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Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus
Comparative Biochemistry and Physiology, Part B 140 (2005) 427 – 435 www.elsevier.com/locate/cbpb
Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus Ji Hee Hana, Kwang Sik Leea, Jianhong Lia,1, Iksoo Kimb, Yeon Ho Jec, Doh Hoon Kima, Hung Dae Sohna, Byung Rae Jina,* b
a College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea c School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
Received 6 August 2004; received in revised form 9 November 2004; accepted 14 November 2004
Abstract A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30–40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. D 2004 Elsevier Inc. All rights reserved. Keywords: Araneus ventricosus; Baculovirus; cDNA; Chitinase; N-Glycosylation; Glycosyl hydrolase; Insect cells; Spider
1. Introduction Chitin is one of the most abundant polysaccharides in nature and is a linear polymer of h (1Y4) linked N– acetylglucosamine (GlcNAc) residues. It is one of the most unique biochemical constituents found in the exoskeletons and gut linings of arthropods and fungi. Chitinolytic enzymes that catalyze the hydrolysis of chitin have been found in chitin-containing organisms as well as in microorganisms that do not have chitin. The enzymes, chitinases, from various organisms have various biological functions. Chitinase plays roles in the molting process of invertebrates, * Corresponding author. Tel./fax: +82 51 200 7594. E-mail address: [email protected] (B.R. Jin). 1 Present address: College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.11.009
including spiders, the digestion of chitinous food, and defense against chitin-bearing pathogens (Flach et al., 1992; Kramer and Muthukrishnan, 1997). Chitin degradation is a complex process and is catalyzed by a two-component chitinolytic enzyme system, chitinase (EC 3.2.1.14), and h-N-acetylglucosaminidase (EC 3.2.1.30) (Fukamizo and Kramer, 1985a,b). The two enzymes exhibit a synergism in the hydrolysis process of chitin. Chitinase activity has been found with bacteria, fungi, plants, invertebrates—including spiders (Mommsen, 1978, 1980)—and vertebrates, and chitinase genes have been also isolated from several organisms. Chitinase genes offer several opportunities for gene manipulation in a variety of purposes; that is, the enhancement of host plant resistance and pathogenicity in transgenic plants and biological control agents (Bonning and Hammock, 1996; Estruch et al., 1997; Kramer and Muthukrishnan, 1997).
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Furthermore, a recent study showed the possible application of tick chitinase in the control of tick vector (You et al., 2003). In order to extend our understanding of the chitinase genes in arthropods, a novel fat body-specific chitinase cDNA from the spider Araneus ventricosus was isolated and characterized in this research. The spider, A. ventricosus, is the abundant species in Korea (Kim et al., 1997). The body size of A. ventricosus is relatively larger than that of the other species. The mechanical properties of the web of A. ventricosus have been reported (Kim et al., 1997). The spider chitinase cDNA was expressed in baculovirusinfected insect cells and the activity of the recombinant enzyme was determined. In addition, we showed that Nglycosylation in A. ventricosus chitinase is not essential for chitinolytic activity.
2. Materials and methods 2.1. Animals Spiders, A. ventricosus, were collected at Namhae, Kyungnam province in Korea. The live spiders were directly used in this study. 2.2. cDNA library screening, nucleotide sequencing, and data analysis A cDNA library (Chung et al., 2001) was constructed using whole bodies of A. ventricosus. Sequencing of randomly selected clones harboring cDNA inserts was performed to generate the expressed sequence tags (ESTs). For DNA sequencing, plasmid DNA was extracted by Wizard mini-preparation kit (Promega, Madison, WI). Sequence of each cDNA clone was determined using an automatic sequencer (model 310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster City, CA). The sequences were compared using the DNASIS and BLAST programs provided by the NCBI, GenBank, EMBL, and SwissProt databases were searched for sequence homology using a BLAST algorithm program (www.ncbi.nlm. nih.gov/BLAST). MacVector (ver. 6.5, Oxford Molecular) was used to align the amino acid sequences of chitinase gene. With the GenBank-registered chitinase amino acid sequences, phylogenetic analysis was performed using PAUP (Phylogenetic Analysis Using Parsimony) version 4.0 (Swofford, 2000). 2.3. RNA isolation and Northern blot analysis Mature adults of the spider A. ventricosus were dissected under a stereo-microscope (Zeiss, Jena, Germany), individual samples such as fat body, midgut, and silk gland were harvested, and washed twice with PBS (140 mM NaCl, 27 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). Total
RNA was isolated from the whole body, midgut, fat body, and silk gland of A. ventricosus by using the Total RNA Extraction Kit (Promega). Total RNA (10 Ag/lane) from A. ventricosus was denatured by glyoxylation (McMaster and Carmichael, 1977), transferred onto a nylon blotting membrane (Schleicher and Schuell, Dassel, Germany), and hybridized at 42 8C with a probe in a hybridization buffer containing 5 SSC, 5 Denhardt’s solution, 0.5% SDS, and 100 Ag/ml denatured salmon sperm DNA. The 1515-bp A. ventricosus chitinase cDNA clone was labeled with [a-32P] dCTP (Amersham, Arlington Heights, IL, USA) using the Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA, USA) for use as a probe for hybridization. After hybridization, the membrane filter was washed three times for 30 min each in 0.1% SDS and 0.2 SSC (1SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 65 8C and exposed to autoradiography film. For rehybridization, the membrane was washed for 20 min at room temperature in sterile millipore water. Then, the membrane was washed overnight at 65 8C in 50 mM TrisHCl (pH 8.0), 50% dimethylformamide and 1% SDS in order to remove the hybridized probe. The membrane was then rehybridized to [a-32P] dCTP-labeled 28S rRNA probe (Lee et al., 2003). The 28S rRNA gene was used as an internal loading control. 2.4. Genomic DNA isolation and Southern blot analysis Genomic DNA was extracted from the fat body of A. ventricosus adult using a Wizardk Genomic DNA Purification Kit, according to the manufacturer’s instructions (Promega). Genomic DNA from A. ventricosus was digested with EcoRI and HindIII, and electrophoresed in 1.0% agarose gel. The DNA from the gel was transferred onto a nylon blotting membrane (Schleicher and Schuell) and hybridized at 42 8C with a probe in a hybridization buffer. Hybridization condition, fragment labeling, and filter washing were as described for the Northern blot analysis. 2.5. Cell culture and construction of recombinant virus Spodoptera frugiperda IPLB Sf21-AE (Vaughn et al., 1977) clone 9 (Sf9) cells were maintained at 27 8C in TC100 medium (GIBCO BRL LIFE Technologies), supplemented with 10% fetal bovine serum (FBS; GIBCO BRL LIFE Technologies) as described by standard methods (O’Reilly et al., 1992). Wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant AcNPV were propagated in Sf9 cells. The titer was expressed as plaque forming units (PFU) per ml (O’Reilly et al., 1992). The 1515-bp A. ventricosus chitinase cDNA from pBlueScript–AvChit was digested with SacI and KpnI, and then inserted into the SacI and KpnI sites of pBacPAK9 (Clontech, Palo Alto, CA) to produce baculovirus transfer
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vector pBacPAK9–AvChit. In the transfer vector, the chitinase cDNA is under the control of the AcNPV polyhedrin promoter. For the construction of recombinant virus, 35-mm cell culture dishes were seeded with 1.0–1.5106 cells and incubated at 27 8C for 1 h to allow cell attachment. Total of 1 Ag of BacPAK6 viral DNA (Clontech) and 5 Ag of pBacPAK9–AvChit in 20 mM HEPES buffer in a final volume of 50 Al were mixed in a polystyrene tube. Moreover, 50 Al of 100 Ag/ml Lipofectink (GIBCO BRL LIFE Technologies) was gently mixed with the DNA solution and the mixture was incubated at room temperature for 30 min. The cells were washed twice with 2 ml serumfree TC100 medium and refed with 1.5 ml serum-free TC100 medium. The Lipofectin-DNA complexes were added dropwise to the medium covering the cells while the dish was gently swirled. After incubation at 27 8C for 5 h, TC100 medium containing antibiotics and 10% FBS was added to each dish and incubation at 27 8C was continued. At 5 days p.i., the supernatant was harvested, clarified by centrifugation at 2,000 rpm for 5 min, and stored at 4 8C. Recombinant AcNPV was plaque purified on 6-well plates seeded with 1.5106 Sf9 cells as described previously (O’Reilly et al., 1992). 2.6. Preparation of polyclonal antibody and Western blot analysis Recombinant A. ventricosus chitinase was electroeluted from the SDS-polyacrylamide gel electrophoresis (PAGE) gel, mixed with equal volume of Freund’s complete adjuvant (a total of 200 Al, Sigma) and injected into Balb/c mice, respectively. Three successive injections were performed with 1-week interval beginning a week after the first injection with antigens mixed with equal volume of Freund’s incomplete adjuvant (a total of 200 Al, Sigma). Bloods were collected 3 days after the last injection and centrifuged at 13,000g for 5 min. The supernatant antibodies were stored at 70 8C until use. For Western blot analysis, the protein samples were subjected to 10% SDS-PAGE (Laemmli, 1970). Proteins were blotted to a sheet of nitrocellulose membrane (Sigma, 0.45 Am of pore size) (Towbin et al., 1979). The blotting was performed in transfer buffer (25 mM Tris and 192 mM glycine in 20% methanol) at 30 V overnight at 4 8C. After blotting, the membrane was blocked by incubation in 1% BSA solution for 2 h at room temperature. The blocked membrane was incubated with antiserum solution (1:1000 v/v) for 1 h at room temperature and washed in TBST (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.05% Tween 20). The membrane was then incubated with anti-mouse IgG horseradish peroxidase (HRP) conjugate and HRP-streptavidin complex. After repeated washing, the membrane was incubated with ECL detection reagents (Amersham Pharmacia Biotech) and exposed to autoradiography film.
