Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans

Insect Biochemistry and Molecular Biology 32 (2002) 979–989 www.elsevier.com/locate/ibmb

Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans J. Yan a,b, Q. Cheng a,b, S. Narashimhan c, C.-B. Li a,b, S. Aksoy a,∗ a

Department of Epidemiology and Public Health, Section of Vector Biology, Yale University School of Medicine, New Haven, CT 06510, USA b Institute of Genetics, Fudan University, Shanghai, People’s Republic of China c Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510, USA Received 12 October 2001; received in revised form 30 November 2001; accepted 20 December 2001

Abstract A chitinase cDNA, GChit1 was isolated from Glossina morsitans morsitans and shown to be specifically expressed in fat body tissue. GChit1 is encoded by a 1.6 kb mRNA with a putative open reading frame (ORF) of 460 amino acids (predicted pI ⫽ 7.5, m.w. ⫽ 51kDa) that contains a signal peptide domain and two potential N-linked glycosylation sites. The ORF exhibits homology to various chitinases characterized from insects. It has the conserved catalytic site residues and the cysteine-rich 3⬘-end domain associated with chitin binding although the serine/threonine rich domain is apparently missing. Southern blot data indicate that GChit1 is present as a single-copy locus in the Glossina genome. Northern analysis indicates that transcripts for GChit1 can be detected only from the fat body of adult flies. Similarly, chitinase activity could be detected in fat body but not in the gut or salivary gland tissues. The full-length cDNA was expressed in vitro in Drosophila S2 cells and the molecule was produced in a soluble form. Polyclonal antibodies raised against recGChit1 could recognize a protein of about 50 kDa in adult fat body extracts. In addition to fat body, chitinase protein was detected by Western analysis from the milk gland tissue of pregnant females as well as from the intrauterine larval and pupal developmental stages. No chitinase specific mRNA transcripts could be observed, however from larvae and pupae. The intrauterine larva of tsetse may receive the protein from its mother via the milk gland route. The molecular characteristics of GChit and its product and the potential role of this chitinase in tsetse biology are discussed.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Tsetse; Fat body; Chitinase; Trypanosomiasis; Ecdysis

1. Introduction Chitin, a polysaccharide composed of β, 1-4-linked N-acetyl-D-glucosamine is present in the exoskeleton of arthropods and requires degradation for the proper development of different larval stages. In addition, it is a component of the gut lining or peritrophic matrix (PM) of insects, a sleeve-like extracellular layer that surrounds the food bolus and needs to be degraded late in digestion for efficient absorption of nutrients. The chitinase enzyme responsible for the degradation of chitin has now ∗ Corresponding author. Tel.: +203-737-2180; fax: +203-785-4782. Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 4MU, 4-methyl umbelliferone; PBS, phosphate buffered saline; RT-PCR, reverse transcriptase-polymerase chain reaction; BSA, bovine serum albumin; DTT, dithiothreitol E-mail address: [email protected] (S. Aksoy).

been biochemically and molecularly characterized from the molting fluid of the tobacco hornworm, Manduca sexta (Bade and Stinson, 1978; Kramer et al., 1993) and the silk worm Bombyx mori (Abdel-Banat and Koga, 2001; Abdel-Banat et al., 1999; Koga et al., 1997). In the mosquito Anopheles gambiae, a gut specific chitinase gene product has been characterized and is thought to be a regulator of PM structure and function (Shen and Jacobs-Lorena, 1997). Molecular studies have shown the presence of similar chitinase genes in other arthropods (de la Vega et al., 1998). Chitinases have also now been characterized from a variety of parasitic eukaryotes including Plasmodium (Shahabuddin and Vinetz, 1999; Vinetz et al., 2000), the nematode Brugia malayi (Fuhrman et al., 1992) and the kinetoplastid Leishmania (Shakarian and Dwyer, 1998) where the protein is thought to be involved in the transmission of these agents in their insect vector, presumably by degrading

