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Insect Biochemistry and Molecular Biology xxx (2010) 1e8

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Insect Biochemistry and Molecular Biology

journal homepage: www.elsevier .com/locate/ ibmb

Functional expression in insect cells of glycosylphosphatidylinositol-linkedalkaline phosphatase from Aedes aegypti larval midgut: A Bacillusthuringiensis Cry4Ba toxin receptor

Manasave Dechklar, Kasorn Tiewsiri 1, Chanan Angsuthanasombat*, Kusol Pootanakit*

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand

a r t i c l e i n f o

Article history:Received 24 August 2010Received in revised form18 November 2010Accepted 29 November 2010

Keywords:Bacillus thuringiensisCry d-endotoxinMembrane-bound alkaline phosphataseSpodoptera frugiperdaBaculovirus systemCytotoxicity assay

* Corresponding authors. Tel.: þ66 2 800 3624x124E-mail addresses: [email protected] (C. An

mahidol.ac.th (K. Pootanakit).1 Present address: Department of Entomology, Co

14456-0462, USA.

0965-1748/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.ibmb.2010.11.006

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a b s t r a c t

Bacillus thuringiensis produces insecticidal crystal (Cry) proteins which bind to cell surface receptorson the brush border membrane of susceptible midgut larvae. The toxinereceptor interaction generatespores in midgut epithelial cells resulting in cell lysis. Here, a cDNA encoding membrane-bound alkalinephosphatase from Aedes aegypti (Aa-mALP) midgut larvae, based on the sequence identity hit to Bombyxmori membrane-bound ALP, was amplified by RT-PCR and transiently expressed in Spodoptera frugiperda(Sf9) insect cells as a 58-kDa membrane-bound protein via the baculovirus expression system andconfirmed by digestion with phosphatidylinositol-specific phospholipase C and LC-MS/MS analysis.Immunolocalization results showed that Cry4Ba is able to bind to only Sf9 cells-expressing Aa-mALP.Moreover, these cells were shown to undergo cell lysis in the presence of 100 mg/ml trypsin-treated toxin.Finally, trypan blue exclusion assay also demonstrated an increase in cell death in recombinant cellstreated with Cry4Ba. Overall results indicated that Aa-mALP proteinwas responsible for mediating Cry4Batoxicity against Sf9 cells, suggesting its role as a receptor for Cry4Ba toxin in A. aegypti mosquito larvae.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Bacillus thuringiensis (Bt) is a spore-forming bacterium thatproduces two main families of crystalline inclusion bodies: crystal(Cry) and cytolytic (Cyt) d-endotoxins during sporulation (Aronsonet al., 1986; Höfte and Whiteley, 1989; Crickmore et al., 1998). Afteringestion by susceptible larvae, crystal protoxins are solubilized torelease activated Cry toxins by larval midgut proteases. The acti-vated Cry toxins then bind specifically to protein receptors in themidgut epithelial cell. The toxinereceptor interaction facilitatestoxin insertion into the membrane, leading to ion-permeable poreformation in the midgut cell. The ion-leakage pores cause osmoticlysis of the insect midgut cells, resulting in the death of the larva(Knowles and Ellar, 1987; Knowles, 1994).

The binding of Cry toxins to specific receptors inmidgut larvae isone of the critical steps leading to the action of Cry toxin activity.Many studies suggested multiple Cry toxin receptors: cadherin-like

9; fax: þ66 2 441 9906.gsuthanasombat), mbkpn@

rnell University, Geneva, NY

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., et al., Functional expression(2010), doi:10.1016/j.ibmb.20

proteins (Vadlamudi et al., 1995; Nagamatsu et al., 1998a, 1998b;Xie et al., 2005; Flannagan et al., 2005; Chen et al., 2009a), GPI(glycosylphosphatidylinositol)-anchored aminopeptidase-N (APN)(Knight et al., 1994, 1995; Masson et al., 1995; Luo et al., 1996, 1997;Yaoi et al., 1997; Garner et al., 1999; Chen et al., 2009b), GPI-anchored alkaline phosphatase (ALP) (Jurat-Fuentes and Adang,2004; Fernandez et al., 2006; Arenas et al., 2010) and glycolipids(Griffitts et al., 2005).