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2.7. Determination of chitinase activity Chitinase activity was detected from the gel after SDS-PAGE by the method of Trudel and Asselin (1989). Gel was incubated in 150 mM sodium acetate buffer at pH 5.0 for 5 min. The gel was then put on a glass plate and covered with a 7.5% polyacrylamide overlay gel containing 0.01% (w/v) glycol chitin in 100 mM sodium acetate buffer (pH 5.0). Gel was incubated at 37 8C for 1 h under the moist conditions. Lytic zones were visualized by placing the gels on a UV illuminator and were photographed. 2.8. Tunicamycin treatment The addition of N-linked carbohydrate by infected insect cells was verified by culture in the presence of tunicamycin (5 Ag/ml, Sigma) to prevent the addition of N-linked carbohydrate (Hasemann and Capra, 1990). Sf9 cells were mock infected or infected with wild-type AcNPV or recombinant viruses in a 35-mm-diameter dish (1106 cells) and were incubated for 2 h at 27 8C. The supernatants were replaced with 5 ml of supplemented TC100 medium containing 5 Ag tunicamycin/ml medium. After incubation 27 8C, total cellular lysates was harvested from infected cells at 24, 48, and 72 h p.i. Total cellular lysates were subjected to 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity were identified as dark lytic zones under UV illumination (Trudel and Asselin, 1989).
3. Results 3.1. Cloning and sequencing of AvChit1 cDNA A cDNA library was constructed using whole bodies of A. ventricosus (Chung et al., 2001). Sequencing of randomly selected clones harboring cDNA inserts was performed to generate the A. ventricosus ESTs. One clone, which is 1515 bp long had a full-length coding sequence similar to that of previously reported chitinases. The deduced amino acid sequences of a cDNA encoding the A. ventricosus chitinase (AvChit1) are presented in Fig. 1. The AvChit1 cDNA contains an open reading frame (ORF) of 1293 bp encoding 431 amino acid residues. A multiple sequence alignment of the deduced protein sequence of AvChit1 with other chitinase sequences is shown in Fig. 1. Alignment of the AvChit1 sequences with those of chitinase from several other species indicates the extent of the identity that exists. AvChit1 conserved the chitinase family 18 active site signature, which is a consensus sequence, [LIVMFY]-[DN]-G[LIVMF]-[DN]-[LIVMF]-[DN]-X-E (Van Scheltinga et al., 1994). The deduced AvChit1 N-terminal sequence con-
430 J.H. Han et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 427–435 Fig. 1. Alignment of the amino acid sequence of AvChit1 cDNA with known chitinases. Residues are numbered according to the aligned chitinase sequences, and invariant residues are shaded black. Dots represent gaps introduced to improve alignment. Chitinase family 18 active site signature is marked by asterisk. The arrow shows the end of the signal peptides. The N-glycosylation site is indicated with the cross. The GenBank accession numbers of AvChit1 cDNA is AY120879. The abbreviation and GenBank accession number for the chitinase sequences aligned are: AvChit1, Araneus ventricosus chitinase (AY120879; this study); Gchit1, Glossina morsitans morsitans chitinase (AF337908); Llchit1, Lutzomyia longipalpis chitinase (AY148807); AgChi-1, Anopheles gambiae chitinase (AF008575); PcChit1, Phaedon cochleariae chitinase (Y18011), DmCHT1, Drosophila melanogaster chitinase (Q9W5U3); HsChit, Homo sapiens chitotriosidase (U62662).
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tains a highly hydrophobic amino acid stretch that is likely to function as a signal peptide. Using Von Heijne’s (1986) rule, we predict that the signal peptide is cleaved after Ala-19. AvChit1 possesses one putative N-glycosylation site at the amino acid residues 260–263 (NGTP). A phylogenetic analysis using the deduced amino acid sequences of known chitinase genes revealed that the chitinases are divided into three groups (Fig. 2A). The AvChit1 formed a subgroup together with chitinase genes from insect species such as Glossina morsitans morsitans, Lutzomyia longipalpis, Anopheles gambiae, Phaedon cochleariae, and Drosophila melanogaster, and a human chitotriosidase, which lack both the serine/threonine and chitin binding domains. Similarly, the deduced protein sequence of the AvChit1 showed 43% protein sequence identity to both G. morsitans morsitans chitinase and human chitotriosidase, and 30–40% to insect chitinases within the subgroup (Fig. 2B).
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3.2. Tissue-specific expression of AvChit1 The tissue specific nature of AvChit1 expression was determined from fat body, midgut, silk gland and epidermis by Northern blot analysis (Fig. 3). AvChit1 was found to be expressed only in fat body and not in midgut and epidermis of A. ventricosus, evidencing the fat body as a specific site for AvChit1 synthesis. Furthermore, tissue specific expression of AvChit1 was analyzed from the protein samples of epidermis, fat body, and midgut of A. ventricosus. Each protein sample was subjected to 10% SDS-PAGE (Fig. 4A) and Western blot analysis (Fig. 4B). A signal band of 61 kDa was detected specifically from the fat body in the Western blot analysis, but not in the epidermis and midgut protein samples. The protein band with a 61 kDa also showed chitinolytic activity (lane 2 of Fig. 4C). The result is in good agreement with the Northern blot hybridization result that AvChit1 showed the
Fig. 2. Relationships among amino acid sequences of AvChit1 and known chitinases. (A) A maximum parsimony analysis for the amino acid sequences of AvChit1 and known chitinases. The tree was obtained by bootstrap analysis with the option of heuristic search and the numbers on the branches represent bootstrap values for 1000 replicates. Outgroup was chosen as bacterium Streptomyces olivaceoviridis exochitinase (SoChit; Q05638). The abbreviation and GenBank accession number for the chitinase sequences analyzed are: AvChit1, A. ventricosus chitinase (AY120879; this study); GChit1, G. morsitans morsitans chitinase (AF337908); Llchit1, L. longipalpis chitinase (AY148807); AgChi-1, A. gambiae chitinase (AF008575); PcChit1, P. cochleariae chitinase (Y18011); DmCHT1, D. melanogaster chitinase (Q9W5U3); HsChit, H. sapiens chitotriosidase (U62662); MmAMCase, Mus musculus acidic mammalian chitinase (AF290003); HsAMCase, H. sapiens acidic mammalian chitinase (AF290004); BtAMCase, Bos taurus acidic mammalian chitinase (Q95M17); fChi2, Paralichthys olivaceus chitinase (AB121733); HlChit, Haemaphysalis longicornis chitinase (ABO74977). (B) Pairwise identities and similarities of the deduced amino acid sequence of AvChit1 cDNA among chitinase sequences.
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analyze the protein synthesis in Sf9 cells infected with the recombinant virus. The recombinant AvChit1 was present as a single band of about 61 kDa polypeptide in the cells infected with the recombinant virus, but not in the cells infected with the wild-type AcNPV or mock-infected cells. In SDS-polyacrylamide gels containing 0.01% glycol chitin, the protein bands with chitinolytic activity were directly detected as dark lytic zones under UV illumination. The chitinolytic activity was detected in the recombinant AvChit1 with a 61 kDa from recombinant virus-infected insect cells (Fig. 6C). Fig. 3. Northern blot analysis of AvChit1 gene. Total RNA was isolated from the fat body (lane 1), midgut (lane 2), silk gland (lane 3), and epidermis (lane 4), respectively. The RNA was separated by 1.0% formaldehyde agarose gel electrophoresis (upper panel), transferred on to a nylon membrane, and hybridized with the appropriate radiolabelled probe (middle panel). The 28S rRNA gene was used as an internal loading control (lower panel). Transcripts are indicated by arrow on the right side of the panel.
tissue-specific expression in the fat body. However, the protein bands displaying chitinolytic activity in enzyme assay were detected in both epidermis (approximately 60 kDa) and midgut (approximately 40 kDa; Fig. 4C). This result suggests the presence of other chitinase genes in both epidermis and midgut as well as in fat body of A. ventricosus. In addition, the chitinolytic activity assay suggests that A. ventricosus has another chitinase (approximately 47 kDa) in the fat body (lane 2 of Fig. 4C). 3.3. Copy number of AvChit1 gene To determine the number of AvChit1 gene in the A. ventricosus genome, genomic DNA was digested with restriction enzymes, which do not cut within the chitinase gene, blotted, and hybridized with the AvChit1 cDNA. Single hybridizing bands were detected with all enzymes indicating the AvChit1 is a single gene (Fig. 5). 3.4. Expression of AvChit1 cDNA in baculovirus-infected insect cells To assess AvChit1 cDNA, the 1515 bp for AvCht1 cDNA was inserted into baculovirus transfer vector. The baculovirus transfer vector was used to generate a recombinant virus expressing AvChit1. Transfer vector pBacPAK9– AvChit was constructed by insertion of AvChit1 cDNA under the control of AcNPV polyhedrin promoter of pBacPAK9 (data not shown). Recombinant AcNPV, which we have termed AcNPV–AvChit1, was produced in insect Sf9 cells by cotransfection with wild-type AcNPV DNA and the transfer vector. To examine the expression of AvChit1 cDNA by recombinant virus in insect cells, SDS-PAGE (Fig. 6A) and Western blot analysis (Fig. 6B) were performed to
Fig. 4. Tissue-specific expression of AvChit1. The protein samples were collected from epidermis (lane 1), fat body (lane 2), and midgut (lane 3) of A. ventricosus. The protein samples were subjected to 10% SDS-PAGE (A), electroblotted and incubated with recombinant AvChit1 antibody (B). The same protein samples as in panel (A) were separated by 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity are identified as dark lytic zones under UV illumination (C). The AvChit1 (solid arrow) and signal bands showing chitinolytic activity (open arrow) are indicated on the right side of the panel. Molecular weight standards were used as size marker.