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the chitin-containing barriers in the gut. A chitinase has also been cloned from the venom of the wasp Chelonus sp with no known function (Krishnan et al., 1994). The molecular identification of chitinase genes and their putative products from different organisms suggests that they represent a family of proteins with conserved motifs expressed in different tissues where they might fulfill different functions. We are interested in the biology of the tsetse fly (genus Glossina) since they are the sole vectors of sleeping sickness disease in humans and nagana in animals. The disease is deadly with no foreseeable mammalian vaccines or effective and affordable drugs for chemotherapy in sight. Transmission of the disease agent, the protozoan African trypanosome begins when the tsetse fly takes a blood meal from an infected host. The parasite undergoes development in the gut as well as in the salivary glands or hypopharynx of the fly before it is eventually transmitted to its mammalian host through the bite of the fly (Vickerman et al., 1988). Unlike the majority of insects, tsetse adults have a continuously synthesized PM similar to the larval stages of most insects and its PM has been shown, by histochemistry to be formed of a mixture of glycosaminoglycans, glycoproteins and chitin (Lehane et al., 1996). Attempts to purify a chitinase activity or a chitinase gene product from Trypanosoma brucei sp. have not yet been successful (Arnold et al., 1992; Schlein and Jacobson, 1993) and no chitinase activity or encoding sequences have been reported from tsetse. Tsetse midgut symbiotic bacterium Sodalis has been shown to have chitinolytic activity in vitro (Welburn et al., 1993), but it remains to be seen whether this activity is secreted in to the fly gut. Here we report on the selection and molecular characterization of a chitinase cDNA, GChit1 from Glossina morsitans morsitans. Unlike other insect chitinases described to date, GChit1 is expressed preferentially in fat body tissue of tsetse. We describe the characteristics of the cDNA and its putative product and report on its tissue-specific expression profile. The cDNA was expressed in an eukaryotic expression system and the recombinant product was used to raise polyclonal sera. We evaluate the presence of this chitinase protein and chitinase activity in different fly tissues and developmental stages and discuss its potential function(s) in tsetse biology.

2. Materials and methods 2.1. Insects The G. m. morsitans puparia obtained from the Tsetse Research Laboratory (Bristol University, Bristol, United Kingdom) were originally established from puparia collected in Zimbabwe. The colony is maintained in the

insectary at Yale University’s Laboratory of Epidemiology and Public Health at 24–26 °C with 55% humidity and the flies are fed every other day on defibrinated bovine blood (Crane Laboratories, Syracuse, NY) using an artificial membrane system (Moloo, 1971). 2.2. RNA extraction, RT-PCR and isolation of chitinase cDNA clone Total RNA was isolated from fat body of teneral flies (Chomczynski and Sacchi, 1987) and cDNA was prepared with a first strand DNA syntheses kit (Pharmacia) and subjected to RT-PCR amplification. A degenerate sense primer, 5⬘-GGYGGYTGGAATGARGG-3⬘ and an anti-sense primer, 5⬘-GAYTTRGATTGGGAATAYCC3⬘ were designed corresponding to the conserved amino acid sequences from several arthropod chitinases, GGWNEG and DGLDLDW, respectively. PCR amplification was performed using the following conditions: 94 °C for 3 min for one cycle; 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s for 35 cycles; 72 °C for 5 min for one cycle. PCR products were separated in 1% agarose gel and the major band of about 130 bp was excised, extracted and subcloned in pGEM-T vector (Promega, CAT# A3600). The DNA sequence of the insert was obtained and its putative translation product was found to display high similarity to various putative chitinases. Using this PCR-fragment as the hybridization probe, a fat body cDNA library constructed in the λ-ZAP vector as previously described was screened (Hao et al., 2001). The isolated positive clones were converted to plasmids by in vitro excision using a helper phage according to manufacturer’s direction. 2.3. Sequencing and sequence data analysis The insert of the longest clone (pGChit1) was fully sequenced on both strands using an automated sequencer (Keck Center, Yale University) and the sequence was deposited into the public database, (Genbank Accession # AF337908). Comparative sequence analysis and alignments were carried out by using programs in the DNAStar software (Lasergene, Madison WI). The putative open reading frame was analyzed by SignalP v1.1 (Center for Biological Sequence Analysis, Technical University of Denmark, http://genome.cbs.dtu.dk/htbin/ nph), and PSORT II analysis (Prediction of Protein Sorting Signals and Localization Sites in Amino Acid Sequences, http://psort.nibb.ac.jp). Database searches were performed on the NCBI wwwserver with the BLAST program. 2.4. Southern blot analysis G. m. morsitans genomic DNA was digested with restriction enzymes EcoRI, BamHI and PstI, respect-

