Characterization of an exochitinase from Epiphyas postvittana nucleopolyhedrovirus (family Baculoviridae)

Vivienne L. Young1, Robert M. Simpson2 and Vernon K. Ward1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Baculovirus chitinases and other family 18 glycohydrolases have been shown to possess both exo- and endochitinase activities when assayed against fluorescent chito-oligosaccharides. Homology modelling of the chitinase of Epiphyas postvittana nucleopolyhedrovirus (EppoNPV) against Serratia marcescens chitinase A indicated that the enzyme possesses an N-terminal polycystic kidney 1 (PKD1) domain for chitin-substrate feeding and an {alpha}/{beta} TIM barrel catalytic domain characteristic of a family 18 glycohydrolase. EppoNPV chitinase has many features in common with other baculovirus chitinases, including high amino acid identity, an N-terminal secretion signal and a functional C-terminal endoplasmic reticulum-retention sequence. EppoNPV chitinase displayed exo- and endochitinolytic activity against fluorescent chito-oligosaccharides, with Km values of 270±60 and 240±40 µM against 4MU-(GlcNAc)2 and 20±6 and 14±7 µM against 4MU-(GlcNAc)3 for native and recombinant versions of the enzyme, respectively. In contrast, digestion and thin-layer chromatography analysis of short-chain (GlcNAc)2–6 chito-oligosaccharides without the fluorescent 4-methylumbelliferone (4MU) moiety produced predominantly (GlcNAc)2, indicating an exochitinase, although low-level endochitinase activity was detected. Digestion of long-chain colloidal {beta}-chitin and analysis by mass spectrometry identified a single 447 Da peak, representing a singly charged (GlcNAc)2 complexed with a sodium adduct ion, confirming the enzyme as an exochitinase with no detectable endochitinolytic activity. Furthermore, (GlcNAc)3–6 substrates, but not (GlcNAc)2, acted as inhibitors of EppoNPV chitinase. Short-chain substrates are unlikely to interact with the aromatic residues of the PKD1 substrate-feeding mechanism and hence may not accurately reflect the activity of these enzymes against native substrates. Based upon these results, the chitinase of the baculovirus EppoNPV is an exochitinase.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Chitin is the most abundant nitrogen-containing polysaccharide in the environment, consisting of 1,4-{beta}-linked N-acetylglucosamine (GlcNAc) subunits. The breakdown of chitin is an important process and, as a consequence, numerous organisms possess chitinases (EC 3.2.1.14). Chitinases play a critical role in insect growth and development (Zheng et al., 2002; Merzendorfer & Zimoch, 2003), plant defence (Kasprzewska, 2003) and baculovirus pathogenicity (Thomas et al., 2000). Chitinases are members of the family 18 and 19 glycohydrolases, which are differentiated by their amino acid sequence, structure and reaction mechanism (Henrissat, 1991; Henrissat & Bairoch, 1993). Chitinase degrades the chitin chain by three primary mechanisms: endochitinases cleave randomly within the chain (McCreath & Gooday, 1992), exochitinases progressively cleave off two subunits from the reducing or non-reducing ends of the chitin chain (Brurberg et al., 2001) and N-acetylglucosaminidases remove one subunit from the chitin chain (Williams et al., 2002).

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 {alpha}/{beta} 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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Fig. 1. Schematic of aromatic residue subsites of an exochitinase that interact with the hydrophobic face of alternating GlcNAc subunits. Arrow denotes cleavage site. NR, Non-reducing end; R, reducing end. Hydrophobic residue subsites are designated –5 to +2. Chitin subunits are shown as hexagons.

 
A number of chitinases have been identified in baculoviruses, including the chitinase from the type species Autographa californica nucleopolyhedrovirus (AcMNPV) (Hawtin et al., 1995). The enzyme was found associated with viral polyhedra and is presumably released during polyhedral dissolution in the alkaline midgut of insects. This may aid in degrading the chitinous peritrophic membrane lining the insect larval midgut at an early stage of viral infection, allowing the virus more efficient access to the midgut epithelial cells (Hawtin et al., 1997). Chitinase is also expressed late in infection, causing dissolution of the host and assisting release of progeny virus into the environment (Hawtin et al., 1997). AcMNPV chitinase contains an endoplasmic reticulum (ER)-retention sequence at the C terminus (Thomas et al., 1998; Saville et al., 2002, 2004), probably involved in retaining the enzyme inside the cell until late in infection. End-point analysis using the synthetic fluorescent chito-oligosaccharides 4-methylumbelliferyl-(GlcNAc)1–4 [4MU-(GlcNAc)x] showed that the enzyme was active against all of the substrates, excluding 4MU-GlcNAc, over a wide pH range. Therefore, it was proposed that the baculovirus chitinases were unique, as both exo- and endochitinase activities were produced in the same enzyme (Hawtin et al., 1995, 1997; Rao et al., 2004).