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Fig. 5. Southern blot analysis of A. ventricosus genomic DNA for AvChit1 gene. Genomic DNAs were digested with two restriction enzymes, EcoRI (lane 1) and HindIII (lane 2), and hybridized with radiolabelled AvChit1 cDNA. Size markers are shown on the left.
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Fig. 7. N-linked glycosylation of recombinant AvChit1 by tunicamycin treatment. Sf9 cells were infected with recombinant AcNPV at a MOI of 5 PFU per cell. Cells were treated without (lanes 1, 2, and 3) or with (lanes 4, 5, and 6) tunicamycin (5 Ag/mL). Total cellular lysates were collected at 1 (lanes 1 and 4), 2 (lanes 2 and 5), and 3 (lanes 3 and 6) days p.i. Total cellular lysates were subjected to 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity are identified as dark lytic zones under UV illumination. The N-glycosylated AvChit1 (Chi+) and carbohydrate-deficient AvChit1 (Chi ) are indicated by arrow on the right side of the panel.
3.5. N-Glycosylation of recombinant AvChit1 In order to assess whether the expected addition of an Nlinked carbohydrate moiety in the potential N-glycosylation site at Asn260 is being accomplished or not, infected cells were incubated in the presence of tunicamycin, which is a specific inhibitor of the addition of N-linked oligosaccharides. Then, total cellular lysates were subjected to SDSPAGE and chitinolytic activity assay (Fig. 7). Fig. 7 shows an apparent shift in molecular weight of the AvChit1 in the tunicamycin treated Sf9 cells. This result shows that the 61 kDa band and 58 kDa band corresponds to the N-glycosylated and nonglycosylated AvChit1. It indicates that AvChit1 adds an N-linked oligosaccharide. Furthermore, chitinolytic activity was detected in the protein bands of both N-glycosylated and nonglycosylated AvChit1, suggesting that the carbohydrate moieties are not essential for chitinolytic activity.
4. Discussion
Fig. 6. SDS-PAGE, Western blot analysis and chitinolytic activity assay of the recombinant AvChit1 expressed in baculovirus-infected insect cells. Sf9 cells were mock-infected (lane 1) or infected with wild-type AcNPV (lane 2) and recombinant AcNPV (lanes 3, 4, and 5) at a MOI of 5 PFU per cell. Cells were collected at 1 (lane 3), 2 (lanes 2 and 4), and 3 (lane 5) days p.i. Total cellular lysates were subjected to 10% SDS-PAGE (A), electroblotted and incubated with recombinant AvChit1 antibody (B). The same cellular lysates as in (A) were separated by 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity are identified as dark lytic zones under UV illumination (C). The recombinant AvChit1 is indicated by arrow on the right side of the panel. Molecular mass standards were used as size marker.
We have described an A. ventricosus cDNA, which shows sequence similarity to members of family 18 of the glycosyl hydrolase superfamily that comprises microbial, insect, nematode, mammal as well as some plant chitinases. Family 18 glycosyl hydrolases possess a consensus pattern in their primary amino acid sequence, (LIVMEY)(DN) G(LIVMF)(DN)(LIVMF)(DN)XE, in which glutamic acid is the critical active site residue. To test whether AvChit1 belongs to family 18 glycosyl hydrolases, we compared the inferred amino acid sequence with that of other members of the family. The AvChit1 ORF of 431 amino acid residues exhibits homology to various chitinases characterized from insects (Shen and Jacobs-Lorena, 1997; Girard and Jouanin, 1999; Hoskins et al., 2002; Yan et al., 2002; RamalhoOrtigao and Traub-Cseko, 2003) and to human chitotriosidase (Boot et al., 1995). The AvChit1 has the conserved catalytic site residues although the cysteine-rich 3V-end
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domain associated with chitin binding and the serine/ threonine rich domain are missing. The consensus pattern of family 18 glycosyl hydrolases is present in AvChit1, suggesting that this gene encodes a chitinase. One of the structural features observed in chitinases from several animals and microorganisms is a multidomain architecture that includes catalytic domains, a cysteine-rich chitin binding domain distinct from the catalytic domains, and a serine/ threonine-rich domain. The serine/threonine-rich domain is a characteristic of proteoglycans such as syndecans and mucines (Rayms-Keller et al., 2000). The C-terminal cysteine rich domain is found in insect and nematode chitinases as well as in several peritrophic matrix proteins (Barry et al., 1999). The function of this domain is presumably substrate binding, thereby facilitating the hydrolytic process (Yan et al., 2002). AvChit1 lacks both the serine/threonine-rich domain and chitin-binding domain, but has the catalytic consensus motifs. Recent works reported that some chitinases are missing for the serine/threonine rich domain (Yan et al., 2002) or both the serine/threonine and chitin binding domains (De la Vega et al., 1998; Girard and Jouanin, 1999). The expression of AvChit1 at transcriptional level performed by Northern blot analysis suggested that the AvChit1 gene shows the tissue-specific expression in the fat body. Similarly, enzyme assay and Western blot analysis confirmed a chitinolytic activity in the fat body and the presence of about 61 kDa AcChit1 polypeptide. It is likely that the fat body is a main site for AvChit1 synthesis. A fat body-specific chitinase cDNA was isolated from the tsetse fly, where the gene was suggested to play an immune related role (Yan et al., 2002). The AvChit1 is similar to the beetle chitinase from P. cochleariae, which is also missing for both the serine/threonine and chitin binding domains, but still enzymatically active (Girard and Jouanin, 1999). The expression profile of AvChit1 gene in A. ventricosus analyzed by Northern blot clearly showed a fat bodyspecific expression, similarly to that described in the result shown in the Western blot analysis. In addition, the genomic organization of the AvChit1 gene in A. ventricosus analyzed by Southern blot hybridization clearly showed a single band under high-stringency conditions when two restriction enzymes, which have no internal digesting site within AvChit1 cDNA, were employed, suggesting that the AvChit1 gene exists as a single copy in A. ventricosus genome. However, the protein bands displaying chitinolytic activity in enzyme assay were detected in both epidermis and midgut. This result strongly suggests that A. ventricosus has other chitinase genes in both epidermis and midgut as well as in fat body of A. ventricosus. Especially, the strong signal band with chitinolytic activity in the midgut suggests that the midgut also is a main site for chitinase synthesis. In addition, the result suggests the possible presence of another chitinase in the fat body that was recognized by the chitinolytic activity assay. Chitinases belonging to multigene families have been found in Aedes aegypti, A.