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ively, and transferred to a nylon membrane (HybondN+, Amersham Pharmacia Biotech). The plasmid pGChit1 DNA was used to generate a P32-dATP labeled RNA probe using the RNA labeling Kit (Amersham Pharmacia Biotech, RNP3100). The hybridization was performed for 20 h at 60 C in Church buffer (7% SDS/0.5 M sodium phosphate, pH7.2/1% BSA, 1 mM EDTA). The membrane was washed sequentially with low stringency buffer (5% SDS/40 mM phosphate buffer, pH 7.2/1 mM EDTA) and high stringency buffer (1% SDS/40 mM phosphate buffer, pH 7.2/1 mM EDTA) at 60 °C, and exposed to Kodak BioMax MR film. 2.5. Northern blot hybridization The expression of GChit1 cDNA in different tissues and various developmental stages was determined by Northern analysis. Total RNA was prepared from dissected salivary gland, gut, fat body and proventriculus tissues of 50 two week old flies 24 h after blood feeding, respectively. RNA was also prepared from three week old pupae. First/second and third instar larvae were obtained by dissection from pregnant mothers for RNA preparation and fat body tissue of the corresponding mothers was also dissected for analysis. Fat body was collected from teneral flies as well as from flies 2, 12, 24 and 48 h post blood feeding. Ten micrograms of total RNAs were separated in 1.2% agarose gel containing 2.2 M Formaldehyde, transferred to a nylon membrane (Hybond-N+, Amersham), and hybridized to GChit1 specific RNA probe prepared as described above. The filter was later stripped, and a labeled tsetse tubulin RNA probe (Gtub: GenBank accession # AF330159) was hybridized to quantitate the input RNA in each sample. 2.6. Analysis of chitinase activity in fly tissues using glycol-chitin overlay assay The gut and salivary gland tissues were dissected from adult G. m. morsitans, homogenized in 100 µl Tris–HCl pH 7.5, centrifuged at 10 K for 5 min and the supernatant fractions were collected. Similarly fat body was collected from teneral flies (24 h after emergence), and from flies 12, 24 and 80 h after acquiring a blood meal. The protein extracts (5 µg) from each tissue were analyzed on native 8% polyacrylamide gels containing 0.2% glycol chitin in the separating gel to detect chitinase activity (Trudel and Asselin, 1989). Glycol chitin was obtained by acetylation of glycol chitosan by modification of a previous protocol (Molano et al., 1979). Briefly, five grams of glycol chitosan was dissolved in 100 ml of 10% acetic acid by grinding in a mortar and the viscous solution was allowed to stand overnight at 22 °C. Methanol (450 ml) was slowly added and the solution was vacuum filtered and 7.5 ml of acetic anhydride was

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added with magnetic stirring. After 30 min at room temperature, the gel was cut into small pieces, covered with methanol and homogenized in a Waring Blender for 4 min at top speed. This suspension was centrifuged at 27,000g for 15 min at 4 °C and the gelatinous pellet was resuspended in 1 vol of methanol, homogenized, and centrifuged as in the preceding step. The pellet was resuspended in distilled water (500 ml) containing 0.02% (W/V) sodium azide and homogenized for 4 min. This was the final 1% (W/V) stock solution of glycol chitin. Following electrophoresis, the gels were immersed in 100 mM sodium acetate (pH 5.0), incubated at 37 °C for 2 h and stained with the fluorescent brightener, Calcofluor White M2R (Sigma) (0.01%) for 15 min. Bands of chitinase activity were viewed under UV light and photographed (Shen and Jacobs-Lorena, 1997). The experiment was repeated three times, and one representative analysis is shown in Fig. 4. 2.7. Expression of GChit1 in vitro in Drosophila cells The recombinant GChit1 was expressed in vitro in Schneider 2 (S2) Drosophila melanogaster cells using the Drosophila Expression System (Invitrogen, Version D). This vector allows for the inducible expression of the gene of interest as a fusion protein with a 1.8 kDa Bip secretion signal and has a 2.6 kDa V5-His C-terminal tag to facilitate purification of the recombinant protein by nickel-chelating chromatography. Two oligonucleotide primers that could amplify GChit1 cDNA from 112 to 1413 bp were designed: GChit1F 5⬘-TACCT CGAGACTACATTACATTTGACTG-3⬘ and GChit1R 5⬘-CTATAGATCTGCAGCAAACGCAACTG-3⬘. To enable cloning into the vector, XhoI and BglII restriction sites were introduced into the primers, respectively. PCR amplification was performed at 94 °C for 2 min for one cycle and 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min for 35 cycles followed by 72 °C for 5 min for one cycle in a MJ Research (PTC-200) thermocycler. The generated PCR product was gel purified, cleaved by restriction enzymes BglII and XhoI and ligated into the BglII–XhoI restricted expression vector pMT/Bip/V5HisB. The recombinant plasmid pRecGChit1 was sequenced to ensure that GChit1 fragment was in frame with the C-terminal sequence encoding the V5 epitope and polyhistidine tag. Using the Drosophila Expression System Kit (Invitrogen, CAT# K4110-01), S2 cells were stably transfected with pRecGChit1 DNA in combination with the selection vector, pCoHYGRO, according to manufacturer’s directions. For transfection, 300 µl solution A (0.24 M CaCl2/19 µg recombinant DNA) was slowly added to 300 µl of solution B (2×HEPES-Buffered Saline, 50 mM HEPES/1.5 mM NaH2PO4/280 mM NaCl, pH 7.1), incubated at room temperature for 30– 40 min, and was added dropwise to the cells grown at