Here, the focus is on ALP. This is because using toxin overlayassay, Fernandez et al. (2006) identified a 65-kDa GPI-anchored ALPfrom Aedes aegypti BBMV which is shown to interact with Cry11Aa.Moreover, using the same technique, we have identified a protein ofsimilar size (w60 kDa) as a Cry4Ba-binding protein from A. aegyptibrush border membrane vesicles (Moonsom et al., 2007). Sinceamong the known Cry toxin receptors, only ALPs are of around55e65 kDa; thus, our initial postulate is that the 60-kDa proteinthat binds to Cry4Ba may be another ALP as well.

ALPs [EC 3.1.3.1] are found in all animals and as is expected aremainly localized in microvilli of columnar cells (Wolfersberger,1984) and of insect midgut epithelium cells (Eguchi, 1995). ALPscan be divided into two groups: soluble (s-ALP) and membrane-bound (m-ALP) (Eguchi et al., 1972; Azuma and Eguchi, 1989; Itohet al., 1999). In insects, both ALPs are found in larval midgutepithelium cells; however, they are expressed in different cell

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types. The s-ALP is found exclusively in the cavity of goblet cells andin the apical region of the midgut (Azuma and Eguchi, 1989);whereas, m-ALP is localized in the brush border membrane ofcolumnar cells and particularly restricted to the middle andposterior midgut. Moreover, s- and m-ALPs show distinct differ-ences in enzymatic activity (such as optimal pH) and also thestructure of sugar side chain (Okada et al., 1989; Azuma et al., 1991),suggesting that they perform different functions in vivo.

Therefore, to determine if A. aegypti m-ALP (Aa-mALP) canindeed serve as a Cry4Ba toxin receptor, Aa-mALP was initiallyidentified through BLASTP (Basic Local Alignment Search ToolProgram) search of A. aegypti genome database via the deducedamino acid sequence of Bombyx mori m-ALP; and then heterolo-gously expressed in Spodoptera frugiperda (Sf9) cells and tested forCry4Ba toxin binding and cytotoxicity.

2. Materials and methods

2.1. Amplification of cDNA encoding ALP protein fromA. aegypti larval midgut (Aa-mALP)

Two gene-specific primers, Topo-ALP-forward (50-CACCATGACTTTGTATCGGTCTCG-30) and Topo-ALP-reverse (50-TTATTAGGCAAAAACTCGCACCATC-30), were designed to amplify the codingsequence of Aa-mALP. For Topo-ALP-forward primer, 4 additionalbases at the 50-end, CACC, were added for directional cloning intopENTR/SD-TOPO vector; bases after CACC were designed from thestart codon of the candidate Aa-mALP. The ACCATG sequence(underline) acts as a Kozak sequence which plays an important rolein translational initiation. For Topo-ALP-reverse primer, two stopcodons (bold letters) were added to ensure a complete termination;sequences after TTATTAwere designed based on sequence from thestop codon of the candidate Aa-mALP.

For RNA extraction, total RNAs were extracted from isolatedmidgut tissue of 5th instar A. aegypti larvae using Trizol reagent.Total RNAs were used as template for first-strand cDNA synthesisusing Improm-II reverse-transcriptase (Promega, USA). Hot startPCR was performed (94 �C, 3 min) using high fidelity Pfu DNApolymerase for 25 cycles with the following profile: 93 �C for 30 s,65 �C for 10 s, 68 �C for 1 min. The last PCR cycle was followed bya final extension at 68 �C for 7 min. The expected amplicon waspurified, ligated to pENTR/SD-TOPO vector and transformed intoOne Shot TOP10 competent cells following the manufacturer’sprotocol (Invitrogen, USA). Several recombinant plasmids, pENTR/Aa-mALP, were randomly selected and verified by DNA sequencing(Macrogen, South Korea).