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Analysis of the putative chitinase gene of EppoNPV (orf110).
BLASTX and BLASTP searches were performed on the nucleotide and amino acid sequence of EppoNPV orf110. Highly similar sequences were aligned by using MegAlign (Lasergene; DNAstar Inc.) and the percentage identity was calculated. The ORF110 sequence was submitted to SWISS-MODEL (http://swissmodel.expasy.org) and modelled against SmchiA structures, including 1CTN (Perrakis et al., 1994), 1EDQ (Papanikolau et al., 2003), 1FFQ (Papanikolau et al., 2003), 1FFR (Papanikolau et al., 2001) and 1EHN (Papanikolau et al., 2001). A Rhamachandran plot was generated to check for acceptable {pi} and/or {psi} angles. The sequence was also submitted to PredictProtein (http://cubic.bioc.columbia.edu/predictprotein) to identify conserved domains and motifs.

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 0–400 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 XhoI–EcoRI 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 SalI–EcoRI 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 XhoI–EcoRI 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 NcoI–EcoRI 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 ml–1 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 ml–1] and sonicated. The soluble fraction of the cell lysate was applied to an Ni–NTA 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 ml–1) was incubated for 20 min (partial digestion) or 3 h (full digestion) at 37 °C with the (GlcNAc)2–6 native chito-oligosaccharides at a final concentration of 0·5 mg ml–1. 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 ({beta}-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 min–1 into a ThermoFinnigan LCQ mass spectrometer with ESI.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sequence and protein analysis of EppoNPV ORF110
The 1659 bp orf110 of EppoNPV encoded a putative chitinase with 78·2 % amino acid identity to AcMNPV chitinase, 56 % identity to Cydia pomonella chitinase and 51 % identity to SmChiA. The enzyme contained a predicted ER-retention sequence (RDEL) at the C terminus, an N-terminal secretion signal, a polycystic kidney 1 (PKD1) domain and a catalytic domain. The highly conserved family 18 glycohydrolase motif (SIGGWT) and the consensus Prosite motif PDOC00839 (FDGVDIDWE) containing the critical glutamate residue proposed to act as a proton donor were identified in the catalytic domain (GenBank accession no. AAK85674). Primary sequence alignment also identified a number of conserved aromatic residues known to be important in the chitin-feeding mechanism and substrate binding of SmChiA (Uchiyama et al., 2001).

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 {pi}/{psi} angle conformations. Analysis of the proposed structure (Fig. 2) showed that the catalytic domain formed an eight-stranded {alpha}/{beta} 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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Fig. 2. Ribbon structure (left) and computed molecular surface (right) of modelled EppoNPV chitinase. The sequence was submitted to the SWISS-MODEL server and modelled against chitinase structures 1EDQA, 1FFGA, 1CTN, 1EHNA and 1FFRA. The structure of SmChiA (1CTN) is included for comparison. The catalytic domain, N-terminal immunoglobulin (Ig)-like fold and conserved aromatic residues are indicated. Hydrophobic binding-site designations are shown (–13 to +2) and contributing hydrophobic residues are indicated. The catalytic pocket includes the sites designated –1, +1 and +2.

 
Purification of EppoNPV chitinase from virus-infected insects
Active EppoNPV chitinase was partially purified from infected insect larvae (Fig. 3a). orf110 of EppoNPV encodes a 62 kDa protein with an N-terminal leader sequence that is predicted to be cleaved after aa 18 to generate a mature protein of 60 kDa. The N-terminal 6 aa of the mature protein, commencing at aa 19, are predicted to be LPGVPV. N-terminal sequencing of the 60 kDa band identified the first six residues as LPGVPV, matching the amino acids found directly after the predicted secretion-signal cleavage site in the amino acid sequence of EppoNPV chitinase. This confirmed that the purified protein was EppoNPV chitinase and that the N-terminal secretion signal is cleaved upon translation and transport of the protein.



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Fig. 3. SDS-PAGE of purified native and recombinant EppoNPV chitinase. (a) Native EppoNPV chitinase expressed in EppoNPV-infected insects and purified. (b) Recombinant EppoNPV chitinase expressed in E. coli and purified by using nickel-affinity chromatography. Molecular mass markers are indicated in kDa.

 
Cellular localization of EppoNPV chitinase
Localization studies of the EppoNPV chitinase (ChiF) revealed that the chitinase activity remained in the intracellular fraction, despite the presence of an N-terminal secretion signal (Fig. 4a). Upon removal of the putative ER-retention sequence, RDEL, at the C terminus of the protein, the enzyme relocated to the extracellular fraction. This suggested that the N-terminal signal sequence is functional, but the retention sequence causes the enzyme to remain in the ER. The addition of a 6-His tag at the C terminus, directly after the RDEL sequence (ChiHIS), caused the enzyme to localize to the extracellular fraction (Fig. 4b), confirming that the retention sequence has to be at the C terminus to be recognized.