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22–24 °C to a density of 4 × 106cells / ml. Forty eight hours after transfection, the cells were centrifuged and resuspended in complete DES Expression Medium containing 300 µg/ml hygromycin B. The selection medium was replaced every 4–5 days until resistant cells started to grow after about 3–4 weeks. Twenty-four different transformed lines were initially selected and maintained for expression analysis. The transformed lines were induced with copper sulfate (500 µM), and 48 h post induction, the expression of recGChit1 was analyzed in cell pellets and supernatants by Western blot analysis. The harvested cells were pelleted, the supernatant was transferred to a new tube and the cells were washed in PBS (137 mM NaCl/2.7 mM KCl/10 mM Na2HPO4/1.8 mM KH2PO4, pH 7.4) and resuspended into 50 µl lysis buffer (50 mM Tris, pH 7.8/150 mM NaCl/1% Nonidet P-40). Both the supernatant and cell lysates were analyzed by 12% SDSPAGE analysis and proteins were electroblotted to nitrocellulose. The membrane was blocked with 5% dried milk in PBS and 0.05% Tween 20 (PBST), and incubated with mouse monoclonal anti-V5 antibody (Invitrogen, Cat # R960-25) at 1:5000 dilution in 1% BSA/PBST for 1 h at room temperature. The membrane was then incubated with HRP-labeled anti-mouse IgG at 1:4000 dilution in 1% BSA/PBST for 1 h at room temperature. The protein was visualized using chemiluminescence detection (NEN Life Science, Western Blot Chemiluminescence Reagent Plus, CAT# NEL103). Two cell lines that exhibited high levels of expression of the cross reacting protein (V5 epitope) were selected for further analysis. 2.8. Chitinase assay using 4-MU-(NAG)3 as substrate The two cell lines were expanded to 250 ml cultures in DES Serum-Free Medium. Five days after induction with copper sulfate (500 µM), the culture supernatants were collected, concentrated and desalted through a Centricon PLUS-20 centrifugal filter device with ultracel PL membranes (Fisher Scientific, CAT# VFC2LGC08) to a final volume of 1 ml. The Xpress Protein Purification Kit (Invitrogen, CAT# K854-01) was then used to purify the recombinant His-tagged protein according to manufacturer’s directions. The protein concentration was determined using the BCA protein assay reagent (Pierce) with BSA as a standard. The samples were boiled for 3 min in 1×SDS gel-loading buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% Bromophenol Blue, 0.1% DTT). SDS-PAGE was performed in 8% polyacrylamide gels containing 0.1% SDS. The pH profile of the recombinant 6×His-tagged chitinase protein and its inhibition by the chitinase inhibitor allosamidin were determined in individual wells of a 96well black microfluorimetry plate (microfluor B, CAT# 011-010-7205, Dynatek, Chantilly, VA) (Hollis et al.,

1997). 4-Methylumbelliferyl-N,N⬘,N⬙-triacylchitotrioside [(GlcNAc)3-UMB)] from Sigma was used as the substrate. For determination of pH optimum, the assays were conducted in a final volume of 100 µl with 10 µl enzyme (0.5 µg), 40 µM substrate, 25 µl 0.2 M universal buffer (Frugoni, 1957) with the pH ranging from 4.0 to 9.0, in 0.5 unit increments. The reactions carried out in duplicates were incubated at 25 °C for 30 min and stopped by adding 120 µl of 1M glycine–NaOH buffer (pH 10). The free methylumbelliferone released by enzymatic hydrolysis was determined by fluorescence spectrophotometry (Model HTS7000, Perkin–Elmer Spectrophotometer) using an excitation wavelength of 365 nm and an emission wavelength of 450 nm. Results are reported as initial rates of substrate hydrolysis in relative fluorescence units. The inhibitor allosamidin was prepared as 4 mM stock in water and diluted prior to use and its final concentration tested ranged from 0.0 to 0.25 µM. Allosamidin was kindly provided by S. Sakuda at University of Tokyo (Sakuda et al., 1987). 2.9. Antibody preparation and immunoblotting Four mice were immunized to produce polyclonal antisera according to the following schedule: each mouse received an initial injection of 100 µl (1.5 µg/µl) purified recGChit1 protein and an equal volume of adjuvant followed by six boosts at two-week intervals. Complete Freund’s adjuvant was used for the initial immunization and incomplete Freund’s adjuvant for all boosts. For immunoblotting, protein extracts were prepared from dissected first/second and third-instar larva, two and three week old pupae. Protein extracts were also prepared from dissected midgut, fat body tissues 48 h postfeeding and milkgland tissue was collected from pregnant females. Fat body from teneral and blood fed flies (48 h post feeding) as well as fat body from trypanosome infected flies were analyzed. To study the parasite responsive nature of chitinase expression in fat body, a group of 50 teneral flies were given a T. b. rhodesiense procyclic trypanosome containing blood meal (105 cells/ml). Twelve hours after the infectious meal, 10 flies were dissected and fat body was collected. On day 20, the rest of the flies were dissected and their infection status was scored microscopically. Fat body tissue was collected from flies harboring gut infections. For Western analysis 10 µg of protein extract from each sample were separated on 8% SDS-PAGE gels and electroblotted to nitrocellulose. An identical SDS-PAGE gel was prepared and analyzed by Coomasie-blue staining to ensure both the quality and quantity of protein analyzed from each sample (data not shown). After blocking as described above, the blots were incubated with preimmune sera (1:2000 dilution) as well as with primary polyclonal antisera (1:4000 dilution) and then with HRPlabeled anti-mouse IgG (1:4000 dilution) for 1 h each at