gambiae, and Drosophila (De la Vega et al., 1998). Several arthropod chitinase genes have been identified from midgut (Shen and Jacobs-Lorena, 1997; Ramalho-Ortigao and Traub-Cseko, 2003), epidermis (Fitches et al., 2004), and fat body (Yan et al., 2002). A recent study on immunohistochemical localization showed a strong reactivity in the cuticle epidermis and midgut of H. longicornis, illustrating that the H. longicornis chitinase is abundantly expressed in those tissues (You et al., 2003). From these data, our present result suggests that the spider, A. ventricosus, encodes at least three or four chitinase genes. To further confirm the AvChit1 gene, the recombinant AvChit1 was expressed in baculovirus-infected insect cells and assayed for chitinolytic activity. The recombinant AvChit1 is detected as a single band with about 61 kDa polypeptide in SDS-PAGE, and expression level and chitinolytic activity of the recombinant AvChit1 in baculovirus-infected insect cells clearly increased as culture period extends. However, the size of recombinant AvChit1 observed in the SDS-PAGE (61 kDa) did not correspond to the calculated molecular mass of the AvChit1 cDNA (48 kDa). The difference between the calculated and observed molecular mass on SDS-PAGE was larger than 10 kDa. Similarly, slower migration than predicted from the calculations in SDS-PAGE also was reported for the translation products of other chitinases (Kramer et al., 1993; Kim et al., 1998; Shinoda et al., 2001; Wu et al., 2001; You et al., 2003). This possibly may have caused by differences at the level of post-translational modification (Wu et al., 2001; You et al., 2003). In order to assess whether the expected addition of an Nlinked carbohydrate moiety in the potential N-glycosylation site at Asn260 is being accomplished or not, the recombinant virus-infected cells were incubated in the presence of tunicamycin, which is a specific inhibitor of the addition of N-linked oligosaccharides. An apparent shift in the molecular weight of the recombinant AvChit1 in the Sf9 cells treated with tunicamycin suggests that the 61 kDa polypeptide and 58 kDa polypeptide respectively correspond to the N-glycosylated and nonglycosylated recombinant AvChit1. The result indicates that the AvChit1 is truly N-glycosylated. Furthermore, the chitinolytic activity between Nglycosylated and nonglycosylated recombinant AvChit1 revealed no substantial difference in the enzyme activity, suggesting that the carbohydrate moieties are not essential for AvChit1 chitinolytic activity. A N-glycosylation study in recombinant Manduca sexta chitinase expressed in baculovirus-infected insect cells appears to suggest that the role of N-glycosylation is required for secretion of the protein into the medium (Gopalakrishnan et al., 1995). In addition, Nglycosylation in H. longicornis recombinant chitinase expressed in insect cells has been observed (You et al., 2003). In conclusion, the gene cloning, expression and molecular characterization of a novel fat body-specific chitinase in A. ventricosus are reported in this study. We showed that the
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N-glycosylation in AvChit1 is not essential for chitinolytic activity. These results may provide information for manipulating the chitinase as a potential control agent in the management of insect pests and plant pathogens. Acknowledgements This work was supported by a grant from Ministry of Agriculture and Forestry, Republic of Korea. References Barry, M.K., Triplett, A.A., Christensen, A.C., 1999. A peritrophin-like protein expressed in the embryonic tracheae of Drosophila melanogaster. Insect Biochem. Mol. Biol. 29, 319 – 327. Bonning, B.C., Hammock, B.D., 1996. Development of recombinant baculovirus for insect control. Annu. Rev. Entomol. 41, 191 – 210. Boot, R.G., Renkema, G.H., Strijland, A., Van Zonneveld, A.J., Aerts, J.M.F.G., 1995. Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages. J. Biol. Chem. 270, 26252 – 26256. Chung, E.H., Lee, K.S., Han, J.H., Sohn, H.D., Jin, B.R., 2001. Analysis of expressed sequence tags of the spider, Araneus ventricosus. Int. J. Ind. Entomol. 3, 191 – 199. De la Vega, H., Specht, C.A., Liu, Y., Robbins, P.W., 1998. Chitinases are a multi-gene family in Aedes, Anopheles and Drosophila. Insect Mol. Biol. 7, 309 – 319. Estruch, J.J., Carozzi, N.B., Desai, N., Duck, N.B., Warren, G.W., Koziel, M.G., 1997. Transgenic plants: an emerging approach to pest control. Nat. Biotechnol. 15, 137 – 141. Fitches, E., Wilkinson, H., Bell, H., Bown, D.P., Gatehouse, J.A., Edwards, J.P., 2004. Cloning, expression and functional characterization of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase. Insect Biochem. Mol. Biol. 34, 1037 – 1050. Flach, J., Pilet, P.E., Jolles, P., 1992. What’s new in chitinase research? Experientia 48, 701 – 716. Fukamizo, T., Kramer, K.J., 1985a. Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem. 15, 1 – 7. Fukamizo, T., Kramer, K.J., 1985b. Mechanism of chitin hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem. 15, 141 – 145. Girard, C., Jouanin, L., 1999. Molecular cloning of a gut-specific chitinase cDNA from the beetle Phaedon cochleariae. Insect Biochem. Mol. Biol. 29, 549 – 556. Gopalakrishnan, B., Muthukrishnan, S., Kramer, K.J., 1995. Baculovirusmediated expression of a Manduca sexta chitinase gene: properties of the recombinant protein. Insect Biochem. Mol. Biol. 25, 255 – 265. Hasemann, C.A., Capra, J.D., 1990. High-level production of a functional immunoglobulin heterodimer in a baculovirus expression system. Proc. Natl. Acad. Sci. U. S. A. 87, 3942 – 3946. Hoskins, R.A., Smith, C.D., Carlson, J.W., Carvalho, A.B., Halpern, A., Kaminker, J.S., Kennedy, C., Mungall, C.J., Sullivan, B.A., Sutton, G.G., Yasuhara, J.C., Wakimoto, B.T., Myers, E.W., Celniker, S.E., Rubin, G.M., Karpen, G.H., 2002. Heterochromatic sequences in a Drosophila whole genome shotgun assembly. Genome Biol. 3, Research, 0085.1 – 0085.16. Kim, J.P., Lee, Y.B., Jang, S.J., Kim, M.A., 1997. The studies of properties of matter and chemical qualitative analysis on web of Araneus ventricosus L. KOCH, 1878. Kor. J. Arachnol. 13, 87 – 91. Kim, M.G., Shin, S.W., Bae, K.S., Kim, S.C., Park, H.Y., 1998. Molecular cloning of chitinase cDNAs from the silkworm, Bombyx
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mori and the fall webworm, Hyphantria cunea. Insect Biochem. Mol. Biol. 28, 163 – 171. Kramer, K.J., Muthukrishnan, S., 1997. Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem. Mol. Biol. 27, 887 – 900. Kramer, K.J., Corpuz, L., Choi, H.K., Muthukrishnan, S., 1993. Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta. Insect Biochem. Mol. Biol. 23, 691 – 701. Laemmli, U.K., 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lee, K.S., Chung, E.H., Han, J.H., Sohn, H.D., Jin, B.R., 2003. cDNA cloning of a defender against apoptotic cell death 1 (DAD1) homologue, responsive to external temperature stimulus from the spider, Araneus ventricosus. Comp. Biochem. Physiol., B 135, 117 – 123. McMaster, G.K., Carmichael, G.G., 1977. Analysis of single- and double-stranded nucleic acids on polyacryamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. U. S. A. 74, 4835 – 4838. Mommsen, T.P., 1978. Digestive enzymes of a spider (Tegenaria atrica Koch): II. Carbohydrates. Comp. Biochem. Physiol., A 60, 371 – 375. Mommsen, T.P., 1980. Chitinase and h-N-acetylglucosaminidase from the digestive fluid of the spider, Cupiennius salei. Biochim. Biophys. Acta 612, 361 – 372. O’Reilly, D.R., Miller, L.K., Luckow, V.A., 1992. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman, New York. Ramalho-Ortigao, J.M., Traub-Cseko, Y.M., 2003. Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. Insect Biochem. Mol. Biol. 33, 279 – 287. Rayms-Keller, A., McGaw, M., Oray, C., Carlson, J.O., Beaty, B.J., 2000. Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA. Insect Mol. Biol. 9, 419 – 426. Shen, Z., Jacobs-Lorena, M., 1997. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J. Biol. Chem. 272, 28895 – 28900. Shinoda, T., Kobayashi, J., Matsui, M., Chinzei, Y., 2001. Cloning and functional expression of a chitinase cDNA from the common cutworm, Spodoptera litura, using a recombinant baculovirus lacking the virusencoded chitinase gene. Insect Biochem. Mol. Biol. 31, 521 – 532. Swofford, D.L., 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4. Sinauer Sunderland, Massachuset. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350 – 4354. Trudel, J., Asselin, A., 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 178, 362 – 366. Van Scheltinga, A.C.T., Kalk, K.H., Beintema, J.J., Dijkstra, B.W., 1994. Crystal structures of hevamine, a plant defense protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure 2, 1181 – 1189. Vaughn, J.L., Goodwin, R.H., Thompkins, G.J., McCawley, P., 1977. The establishment of two insect cell lines from the insect Spodoptera Frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213 – 217. Von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683 – 4690. Wu, Y., Gillian, G., Underwood, A.P., Sakuda, S., Bianco, A.E., 2001. Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus. J. Biol. Chem. 276, 42557 – 42564. Yan, J., Cheng, Q., Narashimhan, S., Li, C.B., Aksoy, S., 2002. Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans. Insect Biochem. Mol. Biol. 32, 979 – 989. You, M., Xuan, X., Tsuji, N., Kamio, T., Taylor, D., Suzuki, N., Fujisaki, K., 2003. Identification and molecular characterization of a chitinase from the hard tick Haemaphysalis longicornis. J. Biol. Chem. 278, 8556 – 8563.