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Fig. 4. Localization of EppoNPV chitinase and substrate specificity. Chitinase-expressing clones were transfected into Sf9 insect cells and cellular (a) and extracellular (b) fractions were assayed against fluorescent N-acetylglucosaminidase, exochitinase and endochitinase substrates. Activity is presented as relative fluorescence units with empty pA1 plasmid vector-control values subtracted. All assays were performed in triplicate. Black bars, 4MU-GlcNAc (glucosaminidase activity); hatched bars, 4MU-(GlcNAc)2 (exochitinase activity); white bars, 4MU-(GlcNAc)3 (endochitinase activity).

 
Expression and purification of EppoNPV chitinase
The EppoNPV chitinase was expressed as a fusion to Trx in E. coli. The protein was predominantly insoluble, but expression at 16 °C in the presence of sorbitol produced sufficient solubility for purification. Purification of the soluble fraction by immobilized metal-affinity chromatography produced 200 µg purified protein (l culture)–1 (Fig. 3b).

Kinetic analysis of EppoNPV chitinase using fluorescent substrates
Assays using the fluorescent substrates 4MU-(GlcNAc)1–3 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 Michaelis–Menten 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)2–6 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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Fig. 5. Kinetic analysis of native chitinase with 4MU-(GlcNAc)3. (a) Plot of chitinase activity with substrate only and in the presence of 1, 2, 5, 10, 20, 50, 100 and 200 µM (GlcNAc)4. (b) Plot of apparent Michaelis–Menten constants in the presence of the oligosaccharide substrates (GlcNAc)3, (GlcNAc)4, (GlcNAc)5 and (GlcNAc)6.

 

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Table 1. Inhibition of chitinase activity by chito-oligosaccharides

The values given show the Ki for the various substrates (µM). ND, Not determined.

 
Thin-layer chromatography analysis of enzyme digestion
Thin-layer chromatography was used to analyse the full and partial digestion of the short-chain GlcNAc substrates by purified native and recombinant chitinase (Fig. 6). Native and recombinant EppoNPV chitinases showed similar activity profiles. (GlcNAc)2 showed no apparent cleavage, confirming that the chitinase possessed no detectable N-acetylglucosaminidase activity. Digestion of the (GlcNAc)3 substrate produced no detectable (GlcNAc)2, with the substrate remaining apparently uncleaved after 3 h digestion. The (GlcNAc)4 substrate was cleaved completely into (GlcNAc)2 only, while the (GlcNAc)5 substrate was cleaved into (GlcNAc)3 and (GlcNAc)2, suggesting exochitinolytic activity. The (GlcNAc)6 substrate was initially cleaved into (GlcNAc)4 and then to (GlcNAc)2, suggesting an exochitinolytic activity, although after 3 h digestion, a small amount of (GlcNAc)3 was also evident.



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Fig. 6. Thin-layer chromatographic analysis of baculovirus chitinase activity. (a) Partial (20 min) and complete (3 h) digestion of (GlcNAc)2–6 substrates using purified recombinant EppoNPV chitinase. (b) Complete digestion of the GlcNAc substrates using native EppoNPV chitinase. (c) Complete digestion of the GlcNAc substrates using AcMNPV chitinase and Sf21 cell extracts.

 
The AcMNPV chitinase extract was also tested against short-chain substrates with essentially the same results (Fig. 6c). Similar to EppoNPV chitinase, only a minimal amount of (GlcNAc)3, if any, was digested to (GlcNAc)2. After full digestion of (GlcNAc)6, a very small amount of (GlcNAc)3 was present, although it was at too low a level to detect in the scanned plate. The negative control did not digest any of the substrates.

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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Fig. 7. ESI-MS of colloidal chitin digested with recombinant EppoNPV chitinase.

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The 1659 bp orf110 of EppoNPV showed a high level of sequence identity to the chitinase of AcMNPV. It proved to be a canonical baculovirus chitinase with almost all properties being essentially similar to those of the well-studied AcMNPV chitinase. It contained an N-terminal secretion sequence that was cleaved upon translation and a C-terminal ER-retention sequence (RDEL) that was functional when at the C terminus of the protein. ER-retention sequences have also been identified in the chitinases of Bombyx mori NPV (Gomi et al., 1999) and Choristoneura fumiferana MNPV (GenBank accession no. NC_004778) and are likely to be a common feature of many baculovirus chitinases. AcMNPV chitinase has been reported to have activity at high pH; in contrast, EppoNPV chitinase showed no such high-pH activity. This is possibly due to the enhancement of fluorescence of umbelliferone at high pH levels in an end-point assay, compared with the use of a standard 4MU curve at each pH value and the measurement of initial velocity kinetics, as undertaken in this study. Furthermore, the use of the fluorescent end-point assay did indicate activity to high pH (data not shown).

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 {beta}-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)2–6 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)3–6 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.


   ACKNOWLEDGEMENTS
 
This research was supported by a NERF grant from the New Zealand Foundation for Research Science and Technology. The squid pen chitin was kindly donated by Industrial Research Limited, Lower Hutt, New Zealand.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 13 June 2005; accepted 26 August 2005.



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