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room temperature, respectively. The native protein was visualized using chemiluminescence detection.

3. Results 3.1. Isolation of G. m. morsitans chitinase cDNA Using two degenerate oligonucleotide primers designed against the conserved catalytic domain, RTPCR was performed with fat body cDNA to prepare a probe to isolate Glossina chitinase from a cDNA library. A major product of about 130 bp was obtained and its translated product showed high level of identity (60– 65%) to various arthropod chitinase cDNA products. Using this fragment as the hybridization probe, nine positive clones were selected from approximately 5 × 105 plaques of a G. m. morsitans fat body cDNA library. Partial sequencing and restriction enzyme mapping revealed no heterogeneity among these inserts except for size. The clone with the longest insert (GChit1) was fully sequenced and used for further experiments. 3.2. Nucleotide and predicted amino acid coding sequence of GChit1 The 1551-bp insert of pGChit1 consists of a 29-bp 5⬘untranslated region (UTR), an ORF encoding 460 residues and a 132-bp 3⬘-UTR (Fig. 1). The 24 aa hydrophobic region at the 5⬘-end has signal peptide characteristics. The calculated molecular mass and the pI of the putative mature protein are 51 kDa, and 7.48, respectively. A search of the public databases with the deduced amino acids of the GChit1 cDNA shows the highest level of overall identity with chitinases characterized from other insect species. An alignment of GChit1 with D. melanogaster hypothetical sequences, the chitinases characterized from Aedes aegypti (de la Vega et al., 1998), An. gambiae (Shen and Jacobs-Lorena, 1997), B. mori (Abdel-Banat and Koga, 2001), M. sexta (Kramer et al., 1993), the prawn Penaeus japonicus (Watanabe et al., 1998), the braconid wasp Chelonus sp. (Krishnan et al., 1994) and Mus musculus (Boot et al., 2001) is shown in Fig. 2. The level of overall identity with the D. mel and D. mel2 sequences were 54 and 39% respectively while the % identity with other chitinase sequences varied from 30 to 40. When the same alignment was performed using the 5⬘-end 350 aa of GChit1, the identities were higher and varied from 40 to 50%. While the cysteine-rich domain associated with the C-terminus of many chitinases is also present in GChit1, the serine/threonine-rich central domain is missing. The chitin binding motif, C X2 (F/Y/W) X2 C X2 G X6-9 C X2 (F/Y) (D/N) associated with chitinases as well as several PM proteins is also present in GChit1 (Barry et al.,

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1999). The active site centered on the two catalytic residues Asp153 and Glu154 are conserved in all aligned sequences including GChit1. The tryptophan residue, W153, present in GChit1 has been shown to be essential for the chitinolytic activity as its substitution with a phenylalanine or a glycine residue in the Manduca protein has resulted in diminished activity although it did not affect substrate binding (Huang et al., 2000). GChit1 possesses two putative N-glycosylation sites. 3.3. Genomic organization GChit1 The copy number of the GChit1 locus in G. m. morsitans genome was estimated by Southern blot analysis (Fig. 3A). Genomic DNA was digested with the restriction enzymes, which do have recognition sites within the GChit1 cDNA sequence. Hybridization with GChit1 cDNA showed the presence of only one detectable fragment in each digestion, suggesting thatGChit1 exists as a single copy gene in G. m. morsitans genome. PCR amplification of G. m. morsitans genomic DNA with several GChit1 cDNA specific oligonucleotide primers and their subsequent sequence analysis indicates the presence of at least one intron of 123-bp at the 5⬘-end region of the gene (data not shown). 3.4. Expression of Glossina chitinase mRNA The tissue specific nature of GChit1 expression was determined from salivary glands, proventriculus, midgut and fat body by Northern analysis (Fig. 3B). GChit1 was found to be expressed only in fat body and not in salivary gland, proventriculus or midgut tissues of flies analyzed 24 h after a blood meal (Lanes 1–4, respectively). Analysis of chitinase transcripts in fat body dissected and analyzed 0, 2, 12, 24 and 48 h after the blood meal showed no quantitative differences (Fig. 3C, Lanes 1–5, respectively). The presence of transcript abundance for GChit1 in the different developmental stages of tsetse was also investigated but could not be detected in either the intrauterine larval or pupal stages (Fig. 4B, Lane 1–4, respectively). As a control, GmTub gene was hybridized to the same Northern blots to indicate both the integrity and quantity of the RNA analyzed for each sample. 3.5. Detection of chitinase activity in Glossina tissues Chitinase activity was analyzed by electrophoresis of protein extracts prepared from salivary gland, midgut and fat body tissues in native polyacrylamide gels containing the synthetic substrate glycol chitin. Following electrophoresis, the gel was incubated to allow substrate hydrolysis by chitinase and then stained with a fluorescent dye that binds the substrate. Brightly fluorescent