Cloning and expression of a fat body-specific chitinase cDNA from the spider, Araneus ventricosus Ji Hee Hana, Kwang Sik Leea, Jianhong Lia,1, Iksoo Kimb, Yeon Ho Jec, Doh Hoon Kima, Hung Dae Sohna, Byung Rae Jina,* b
a College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Korea Department of Agricultural Biology, National Institute of Agricultural Science and Technology, Suwon 441-100, Korea c School of Agricultural Biotechnology, Seoul National University, Seoul 151-742, Korea
Received 6 August 2004; received in revised form 9 November 2004; accepted 14 November 2004
Abstract A fat body-specific chitinase cDNA was cloned from the spider, Araneus ventricosus. The cDNA encoding A. ventricosus chitinase (AvChit1) is 1515 bp long with an open reading frame (ORF) of 431 amino acid residues. AvChit1 possesses the chitinase family 18 active site signature and one N-glycosylation site. The deduced amino acid sequence of AvChit1 cDNA showed 43% identity to both Glossina morsitans morsitans chitinase and a human chitotriosidase, and 30–40% to some insect chitinases which lack both the serine/threonine and chitin binding domains. Southern blot analysis of genomic DNA suggested the presence of AvChit1 gene as a single copy. Northern and Western blot analysis and enzyme activity assay showed the tissue-specific expression of AvChit1 in the A. ventricosus fat body. The AvChit1 cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells and the recombinant AvChit1 showed activity in the chitinase enzyme assay using 0.1% glycol chitin as a substrate. Treatment of recombinant virus-infected Sf9 cells with tunicamycin, a specific inhibitor of N-glycosylation, revealed that AvChit1 is N-glycosylated, but the carbohydrate moieties are not essential for chitinolytic activity. D 2004 Elsevier Inc. All rights reserved. Keywords: Araneus ventricosus; Baculovirus; cDNA; Chitinase; N-Glycosylation; Glycosyl hydrolase; Insect cells; Spider
1. Introduction Chitin is one of the most abundant polysaccharides in nature and is a linear polymer of h (1Y4) linked N– acetylglucosamine (GlcNAc) residues. It is one of the most unique biochemical constituents found in the exoskeletons and gut linings of arthropods and fungi. Chitinolytic enzymes that catalyze the hydrolysis of chitin have been found in chitin-containing organisms as well as in microorganisms that do not have chitin. The enzymes, chitinases, from various organisms have various biological functions. Chitinase plays roles in the molting process of invertebrates, * Corresponding author. Tel./fax: +82 51 200 7594. E-mail address: [email protected] (B.R. Jin). 1 Present address: College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. 1096-4959/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2004.11.009
including spiders, the digestion of chitinous food, and defense against chitin-bearing pathogens (Flach et al., 1992; Kramer and Muthukrishnan, 1997). Chitin degradation is a complex process and is catalyzed by a two-component chitinolytic enzyme system, chitinase (EC 3.2.1.14), and h-N-acetylglucosaminidase (EC 3.2.1.30) (Fukamizo and Kramer, 1985a,b). The two enzymes exhibit a synergism in the hydrolysis process of chitin. Chitinase activity has been found with bacteria, fungi, plants, invertebrates—including spiders (Mommsen, 1978, 1980)—and vertebrates, and chitinase genes have been also isolated from several organisms. Chitinase genes offer several opportunities for gene manipulation in a variety of purposes; that is, the enhancement of host plant resistance and pathogenicity in transgenic plants and biological control agents (Bonning and Hammock, 1996; Estruch et al., 1997; Kramer and Muthukrishnan, 1997).
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Furthermore, a recent study showed the possible application of tick chitinase in the control of tick vector (You et al., 2003). In order to extend our understanding of the chitinase genes in arthropods, a novel fat body-specific chitinase cDNA from the spider Araneus ventricosus was isolated and characterized in this research. The spider, A. ventricosus, is the abundant species in Korea (Kim et al., 1997). The body size of A. ventricosus is relatively larger than that of the other species. The mechanical properties of the web of A. ventricosus have been reported (Kim et al., 1997). The spider chitinase cDNA was expressed in baculovirusinfected insect cells and the activity of the recombinant enzyme was determined. In addition, we showed that Nglycosylation in A. ventricosus chitinase is not essential for chitinolytic activity.
2. Materials and methods 2.1. Animals Spiders, A. ventricosus, were collected at Namhae, Kyungnam province in Korea. The live spiders were directly used in this study. 2.2. cDNA library screening, nucleotide sequencing, and data analysis A cDNA library (Chung et al., 2001) was constructed using whole bodies of A. ventricosus. Sequencing of randomly selected clones harboring cDNA inserts was performed to generate the expressed sequence tags (ESTs). For DNA sequencing, plasmid DNA was extracted by Wizard mini-preparation kit (Promega, Madison, WI). Sequence of each cDNA clone was determined using an automatic sequencer (model 310 Genetic Analyzer; Perkin-Elmer Applied Biosystems, Foster City, CA). The sequences were compared using the DNASIS and BLAST programs provided by the NCBI, GenBank, EMBL, and SwissProt databases were searched for sequence homology using a BLAST algorithm program (www.ncbi.nlm. nih.gov/BLAST). MacVector (ver. 6.5, Oxford Molecular) was used to align the amino acid sequences of chitinase gene. With the GenBank-registered chitinase amino acid sequences, phylogenetic analysis was performed using PAUP (Phylogenetic Analysis Using Parsimony) version 4.0 (Swofford, 2000). 2.3. RNA isolation and Northern blot analysis Mature adults of the spider A. ventricosus were dissected under a stereo-microscope (Zeiss, Jena, Germany), individual samples such as fat body, midgut, and silk gland were harvested, and washed twice with PBS (140 mM NaCl, 27 mM KCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). Total
RNA was isolated from the whole body, midgut, fat body, and silk gland of A. ventricosus by using the Total RNA Extraction Kit (Promega). Total RNA (10 Ag/lane) from A. ventricosus was denatured by glyoxylation (McMaster and Carmichael, 1977), transferred onto a nylon blotting membrane (Schleicher and Schuell, Dassel, Germany), and hybridized at 42 8C with a probe in a hybridization buffer containing 5 SSC, 5 Denhardt’s solution, 0.5% SDS, and 100 Ag/ml denatured salmon sperm DNA. The 1515-bp A. ventricosus chitinase cDNA clone was labeled with [a-32P] dCTP (Amersham, Arlington Heights, IL, USA) using the Prime-It II Random Primer Labeling Kit (Stratagene, La Jolla, CA, USA) for use as a probe for hybridization. After hybridization, the membrane filter was washed three times for 30 min each in 0.1% SDS and 0.2 SSC (1SSC is 0.15 M NaCl and 0.015 M sodium citrate) at 65 8C and exposed to autoradiography film. For rehybridization, the membrane was washed for 20 min at room temperature in sterile millipore water. Then, the membrane was washed overnight at 65 8C in 50 mM TrisHCl (pH 8.0), 50% dimethylformamide and 1% SDS in order to remove the hybridized probe. The membrane was then rehybridized to [a-32P] dCTP-labeled 28S rRNA probe (Lee et al., 2003). The 28S rRNA gene was used as an internal loading control. 2.4. Genomic DNA isolation and Southern blot analysis Genomic DNA was extracted from the fat body of A. ventricosus adult using a Wizardk Genomic DNA Purification Kit, according to the manufacturer’s instructions (Promega). Genomic DNA from A. ventricosus was digested with EcoRI and HindIII, and electrophoresed in 1.0% agarose gel. The DNA from the gel was transferred onto a nylon blotting membrane (Schleicher and Schuell) and hybridized at 42 8C with a probe in a hybridization buffer. Hybridization condition, fragment labeling, and filter washing were as described for the Northern blot analysis. 2.5. Cell culture and construction of recombinant virus Spodoptera frugiperda IPLB Sf21-AE (Vaughn et al., 1977) clone 9 (Sf9) cells were maintained at 27 8C in TC100 medium (GIBCO BRL LIFE Technologies), supplemented with 10% fetal bovine serum (FBS; GIBCO BRL LIFE Technologies) as described by standard methods (O’Reilly et al., 1992). Wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) and recombinant AcNPV were propagated in Sf9 cells. The titer was expressed as plaque forming units (PFU) per ml (O’Reilly et al., 1992). The 1515-bp A. ventricosus chitinase cDNA from pBlueScript–AvChit was digested with SacI and KpnI, and then inserted into the SacI and KpnI sites of pBacPAK9 (Clontech, Palo Alto, CA) to produce baculovirus transfer
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vector pBacPAK9–AvChit. In the transfer vector, the chitinase cDNA is under the control of the AcNPV polyhedrin promoter. For the construction of recombinant virus, 35-mm cell culture dishes were seeded with 1.0–1.5106 cells and incubated at 27 8C for 1 h to allow cell attachment. Total of 1 Ag of BacPAK6 viral DNA (Clontech) and 5 Ag of pBacPAK9–AvChit in 20 mM HEPES buffer in a final volume of 50 Al were mixed in a polystyrene tube. Moreover, 50 Al of 100 Ag/ml Lipofectink (GIBCO BRL LIFE Technologies) was gently mixed with the DNA solution and the mixture was incubated at room temperature for 30 min. The cells were washed twice with 2 ml serumfree TC100 medium and refed with 1.5 ml serum-free TC100 medium. The Lipofectin-DNA complexes were added dropwise to the medium covering the cells while the dish was gently swirled. After incubation at 27 8C for 5 h, TC100 medium containing antibiotics and 10% FBS was added to each dish and incubation at 27 8C was continued. At 5 days p.i., the supernatant was harvested, clarified by centrifugation at 2,000 rpm for 5 min, and stored at 4 8C. Recombinant AcNPV was plaque purified on 6-well plates seeded with 1.5106 Sf9 cells as described previously (O’Reilly et al., 1992). 2.6. Preparation of polyclonal antibody and Western blot analysis Recombinant A. ventricosus chitinase was electroeluted from the SDS-polyacrylamide gel electrophoresis (PAGE) gel, mixed with equal volume of Freund’s complete adjuvant (a total of 200 Al, Sigma) and injected into Balb/c mice, respectively. Three successive injections were performed with 1-week interval beginning a week after the first injection with antigens mixed with equal volume of Freund’s incomplete adjuvant (a total of 200 Al, Sigma). Bloods were collected 3 days after the last injection and centrifuged at 13,000g for 5 min. The supernatant antibodies were stored at 70 8C until use. For Western blot analysis, the protein samples were subjected to 10% SDS-PAGE (Laemmli, 1970). Proteins were blotted to a sheet of nitrocellulose membrane (Sigma, 0.45 Am of pore size) (Towbin et al., 1979). The blotting was performed in transfer buffer (25 mM Tris and 192 mM glycine in 20% methanol) at 30 V overnight at 4 8C. After blotting, the membrane was blocked by incubation in 1% BSA solution for 2 h at room temperature. The blocked membrane was incubated with antiserum solution (1:1000 v/v) for 1 h at room temperature and washed in TBST (10 mM Tris–HCl, pH 8.0, 100 mM NaCl, 0.05% Tween 20). The membrane was then incubated with anti-mouse IgG horseradish peroxidase (HRP) conjugate and HRP-streptavidin complex. After repeated washing, the membrane was incubated with ECL detection reagents (Amersham Pharmacia Biotech) and exposed to autoradiography film.
