIlvaine's buffer at pH 6. Conversion of fluorescence units to moles of substrate was determined by using known amounts of 4MU measured under the same conditions as the assay. The pH optimum of the chitinase was determined by using a time-course assay over a pH range: McIlvaine's buffer was used between pH 3 and 7, and 50 mM Tris/HCl between pH 7 and 10. Kinetic parameters were determined by using a time-course assay with the concentration of fluorescent substrates varying between 0·16 and 400 µM and short-chain chitin concentrations varying between 1 and 200 µM. The maximum concentrations for the fluorescent substrates were limited by solubility and the maximum concentrations for the short-chain chitins were limited by substrate availability.
Analysis of chitin-hydrolysis products by thin-layer chromatography.
Purified recombinant chitinase (10 µg ml1) was incubated for 20 min (partial digestion) or 3 h (full digestion) at 37 °C with the (GlcNAc)26 native chito-oligosaccharides at a final concentration of 0·5 mg ml1. The chito-oligosaccharides were detected by using the method described by Walkley & Tillman (1977). Briefly, 10 µl reaction mix was spotted onto a Silica 60 plate (Merck) and resolved in a vertical chromatography tank containing 1-butanol/ethanol/H2O (5 : 3 : 2) as resolving buffer. The plate was developed in 0·5 g diphenylamine, 1 ml aniline and 5 ml orthophosphoric acid made up to 50 ml with acetone. The plate was dried and heated to 120 °C for colour development. Once developed, the image was acquired by using an Epson flatbed scanner. The chito-oligosaccharides were also digested fully (3 h) with purified native EppoNPV chitinase and the AcMNPV chitinase preparation.
Analysis of chitin-hydrolysis products by electrospray ionization mass spectrometry (ESI-MS).
Colloidal chitin was prepared as described by Tanaka et al. (1999). Shrimp pen chitin (
-chitin; 1 g) was incubated in orthophosphoric acid (85 %) for 20 h. The chitin precipitate was rinsed in distilled H2O and then neutralized with 10 M NaOH. The chitin was further rinsed and then resuspended in distilled H2O and stored at 4 °C. Colloidal chitin (90 µg) and chitinase (2 µg) were incubated at 37 °C for 30 min in deionized H2O. The reaction was then centrifuged at 20 000 g for 10 min and the enzyme was removed by using a ZipTip pipette tip containing C18 resin (Millipore). The sample was submitted to the Protein Microchemistry Facility, Department of Biochemistry, University of Otago, New Zealand, and applied by direct infusion at 3 µl min1 into a ThermoFinnigan LCQ mass spectrometer with ESI.
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RESULTS |
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The EppoNPV chitinase structure was modelled by using five known SmChiA structures (Fig. 2). Rhamachandran plots indicated that 98·7 % of the residues in the model had acceptable
/
angle conformations. Analysis of the proposed structure (Fig. 2
) showed that the catalytic domain formed an eight-stranded
/
TIM barrel and the PKD1 domain formed an immunoglobulin-like fold. Conserved aromatic residues were located on the surface of the protein in positions similar to those of the conserved aromatic residues found on the surface of SmChiA and other chitinases (Uchiyama et al., 2001
). The conserved residues included Trp27 and Trp63 along the immunoglobulin-like fold, Trp223, Trp236 and Tyr161 leading into the catalytic cleft and Trp158, Trp529, Trp266, Tyr 410 and Trp388 forming the catalytic binding site (Fig. 2
). The predicted structure confirmed that the enzyme is a family 18 glycohydrolase.
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Kinetic analysis of EppoNPV chitinase using fluorescent substrates
Assays using the fluorescent substrates 4MU-(GlcNAc)13 showed that both the native and recombinant forms of EppoNPV chitinase displayed endo- and exochitinolytic activity, but were inactive with 4MU-(GlcNAc) (data not shown), indicating that the EppoNPV chitinase is not an N-acetylglucosaminidase. The MichaelisMenten constants were 270±60 and 240±40 µM against 4MU-(GlcNAc)2 and 20±6 and 14±7 µM against 4MU-(GlcNAc)3 for the native and recombinant enzymes, respectively. EppoNPV chitinase displayed a pH optimum of approximately 5·5 with little enzyme activity beyond pH 8·5, using a 4MU standard curve for each pH value tested to assess the level of activity.