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Fig. 1. The GChit1 cDNA sequence and deduced amino acid sequence of its product. The putative signal peptide sequence is underlined at its 5⬘-end. The residues underlined by carets identify potential glycosylation sites. The polyadenylation signal AATAAA is underlined preceeding the 3⬘-end poly(A) stretch. The arrows indicate the conserved domains from which the degenerate DNA oligonucleotides were designed. The boxed region shows the catalytic domain and the cysteine residues in reverse-phase form the putative chitin binding domain.

areas represent regions with no active enzyme, and dark areas indicate regions where enzyme activity has degraded the substrate. Chitinase activity could not be detected in either salivary gland or midgut tissues (Fig. 4, Lanes 1–2, respectively). It could however be detected in the fat body extracts and the activity was found to reach its highest level 24 h after blood feeding (Fig. 4, Lane 5). Chitinase activity was barely detectable in fat body dissected from teneral (newly emerged) and starved flies (80 h post blood meal), (Lanes 3 and 6, respectively). Treatment of the tissue homogenates with trypsin before gel analysis did not increase the level of the activity detected at each time suggesting that GChit1 is not a zymogen (data not shown).

3.6. pH profile and allosamidin sensitivity of recGChit1

The pH-dependent activity profiles and allosamidin inhibitory concentration curves for recGChit1 were determined by microfluorimetry using 4-MU-GlcNAc3 as substrate (Fig. 5A). RecGChit1 had a chitinase activity and a broad pH optimum of pH 7.5–8.5, peaking at about pH 8.0. The sensitivity of recGChit1 to the inhibitor allosamidin was also determined (Fig. 5B). The allosamidin concentration of 50 nM was sufficient to block 50% (IC50) of the chitinase activity.

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Fig. 2. Alignment of the deduced GChit1 ORF with related sequences. Conserved residues are indicated with boxes and the underlined box corresponds to the enzyme active site. The abbreviations and GenBank accession numbers for the chitinase sequences analyzed are: GChit1, G. m. morsitans (AF337908); D. mel, D. melanogaster hypothetical protease (AAF46665); D.mel2, D. melanogaster hypothetical protease (AAF54987), AgChit, A. gambiae (AAB87764); AaChit, A. aegypti (AF026491); BmChit, B. mori (U86876); MsChit, M. sexta (U02270); P. japonica, P. japonicus(AB008027); Chelonus sp., braconid wasp Chelonus sp. (U10422); Mus Musculus (AF290003).

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Fig. 3. Autoradiograms showing Southern and Northern analysis with GChit1. (A). G. m. morsitans genomic DNA was cleaved with EcoRI, BamHI and PstI (Lane 1–3, respectively). The southern blot was hybridized to radiolabelled pGChit1 plasmid DNA. (B) Total RNA was isolated and analyzed from proventriculus, salivary glands, midgut and fat body (Lanes 1–4, respectively). For expression analysis in different developmental stages, RNA was isolated from first and third instar larva fat body, their corresponding mothers and from 20–25 days old pupae, Lane 5, first instar larva; Lane 6, fat body from flies from which the first instar larva were collected; Lane 7, third instar larva; Lane 8, fat body from flies from which third instar larva were collected; Lane 9, pupae. The blots were stripped and rehybridized with Glossina tubulin gene (Gtub). (C) Total RNA was isolated from fat body tissue at different times following a blood meal: teneral flies (Lane 1), 2 h after feeding (Lane 2), 12 h after feeding (Lane 3), 24 h after feeding (Lane 4) and 48 h after feeding (Lane 5).

Fig. 4. Chitinase activity in Glossina tissue extracts. 5 µg protein extracted from salivary gland, gut and fat body tissues at designated times after blood meal were analyzed on native 8% polyacrylamide gels containing 0.2% glycol chitin.