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2.7. Determination of chitinase activity Chitinase activity was detected from the gel after SDS-PAGE by the method of Trudel and Asselin (1989). Gel was incubated in 150 mM sodium acetate buffer at pH 5.0 for 5 min. The gel was then put on a glass plate and covered with a 7.5% polyacrylamide overlay gel containing 0.01% (w/v) glycol chitin in 100 mM sodium acetate buffer (pH 5.0). Gel was incubated at 37 8C for 1 h under the moist conditions. Lytic zones were visualized by placing the gels on a UV illuminator and were photographed. 2.8. Tunicamycin treatment The addition of N-linked carbohydrate by infected insect cells was verified by culture in the presence of tunicamycin (5 Ag/ml, Sigma) to prevent the addition of N-linked carbohydrate (Hasemann and Capra, 1990). Sf9 cells were mock infected or infected with wild-type AcNPV or recombinant viruses in a 35-mm-diameter dish (1106 cells) and were incubated for 2 h at 27 8C. The supernatants were replaced with 5 ml of supplemented TC100 medium containing 5 Ag tunicamycin/ml medium. After incubation 27 8C, total cellular lysates was harvested from infected cells at 24, 48, and 72 h p.i. Total cellular lysates were subjected to 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity were identified as dark lytic zones under UV illumination (Trudel and Asselin, 1989).
3. Results 3.1. Cloning and sequencing of AvChit1 cDNA A cDNA library was constructed using whole bodies of A. ventricosus (Chung et al., 2001). Sequencing of randomly selected clones harboring cDNA inserts was performed to generate the A. ventricosus ESTs. One clone, which is 1515 bp long had a full-length coding sequence similar to that of previously reported chitinases. The deduced amino acid sequences of a cDNA encoding the A. ventricosus chitinase (AvChit1) are presented in Fig. 1. The AvChit1 cDNA contains an open reading frame (ORF) of 1293 bp encoding 431 amino acid residues. A multiple sequence alignment of the deduced protein sequence of AvChit1 with other chitinase sequences is shown in Fig. 1. Alignment of the AvChit1 sequences with those of chitinase from several other species indicates the extent of the identity that exists. AvChit1 conserved the chitinase family 18 active site signature, which is a consensus sequence, [LIVMFY]-[DN]-G[LIVMF]-[DN]-[LIVMF]-[DN]-X-E (Van Scheltinga et al., 1994). The deduced AvChit1 N-terminal sequence con-
430 J.H. Han et al. / Comparative Biochemistry and Physiology, Part B 140 (2005) 427–435 Fig. 1. Alignment of the amino acid sequence of AvChit1 cDNA with known chitinases. Residues are numbered according to the aligned chitinase sequences, and invariant residues are shaded black. Dots represent gaps introduced to improve alignment. Chitinase family 18 active site signature is marked by asterisk. The arrow shows the end of the signal peptides. The N-glycosylation site is indicated with the cross. The GenBank accession numbers of AvChit1 cDNA is AY120879. The abbreviation and GenBank accession number for the chitinase sequences aligned are: AvChit1, Araneus ventricosus chitinase (AY120879; this study); Gchit1, Glossina morsitans morsitans chitinase (AF337908); Llchit1, Lutzomyia longipalpis chitinase (AY148807); AgChi-1, Anopheles gambiae chitinase (AF008575); PcChit1, Phaedon cochleariae chitinase (Y18011), DmCHT1, Drosophila melanogaster chitinase (Q9W5U3); HsChit, Homo sapiens chitotriosidase (U62662).
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tains a highly hydrophobic amino acid stretch that is likely to function as a signal peptide. Using Von Heijne’s (1986) rule, we predict that the signal peptide is cleaved after Ala-19. AvChit1 possesses one putative N-glycosylation site at the amino acid residues 260–263 (NGTP). A phylogenetic analysis using the deduced amino acid sequences of known chitinase genes revealed that the chitinases are divided into three groups (Fig. 2A). The AvChit1 formed a subgroup together with chitinase genes from insect species such as Glossina morsitans morsitans, Lutzomyia longipalpis, Anopheles gambiae, Phaedon cochleariae, and Drosophila melanogaster, and a human chitotriosidase, which lack both the serine/threonine and chitin binding domains. Similarly, the deduced protein sequence of the AvChit1 showed 43% protein sequence identity to both G. morsitans morsitans chitinase and human chitotriosidase, and 30–40% to insect chitinases within the subgroup (Fig. 2B).
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3.2. Tissue-specific expression of AvChit1 The tissue specific nature of AvChit1 expression was determined from fat body, midgut, silk gland and epidermis by Northern blot analysis (Fig. 3). AvChit1 was found to be expressed only in fat body and not in midgut and epidermis of A. ventricosus, evidencing the fat body as a specific site for AvChit1 synthesis. Furthermore, tissue specific expression of AvChit1 was analyzed from the protein samples of epidermis, fat body, and midgut of A. ventricosus. Each protein sample was subjected to 10% SDS-PAGE (Fig. 4A) and Western blot analysis (Fig. 4B). A signal band of 61 kDa was detected specifically from the fat body in the Western blot analysis, but not in the epidermis and midgut protein samples. The protein band with a 61 kDa also showed chitinolytic activity (lane 2 of Fig. 4C). The result is in good agreement with the Northern blot hybridization result that AvChit1 showed the
Fig. 2. Relationships among amino acid sequences of AvChit1 and known chitinases. (A) A maximum parsimony analysis for the amino acid sequences of AvChit1 and known chitinases. The tree was obtained by bootstrap analysis with the option of heuristic search and the numbers on the branches represent bootstrap values for 1000 replicates. Outgroup was chosen as bacterium Streptomyces olivaceoviridis exochitinase (SoChit; Q05638). The abbreviation and GenBank accession number for the chitinase sequences analyzed are: AvChit1, A. ventricosus chitinase (AY120879; this study); GChit1, G. morsitans morsitans chitinase (AF337908); Llchit1, L. longipalpis chitinase (AY148807); AgChi-1, A. gambiae chitinase (AF008575); PcChit1, P. cochleariae chitinase (Y18011); DmCHT1, D. melanogaster chitinase (Q9W5U3); HsChit, H. sapiens chitotriosidase (U62662); MmAMCase, Mus musculus acidic mammalian chitinase (AF290003); HsAMCase, H. sapiens acidic mammalian chitinase (AF290004); BtAMCase, Bos taurus acidic mammalian chitinase (Q95M17); fChi2, Paralichthys olivaceus chitinase (AB121733); HlChit, Haemaphysalis longicornis chitinase (ABO74977). (B) Pairwise identities and similarities of the deduced amino acid sequence of AvChit1 cDNA among chitinase sequences.
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analyze the protein synthesis in Sf9 cells infected with the recombinant virus. The recombinant AvChit1 was present as a single band of about 61 kDa polypeptide in the cells infected with the recombinant virus, but not in the cells infected with the wild-type AcNPV or mock-infected cells. In SDS-polyacrylamide gels containing 0.01% glycol chitin, the protein bands with chitinolytic activity were directly detected as dark lytic zones under UV illumination. The chitinolytic activity was detected in the recombinant AvChit1 with a 61 kDa from recombinant virus-infected insect cells (Fig. 6C). Fig. 3. Northern blot analysis of AvChit1 gene. Total RNA was isolated from the fat body (lane 1), midgut (lane 2), silk gland (lane 3), and epidermis (lane 4), respectively. The RNA was separated by 1.0% formaldehyde agarose gel electrophoresis (upper panel), transferred on to a nylon membrane, and hybridized with the appropriate radiolabelled probe (middle panel). The 28S rRNA gene was used as an internal loading control (lower panel). Transcripts are indicated by arrow on the right side of the panel.
tissue-specific expression in the fat body. However, the protein bands displaying chitinolytic activity in enzyme assay were detected in both epidermis (approximately 60 kDa) and midgut (approximately 40 kDa; Fig. 4C). This result suggests the presence of other chitinase genes in both epidermis and midgut as well as in fat body of A. ventricosus. In addition, the chitinolytic activity assay suggests that A. ventricosus has another chitinase (approximately 47 kDa) in the fat body (lane 2 of Fig. 4C). 3.3. Copy number of AvChit1 gene To determine the number of AvChit1 gene in the A. ventricosus genome, genomic DNA was digested with restriction enzymes, which do not cut within the chitinase gene, blotted, and hybridized with the AvChit1 cDNA. Single hybridizing bands were detected with all enzymes indicating the AvChit1 is a single gene (Fig. 5). 3.4. Expression of AvChit1 cDNA in baculovirus-infected insect cells To assess AvChit1 cDNA, the 1515 bp for AvCht1 cDNA was inserted into baculovirus transfer vector. The baculovirus transfer vector was used to generate a recombinant virus expressing AvChit1. Transfer vector pBacPAK9– AvChit was constructed by insertion of AvChit1 cDNA under the control of AcNPV polyhedrin promoter of pBacPAK9 (data not shown). Recombinant AcNPV, which we have termed AcNPV–AvChit1, was produced in insect Sf9 cells by cotransfection with wild-type AcNPV DNA and the transfer vector. To examine the expression of AvChit1 cDNA by recombinant virus in insect cells, SDS-PAGE (Fig. 6A) and Western blot analysis (Fig. 6B) were performed to
Fig. 4. Tissue-specific expression of AvChit1. The protein samples were collected from epidermis (lane 1), fat body (lane 2), and midgut (lane 3) of A. ventricosus. The protein samples were subjected to 10% SDS-PAGE (A), electroblotted and incubated with recombinant AvChit1 antibody (B). The same protein samples as in panel (A) were separated by 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity are identified as dark lytic zones under UV illumination (C). The AvChit1 (solid arrow) and signal bands showing chitinolytic activity (open arrow) are indicated on the right side of the panel. Molecular weight standards were used as size marker.