The activity against varying concentrations of 4MU-(GlcNAc)3 is shown in Fig. 5(a). Some inhibition of activity was observed at higher substrate concentrations. Furthermore, concentrations of 160 and 400 µM 4MU-(GlcNAc)3 showed a more marked loss of enzyme activity (data not shown), suggesting that digestion of the substrate led to product inhibition. As a consequence, native short-chain substrates were tested as inhibitors of EppoNPV chitinase to determine whether chito-oligosaccharides could block enzyme activity (Fig. 5b
). Analysis of (GlcNAc)26 substrates showed that all except the dimer were able to inhibit enzyme activity against the fluorescent substrates, with Ki values ranging from 19 to 320 µM (Table 1
). The (GlcNAc)2 substrate showed minimal inhibitory activity.
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Analysis of chitin-hydrolysis products by ESI-MS
The products produced from the hydrolysis of colloidal chitin with EppoNPV recombinant chitinase were analysed by ESI-MS. The resultant graph showed a clear peak with a mass of 447·27 units (Fig. 7). Ionization was accomplished by the gain of a sodium adduct ion (22·9 Da) present in the sample to produce a singly charged 447·27 peak, indicating that the correct mass of the species is 424·37 Da. The expected size of (GlcNAc)2 is 424·16 Da; thus, the major species produced from the digestion of colloidal chitin is a chitin dimer, confirming that the enzyme is an exochitinase. No products indicative of endochitinase activity were produced, even after a 30 min digestion of the substrate.
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DISCUSSION |
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Modelling of the EppoNPV chitinase against the known structure of SmchiA identified two major domains: a PKD1 domain (Bycroft et al., 1999) and a catalytic domain indicative of the family 18 glycohydrolase. The PKD1 domain is composed of
-sheets stacked like a sandwich, producing an immunoglobulin-like fold. A similar domain found in bacterial enzymes is involved in carbohydrate splitting and has been proposed to guide the substrate into the catalytic groove (Bork & Doolittle, 1992
; Perrakis et al., 1997
). The structure of the catalytic domain forms a deep substrate-binding cleft. The crystal structure of family 18 chitinases indicates that they should cleave chitin in an exo-type fashion, therefore suggesting that the EppoNPV chitinase should be an exochitinase. Analysis of the protein sequence and homology model of EppoNPV chitinase identified the conserved residues Tyr410 and Trp388, which interact to form the +2 subsite. Subsites +1, 1, 3 and 5, which each bind a chitin dimer, are formed by the residues Trp266, Trp529, Trp158 and Trp161, respectively. Aromatic residues representing the odd-numbered sites out to 13 were also identified. Each aromatic residue interacts with the hydrophobic face of every second GlcNAc in a chitin chain, with the GlcNAc subunit in the even-numbered position having its hydrophobic face exposed (Aronson et al., 2003
). The chain is fed into the cleavage site two subunits at a time via this binding mechanism to produce the (GlcNAc)2 dimer product characteristic of an exochitinase.
Contrary to the predicted structure, the use of fluorescent oligosaccharide substrates suggested that the EppoNPV chitinase possessed both endo- and exochitinase activity, as described previously for AcMNPV chitinase (Hawtin et al., 1995). For the substrates to fluoresce, the 4 MU moiety must be cleaved from the chito-oligosaccharide, indicating that the enzyme can cleave both a dimer and a trimer of chitin. If the enzyme was solely an exochitinase, (GlcNAc)2 should be removed from the 4MU-(GlcNAc)3 substrate, producing non-fluorescent 4MU-GlcNAc, which is not a substrate of this enzyme. The cleavage of 4MU-(GlcNAc)3 to release MU indicates that EppoNPV is an endochitinase. However, the digestion of the chito-oligosaccharides (GlcNAc)26 by EppoNPV chitinase gave some conflicting results. The homologue of 4MU-(GlcNAc)2, (GlcNAc)3, was not cleaved. The homologue of 4MU-(GlcNAc)3, (GlcNAc)4, was digested, producing only dimers. Taken together, these data indicate that binding of the (GlcNAc)3 trimer in 4MU-(GlcNAc)3 does not mimic the binding of either (GlcNAc)3 or (GlcNAc)4. Other studies on the family 18 glycohydrolases have shown similar results (Koga et al., 1997
). The presence of the (GlcNAc)3 product from (GlcNAc)6 and the ability to cleave 4MU-(GlcNAc)3 suggest that the enzyme may possess endochitinase activity or have the ability occasionally to cleave trimers from the native chain, although no monomer or trimer products were observed from (GlcNAc)4 digestion.