3.7. Immunoblotting analysis of GChit1 protein in tsetse The recombinant 6×His-tagged protein GChit1expressed in Drosophila cells was secreted into the culture medium (Fig. 6A). The recombinant protein was purified from the culture supernatant and used to raise antibodies. Western blots performed with preimmune sera showed non-specific cross-reactivity with several proteins of about 30–40 kDa in different tissues (Fig. 6B, Lanes 1– 4). Analysis with the GChit1 polyclonal antibody indicated that a protein of around 52 kDa was recognized specifically in fat body (Lane 5) and in milk gland tissue (Lane 6), but was absent in midgut (Lane 7). GChit1

could also be detected from both first/second and second/third larval stages as well as from 15 and 20 day old pupae (Lanes 8–11, respectively). This was unexpected since GChit1 mRNA could not be detected in either the larval or pupal developmental stages at the transcriptional level, by Nothern analysis. Comparative Western analysis of GChit1 in fat body from teneral and blood fed flies, 36 h after feeding, showed increased expression of the protein following the blood meal (Fig. 6C). This is in contrast to the protein activity results, where only low levels of chitinase could be detected in fat body of teneral flies (Fig. 4). Incubation of teneral fat body extracts with trypsin prior to analysis did not result in an increased activity suggesting that GChit1 might not be present as a zymogen (data not shown). Western analysis shows that the chitinase levels in fat body of trypanosome fed or trypanosome infected flies are similar to the levels detected in normal flies suggesting that the expression of GChit1 may not be parasite sensitive (Fig. 6D). While gel activity analysis shows an activity migrating at a higher molecular weight than GChit1 in in vitro cultured midgut symbiont Sodalis (data not shown), no chitinase-specific protein could be detected by Western from dissected midgut tissue. This may be due to either the distant relatedness of Sodalis chitinase to GChit1 based on its sequence analysis, or to its low abundance in midgut.

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Fig. 5. pH optimum of the purified recGChit1 protein and its sensitivity to the inhibitor allosamidin. (A) The chitonolytic activity of the purified 50 kDa recombinant tsetse chitinase recGChit1 was measured with the substrate GlcNAc3 in buffer with pH ranging from 4.0–9.0. Results show initial rates of substrate hydrolysis in relative fluorescence units. (B) Allosamidin sensitivity of the purified recGChit1. The activities of the purified recGChit1 at pH 8.0 were measured with GlcNAc3 in the presence of different concentrations of allosamidin, ranging from 0.0 to 0.25 µM.

Fig. 6. In vitro expression and Western analysis of chitinase in tsetse. (A) Western analysis of cell culture supernatants from the transformed S2 cells 1–5 days after induction with copper sulfate (500 µM), (Lanes 1–5, respectively) analyzed by the V5 epitope showing the increased secretion of a 50 kDa protein. Subsequently the recombinant protein was purified from the culture supernatant and used for raising polyclonal antibodies. (B) Chitinase expression in different tissues and developmental stages. Protein was extracted from fat body, midgut, first/second instar larva, third instar larva, pupae and milkgland tissues. Ten microgram protein samples were loaded in each lane and analyzed initially with preimmune sera : fat body (Lane 1), midgut (Lane 2), first/second (Lane 3) and third instar larva (Lane 4). GChit1 antibodies were used to analyze products in fat body (Lane 5), milk gland (Lane 6), midgut (Lane 7), first/second and third instar larva (Lanes 8 and 9, respectively); 2-week and 3-week old pupae (Lanes 10 and 11, respectively). The putative chitinase protein is marked. (C) Chitinase expression in fat body in response to the blood meal. Protein was extracted from fat body from teneral flies and from flies 36 h after blood feeding. 10 µg protein extract was loaded, fat body from teneral flies (Lane 1); fat body 36 h after blood feeding (Lane 2). (D) Chitinase expression in fat body in response to the presence of trypanosomes in midgut. 10 µg protein extract was analyzed from fat body of normal flies Lane 2); fat body 24 h after obtaining trypanosome containing blood meal (Lane 3), fat body from flies harboring trypanosome gut infections (Lane 4). Lane 1 shows the purified recGChit1 protein.