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Fig. 5. Southern blot analysis of A. ventricosus genomic DNA for AvChit1 gene. Genomic DNAs were digested with two restriction enzymes, EcoRI (lane 1) and HindIII (lane 2), and hybridized with radiolabelled AvChit1 cDNA. Size markers are shown on the left.
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Fig. 7. N-linked glycosylation of recombinant AvChit1 by tunicamycin treatment. Sf9 cells were infected with recombinant AcNPV at a MOI of 5 PFU per cell. Cells were treated without (lanes 1, 2, and 3) or with (lanes 4, 5, and 6) tunicamycin (5 Ag/mL). Total cellular lysates were collected at 1 (lanes 1 and 4), 2 (lanes 2 and 5), and 3 (lanes 3 and 6) days p.i. Total cellular lysates were subjected to 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity are identified as dark lytic zones under UV illumination. The N-glycosylated AvChit1 (Chi+) and carbohydrate-deficient AvChit1 (Chi ) are indicated by arrow on the right side of the panel.
3.5. N-Glycosylation of recombinant AvChit1 In order to assess whether the expected addition of an Nlinked carbohydrate moiety in the potential N-glycosylation site at Asn260 is being accomplished or not, infected cells were incubated in the presence of tunicamycin, which is a specific inhibitor of the addition of N-linked oligosaccharides. Then, total cellular lysates were subjected to SDSPAGE and chitinolytic activity assay (Fig. 7). Fig. 7 shows an apparent shift in molecular weight of the AvChit1 in the tunicamycin treated Sf9 cells. This result shows that the 61 kDa band and 58 kDa band corresponds to the N-glycosylated and nonglycosylated AvChit1. It indicates that AvChit1 adds an N-linked oligosaccharide. Furthermore, chitinolytic activity was detected in the protein bands of both N-glycosylated and nonglycosylated AvChit1, suggesting that the carbohydrate moieties are not essential for chitinolytic activity.
4. Discussion
Fig. 6. SDS-PAGE, Western blot analysis and chitinolytic activity assay of the recombinant AvChit1 expressed in baculovirus-infected insect cells. Sf9 cells were mock-infected (lane 1) or infected with wild-type AcNPV (lane 2) and recombinant AcNPV (lanes 3, 4, and 5) at a MOI of 5 PFU per cell. Cells were collected at 1 (lane 3), 2 (lanes 2 and 4), and 3 (lane 5) days p.i. Total cellular lysates were subjected to 10% SDS-PAGE (A), electroblotted and incubated with recombinant AvChit1 antibody (B). The same cellular lysates as in (A) were separated by 10% SDS-PAGE containing 0.01% glycol chitin. The proteins with chitinolytic activity are identified as dark lytic zones under UV illumination (C). The recombinant AvChit1 is indicated by arrow on the right side of the panel. Molecular mass standards were used as size marker.
We have described an A. ventricosus cDNA, which shows sequence similarity to members of family 18 of the glycosyl hydrolase superfamily that comprises microbial, insect, nematode, mammal as well as some plant chitinases. Family 18 glycosyl hydrolases possess a consensus pattern in their primary amino acid sequence, (LIVMEY)(DN) G(LIVMF)(DN)(LIVMF)(DN)XE, in which glutamic acid is the critical active site residue. To test whether AvChit1 belongs to family 18 glycosyl hydrolases, we compared the inferred amino acid sequence with that of other members of the family. The AvChit1 ORF of 431 amino acid residues exhibits homology to various chitinases characterized from insects (Shen and Jacobs-Lorena, 1997; Girard and Jouanin, 1999; Hoskins et al., 2002; Yan et al., 2002; RamalhoOrtigao and Traub-Cseko, 2003) and to human chitotriosidase (Boot et al., 1995). The AvChit1 has the conserved catalytic site residues although the cysteine-rich 3V-end
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domain associated with chitin binding and the serine/ threonine rich domain are missing. The consensus pattern of family 18 glycosyl hydrolases is present in AvChit1, suggesting that this gene encodes a chitinase. One of the structural features observed in chitinases from several animals and microorganisms is a multidomain architecture that includes catalytic domains, a cysteine-rich chitin binding domain distinct from the catalytic domains, and a serine/ threonine-rich domain. The serine/threonine-rich domain is a characteristic of proteoglycans such as syndecans and mucines (Rayms-Keller et al., 2000). The C-terminal cysteine rich domain is found in insect and nematode chitinases as well as in several peritrophic matrix proteins (Barry et al., 1999). The function of this domain is presumably substrate binding, thereby facilitating the hydrolytic process (Yan et al., 2002). AvChit1 lacks both the serine/threonine-rich domain and chitin-binding domain, but has the catalytic consensus motifs. Recent works reported that some chitinases are missing for the serine/threonine rich domain (Yan et al., 2002) or both the serine/threonine and chitin binding domains (De la Vega et al., 1998; Girard and Jouanin, 1999). The expression of AvChit1 at transcriptional level performed by Northern blot analysis suggested that the AvChit1 gene shows the tissue-specific expression in the fat body. Similarly, enzyme assay and Western blot analysis confirmed a chitinolytic activity in the fat body and the presence of about 61 kDa AcChit1 polypeptide. It is likely that the fat body is a main site for AvChit1 synthesis. A fat body-specific chitinase cDNA was isolated from the tsetse fly, where the gene was suggested to play an immune related role (Yan et al., 2002). The AvChit1 is similar to the beetle chitinase from P. cochleariae, which is also missing for both the serine/threonine and chitin binding domains, but still enzymatically active (Girard and Jouanin, 1999). The expression profile of AvChit1 gene in A. ventricosus analyzed by Northern blot clearly showed a fat bodyspecific expression, similarly to that described in the result shown in the Western blot analysis. In addition, the genomic organization of the AvChit1 gene in A. ventricosus analyzed by Southern blot hybridization clearly showed a single band under high-stringency conditions when two restriction enzymes, which have no internal digesting site within AvChit1 cDNA, were employed, suggesting that the AvChit1 gene exists as a single copy in A. ventricosus genome. However, the protein bands displaying chitinolytic activity in enzyme assay were detected in both epidermis and midgut. This result strongly suggests that A. ventricosus has other chitinase genes in both epidermis and midgut as well as in fat body of A. ventricosus. Especially, the strong signal band with chitinolytic activity in the midgut suggests that the midgut also is a main site for chitinase synthesis. In addition, the result suggests the possible presence of another chitinase in the fat body that was recognized by the chitinolytic activity assay. Chitinases belonging to multigene families have been found in Aedes aegypti, A.