Crystal structures of inactive chitinase mutants complexed with short chito-oligosaccharides indicate that the chitin binds to all of the subsites in the active site (Papanikolau et al., 2001; van Aalten et al., 2001
). However, analysis of the crystal structures of active chitinases complexed with the same chito-oligosaccharides suggest that because the chito-oligosaccharides and fluorescent substrates are short, they lack the ability to bind to the aromatic residues that interact with the longer chitin chains and instead interact with the binding sites that have the strongest chitin-binding affinity (2 to +2) (Aronson et al., 2003
). Aronson et al. (2003)
used HPLC to show that substrates larger than the tetrasaccharide did bind to the four subsites, but allowed the reducing end to extend out from the enzyme. Thus for (GlcNAc)6, the reducing end would extend out of the enzyme by two GlcNAc substrates and cleavage would produce (GlcNAc)4 and (GlcNAc)2. On occasion, only one GlcNAc subunit would extend out from the enzyme, as the substrate could bind to the 3 to +2 subsites, creating (GlcNAc)3 products. This would explain the low amount of (GlcNAc)3 product detected upon digestion of (GlcNAc)6 by EppoNPV chitinase. (GlcNAc)3 is not digested, as the residues may bind to the +1 and +2 subsites, with the third GlcNAc extended from the active site or to the 1 to 3 subsites. For the fluorescent substrates, the 4MU moiety in 4MU-(GlcNAc)2 may have a positive interaction with the +1 site if the substrate binds in the forward direction or the substrate enters the active site in the reverse orientation with MU in the 1 position. Similarly, for 4MU-(GlcNAc)3, the last GlcNAc must extend from the active site, or MU interacts with the +1 subsite and the GlcNAc units bind to the 1 to 3 subsites. The cleavage of 4MU would leave (GlcNAc)3 in the active site to act as a product inhibitor, generating the inhibition seen at higher concentrations of this substrate. The inhibition activity of (GlcNAc)36 chains supports the contention that short chains can bind either side of the cleavage point and hence not be cleaved. The lack of inhibition by (GlcNAc)2 supports the contention that this represents the true product of this enzyme when exposed to a natural substrate.
A clearer picture emerged when a long-chain substrate was assayed. Digestion of colloidal chitin with EppoNPV chitinase confirmed that the enzyme is an exochitinase, with only (GlcNAc)2 dimers generated. In contrast, endochitinases show a range of product sizes by mass spectrometry analysis (Suginta et al., 2004), even after a short period of digestion. These data, combined with the inhibition data, variations in activity between fluorescent and non-fluorescent substrates and studies of others on aberrant binding of short-chain substrates (Aronson et al., 2003
; Papanikolau et al., 2001
), indicate that care should be taken when using short-chain substrates to define chitinase activity.
We conclude that EppoNPV chitinase is a canonical baculovirus chitinase that is analogous to AcMNPV chitinase and thus displays both exo- and endochitinolytic activities using fluorescent oligosaccharides. However, this may not reflect the actual activity of the enzyme in nature when exposed to longer chitin chains. The fluorescent substrates are excellent quantitative substrates, but may not be definitive indicators of exochitinase and endochitinase activity.
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ACKNOWLEDGEMENTS |
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Received 13 June 2005;
accepted 26 August 2005.
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