4. Discussion We have described from G. m. morsitans a cDNA with strong sequence similarity to members of family 18 of the glycosyl hydrolase superfamily, that comprises microbial, insect, nematode, mammal as well as some plant chitinases. In insects, chitinases are important in the gut for degradation of PM to allow for efficient absorption of nutrients, and they also play an important role in ecdysis for cuticle degradation during development. While chitinases have been characterized from larva and insect guts, this is the first insect chitinase to be reported expressed preferentially in fat body, the primary

organ for immune related mechanisms in insects. A chitinase protein expressed only by macrophages has been described from mammals and is thought to play a role in defense mechanisms against chitin-containing pathogens (Boot et al., 1995). 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 that is glycosylated. GChit1 lacks the serine/threonine rich domain which is characteristic of proteoglycans such as syndecans and mucins (Rayms-Keller et al.,

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2000), but has the catalytic and chitin-binding consensus motifs. The C-terminal cysteine rich domain is found in insect and nematode chitinases as well as in several PM proteins (Barry et al., 1999). The function of this domain is presumably to anchor the enzyme tightly onto the substrate, thereby facilitating the hydrolytic process. GChit1 is apparently present as a single locus in tsetse genome although it is possible that there may be other chitinase homologs distinct from GChit1. There are eight putative chitinase encoding loci within the Drosophila genome and at least another seven with the hallmarks of chitinase proteins (de la Vega et al., 1998). The expression profiles of these genes and their potential functions remain to be investigated but they may well fulfill different functions in different tissues or developmental stages. The drosophila sequence showing the greatest similarity to GChit1 lacks both the serine/threonine and chitin binding domains. This is similar to the gut-specific chitinase cDNA characterized from Phaedon cochleariae which has been found to lack again both domains although it is still enzymatically active (Girard and Jouanin, 1999). However, our efforts to select a chitinase coding sequence from midgut cDNA of Glossina using the same approach described here have not been successful. In addition to detecting mRNA, gel activity analysis also supports fat body to be the major source of chitinase protein in tsetse. Comparative analysis of chitinase transcripts and protein levels from trypanosome fed and infected flies, however, does not suggest that GChit1 expression is parasite responsive in tsetse. Although no significant differences were observed in the abundance of chitinase mRNA in fat body during the digestion process, both activity gels and Western analysis show increased activity and protein levels, respectively, following a blood meal. This suggests that the expression of GChit1 may be under post-transcriptional regulation similar to a number of other tsetse gut products such as serine proteases (Yan et al., 2001b), carboxypeptidase, metalloprotease and cathepsin-B (Yan et al., 2001a). Since both the male and female adult tsetse are strict blood feeders and require a meal every 40–50 h, the post-transcriptional regulation of gene expression might facilitate the synthesis of the necessary enzymes rapidly. The gut chitinase activity characterized from An. gambiae has been found to be stored as a zymogen, which requires activation by trypsin (Shen and Jacobs-Lorena, 1997). In similar experiments, however, GChit1 activity in fat body did not increase upon trypsin activation, suggesting that it may be regulated by a different translational control mechanisms. Since GChit1 does not appear to be involved in the blood meal digestion in tsetse gut, we investigated its possible role in ecdysis. Unlike the majority of insects, tsetse reproduction is viviparous and each egg is retained within a uterus and hatches following fertilization and one young larvae matures and is deposited. During its

nine day intrauterine life, the larvae receives nutrients including its symbiotic flora from its mother’s milkgland secretions. We have previously been able to microinject recombinant symbionts into the pregnant female hemolymph and show that the symbionts could be acquired by the developed larva indicating that a route from hemolymph to larva exists (Cheng and Aksoy, 1999). The pregnant females develop an extensive tubular network of milkglands to mediate the transfer of nutrients to their developing larva. It is interesting that we could detect GChit1 by Western analysis in the larval and pupal developmental stages although no GChit1 specific transcripts could be detected in these developmental stages. It remains to be shown however, whether GChit1 is involved in ecdysis in the larvae or whether there are other chitinases also synthesized by the larva. In most oviparous insects, the route of acquisition of the fat body-synthesized major yolk protein vitellogenin by oocytes is well established (Sappington and Raikhel, 1998). The fact that GChit1 is detected in milk gland tissue suggests that the intrauterine larva of tsetse might indeed receive this protein from its mother via the milk gland secretions. Control of trypanosomiasis has been difficult as no effective mammalian vaccines or drugs as yet exist. Hence vector control has been an important tool for reducing disease. Approaches such as Sterile Insect Technique (SIT) which exploit the low reproductive rate of tsetse have been successful since field populations can crash quickly in response to slight adverse effects on fecundity (Aksoy, 2000). It remains to be seen if larval development can be adversely impacted by blocking either GChit1expression in the mother or its transfer to the larva. If the development of tsetse larva indeed relies on the synthesis and acquisition of the maternal GChit1, approaches which aim to block the synthesis or transfer of GChit1 to the larva may be explored as a novel strategy to reduce field populations.

Acknowledgements This research was supported by NIH/NIAID-AI34033 and Li Foundation awards to S.A. We are grateful to Rita Vitorino-Moreira Rio and Patricia Michelle Strickler for critical reading of the manuscript.

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