gambiae, and Drosophila (De la Vega et al., 1998). Several arthropod chitinase genes have been identified from midgut (Shen and Jacobs-Lorena, 1997; Ramalho-Ortigao and Traub-Cseko, 2003), epidermis (Fitches et al., 2004), and fat body (Yan et al., 2002). A recent study on immunohistochemical localization showed a strong reactivity in the cuticle epidermis and midgut of H. longicornis, illustrating that the H. longicornis chitinase is abundantly expressed in those tissues (You et al., 2003). From these data, our present result suggests that the spider, A. ventricosus, encodes at least three or four chitinase genes. To further confirm the AvChit1 gene, the recombinant AvChit1 was expressed in baculovirus-infected insect cells and assayed for chitinolytic activity. The recombinant AvChit1 is detected as a single band with about 61 kDa polypeptide in SDS-PAGE, and expression level and chitinolytic activity of the recombinant AvChit1 in baculovirus-infected insect cells clearly increased as culture period extends. However, the size of recombinant AvChit1 observed in the SDS-PAGE (61 kDa) did not correspond to the calculated molecular mass of the AvChit1 cDNA (48 kDa). The difference between the calculated and observed molecular mass on SDS-PAGE was larger than 10 kDa. Similarly, slower migration than predicted from the calculations in SDS-PAGE also was reported for the translation products of other chitinases (Kramer et al., 1993; Kim et al., 1998; Shinoda et al., 2001; Wu et al., 2001; You et al., 2003). This possibly may have caused by differences at the level of post-translational modification (Wu et al., 2001; You et al., 2003). In order to assess whether the expected addition of an Nlinked carbohydrate moiety in the potential N-glycosylation site at Asn260 is being accomplished or not, the recombinant virus-infected cells were incubated in the presence of tunicamycin, which is a specific inhibitor of the addition of N-linked oligosaccharides. An apparent shift in the molecular weight of the recombinant AvChit1 in the Sf9 cells treated with tunicamycin suggests that the 61 kDa polypeptide and 58 kDa polypeptide respectively correspond to the N-glycosylated and nonglycosylated recombinant AvChit1. The result indicates that the AvChit1 is truly N-glycosylated. Furthermore, the chitinolytic activity between Nglycosylated and nonglycosylated recombinant AvChit1 revealed no substantial difference in the enzyme activity, suggesting that the carbohydrate moieties are not essential for AvChit1 chitinolytic activity. A N-glycosylation study in recombinant Manduca sexta chitinase expressed in baculovirus-infected insect cells appears to suggest that the role of N-glycosylation is required for secretion of the protein into the medium (Gopalakrishnan et al., 1995). In addition, Nglycosylation in H. longicornis recombinant chitinase expressed in insect cells has been observed (You et al., 2003). In conclusion, the gene cloning, expression and molecular characterization of a novel fat body-specific chitinase in A. ventricosus are reported in this study. We showed that the
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N-glycosylation in AvChit1 is not essential for chitinolytic activity. These results may provide information for manipulating the chitinase as a potential control agent in the management of insect pests and plant pathogens. Acknowledgements This work was supported by a grant from Ministry of Agriculture and Forestry, Republic of Korea. References Barry, M.K., Triplett, A.A., Christensen, A.C., 1999. A peritrophin-like protein expressed in the embryonic tracheae of Drosophila melanogaster. Insect Biochem. Mol. Biol. 29, 319 – 327. Bonning, B.C., Hammock, B.D., 1996. Development of recombinant baculovirus for insect control. Annu. Rev. Entomol. 41, 191 – 210. Boot, R.G., Renkema, G.H., Strijland, A., Van Zonneveld, A.J., Aerts, J.M.F.G., 1995. Cloning of a cDNA encoding chitotriosidase, a human chitinase produced by macrophages. J. Biol. Chem. 270, 26252 – 26256. Chung, E.H., Lee, K.S., Han, J.H., Sohn, H.D., Jin, B.R., 2001. Analysis of expressed sequence tags of the spider, Araneus ventricosus. Int. J. Ind. Entomol. 3, 191 – 199. De la Vega, H., Specht, C.A., Liu, Y., Robbins, P.W., 1998. Chitinases are a multi-gene family in Aedes, Anopheles and Drosophila. Insect Mol. Biol. 7, 309 – 319. Estruch, J.J., Carozzi, N.B., Desai, N., Duck, N.B., Warren, G.W., Koziel, M.G., 1997. Transgenic plants: an emerging approach to pest control. Nat. Biotechnol. 15, 137 – 141. Fitches, E., Wilkinson, H., Bell, H., Bown, D.P., Gatehouse, J.A., Edwards, J.P., 2004. Cloning, expression and functional characterization of chitinase from larvae of tomato moth (Lacanobia oleracea): a demonstration of the insecticidal activity of insect chitinase. Insect Biochem. Mol. Biol. 34, 1037 – 1050. Flach, J., Pilet, P.E., Jolles, P., 1992. What’s new in chitinase research? Experientia 48, 701 – 716. Fukamizo, T., Kramer, K.J., 1985a. Mechanism of chitin oligosaccharide hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem. 15, 1 – 7. Fukamizo, T., Kramer, K.J., 1985b. Mechanism of chitin hydrolysis by the binary enzyme chitinase system in insect moulting fluid. Insect Biochem. 15, 141 – 145. Girard, C., Jouanin, L., 1999. Molecular cloning of a gut-specific chitinase cDNA from the beetle Phaedon cochleariae. Insect Biochem. Mol. Biol. 29, 549 – 556. Gopalakrishnan, B., Muthukrishnan, S., Kramer, K.J., 1995. Baculovirusmediated expression of a Manduca sexta chitinase gene: properties of the recombinant protein. Insect Biochem. Mol. Biol. 25, 255 – 265. Hasemann, C.A., Capra, J.D., 1990. High-level production of a functional immunoglobulin heterodimer in a baculovirus expression system. Proc. Natl. Acad. Sci. U. S. A. 87, 3942 – 3946. Hoskins, R.A., Smith, C.D., Carlson, J.W., Carvalho, A.B., Halpern, A., Kaminker, J.S., Kennedy, C., Mungall, C.J., Sullivan, B.A., Sutton, G.G., Yasuhara, J.C., Wakimoto, B.T., Myers, E.W., Celniker, S.E., Rubin, G.M., Karpen, G.H., 2002. Heterochromatic sequences in a Drosophila whole genome shotgun assembly. Genome Biol. 3, Research, 0085.1 – 0085.16. Kim, J.P., Lee, Y.B., Jang, S.J., Kim, M.A., 1997. The studies of properties of matter and chemical qualitative analysis on web of Araneus ventricosus L. KOCH, 1878. Kor. J. Arachnol. 13, 87 – 91. Kim, M.G., Shin, S.W., Bae, K.S., Kim, S.C., Park, H.Y., 1998. Molecular cloning of chitinase cDNAs from the silkworm, Bombyx
435
mori and the fall webworm, Hyphantria cunea. Insect Biochem. Mol. Biol. 28, 163 – 171. Kramer, K.J., Muthukrishnan, S., 1997. Insect chitinases: molecular biology and potential use as biopesticides. Insect Biochem. Mol. Biol. 27, 887 – 900. Kramer, K.J., Corpuz, L., Choi, H.K., Muthukrishnan, S., 1993. Sequence of a cDNA and expression of the gene encoding epidermal and gut chitinases of Manduca sexta. Insect Biochem. Mol. Biol. 23, 691 – 701. Laemmli, U.K., 1970. Cleavage of structure proteins during the assembly of the head of bacteriophage T4. Nature 227, 680 – 685. Lee, K.S., Chung, E.H., Han, J.H., Sohn, H.D., Jin, B.R., 2003. cDNA cloning of a defender against apoptotic cell death 1 (DAD1) homologue, responsive to external temperature stimulus from the spider, Araneus ventricosus. Comp. Biochem. Physiol., B 135, 117 – 123. McMaster, G.K., Carmichael, G.G., 1977. Analysis of single- and double-stranded nucleic acids on polyacryamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. U. S. A. 74, 4835 – 4838. Mommsen, T.P., 1978. Digestive enzymes of a spider (Tegenaria atrica Koch): II. Carbohydrates. Comp. Biochem. Physiol., A 60, 371 – 375. Mommsen, T.P., 1980. Chitinase and h-N-acetylglucosaminidase from the digestive fluid of the spider, Cupiennius salei. Biochim. Biophys. Acta 612, 361 – 372. O’Reilly, D.R., Miller, L.K., Luckow, V.A., 1992. Baculovirus Expression Vectors: A Laboratory Manual. W. H. Freeman, New York. Ramalho-Ortigao, J.M., Traub-Cseko, Y.M., 2003. Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis. Insect Biochem. Mol. Biol. 33, 279 – 287. Rayms-Keller, A., McGaw, M., Oray, C., Carlson, J.O., Beaty, B.J., 2000. Molecular cloning and characterization of a metal responsive Aedes aegypti intestinal mucin cDNA. Insect Mol. Biol. 9, 419 – 426. Shen, Z., Jacobs-Lorena, M., 1997. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae. J. Biol. Chem. 272, 28895 – 28900. Shinoda, T., Kobayashi, J., Matsui, M., Chinzei, Y., 2001. Cloning and functional expression of a chitinase cDNA from the common cutworm, Spodoptera litura, using a recombinant baculovirus lacking the virusencoded chitinase gene. Insect Biochem. Mol. Biol. 31, 521 – 532. Swofford, D.L., 2000. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods), Version 4. Sinauer Sunderland, Massachuset. Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350 – 4354. Trudel, J., Asselin, A., 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis. Anal. Biochem. 178, 362 – 366. Van Scheltinga, A.C.T., Kalk, K.H., Beintema, J.J., Dijkstra, B.W., 1994. Crystal structures of hevamine, a plant defense protein with chitinase and lysozyme activity, and its complex with an inhibitor. Structure 2, 1181 – 1189. Vaughn, J.L., Goodwin, R.H., Thompkins, G.J., McCawley, P., 1977. The establishment of two insect cell lines from the insect Spodoptera Frugiperda (Lepidoptera: Noctuidae). In Vitro 13, 213 – 217. Von Heijne, G., 1986. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14, 4683 – 4690. Wu, Y., Gillian, G., Underwood, A.P., Sakuda, S., Bianco, A.E., 2001. Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus. J. Biol. Chem. 276, 42557 – 42564. Yan, J., Cheng, Q., Narashimhan, S., Li, C.B., Aksoy, S., 2002. Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans. Insect Biochem. Mol. Biol. 32, 979 – 989. You, M., Xuan, X., Tsuji, N., Kamio, T., Taylor, D., Suzuki, N., Fujisaki, K., 2003. Identification and molecular characterization of a chitinase from the hard tick Haemaphysalis longicornis. J. Biol. Chem. 278, 8556 – 8563.