2.2. Transient expression of recombinant Aa-mALP in Sf9 cells

The Aa-mALP cDNA was transferred from pENTR/Aa-mALPplasmid into Baculodirect Linear DNA (Invitrogen, USA) byhomologous recombination for generation of the recombinantexpression baculovirus using the in vitro Lamda Recombination (LR)strategy according to the instruction manual. Briefly, 300 ng ofBaculodirect C-term Linear DNA was mixed with 300 ng of pENTR/Aa-mALP plasmid and LR Clonase II for 18 h at 26 �C. The LR reac-tions were subsequently mixed with Cellfectin reagent for 30 minat room temperature. Then, to the Sf9 cells that were seeded ata density of 8 � 105 cells per well, the transfection mixture wasdiluted with 0.8 ml serum-free medium and dropped directly ontothe cell surface. After incubation at 26 �C for 5 h, the transfectionmixture was removed and 2 ml of growth medium containing100 mM ganciclovir was added. The transfected cells were furtherincubated at 26 �C until cell lysis is observed. The culture mediumwas collected and kept as viral stock. The cell pellet was washed

Please cite this article in press as: Dechklar, M., et al., Functional expressionInsect Biochemistry and Molecular Biology (2010), doi:10.1016/j.ibmb.20

once with 1 ml PBS (pH 7.4) and centrifuged at 2000�g, 5 min, atroom temperature. The pellet was resuspended in PBS and the cellsuspension was analyzed for protein expression by SDS-PAGE(sodium dodecyl sulphate-polyacrylamide gel electrophoresis).

2.3. Mass spectrometry analysis of Aa-mALP

Cell lysates collected from Sf9 cells-expressing recombinant Aa-mALP were separated by SDS-PAGE and transferred to a poly-vinylidene difluoride membrane by wet electroblotting. Themembrane was stained by Ponceau Red (40% methanol and 10%acetic acid in water) for 5 min, destained with distilled water andair-dried at room temperature. The protein band corresponding tothe 58-kDa Aa-mALP was excised from the membrane and subse-quently submitted for mass spectrometry analysis (Genome Insti-tute, BIOTEC, Thailand).

2.4. Alkaline phosphatase and PI-PLC activity assays

Alkaline phosphatase assay was performed according to Jurat-Fuentes and Adang (2004) and Budatha et al. (2007) but withslight modifications. Briefly, after 3-day post-transfection, trans-fected and non-transfected Sf9 cells were collected by centrifuga-tion at 2000�g, washed 2�with PBS, pH 7.4 and then resuspendedin 100 ml PBS. Then, ALP buffer (100 mM TriseHCl, pH 9.5, 5 mMMgCl2 and 100 mM NaCl) containing 5 mM p-nitrophenyl phos-phate (pNPP, New England BioLabs, USA) as an ALP substrate wasadded to w106 Sf9 cells to make a final volume of 100 ml, and thenincubated at 30 �C for 5 min. Finally, the hydrolysis of pNPP to p-nitrophenol product was measured using a spectrophotometer(405 nm), for 5 min at room temperature.

PI-PLC assay was performed following Bottero et al. (1993) andNishikawa et al. (2002), but with slight modifications. Briefly, after3-day post-transfection, Aa-mALP transfected cells were collectedand then incubated with 1 U of Bacillus cereus PI-PLC (Invitrogen,USA) in PBS, pH 7.4 for 2 h at 4 �C with gentle agitation. Next, thecell pellet and supernatant were collected by centrifugation at2000�g, 10 min, 4 �C. The supernatants were re-centrifuged againat 8000�g, 10 min, 4 �C to harvest the clear suspension. Both thecell pellet and the supernatant fractions were analyzed by SDS-PAGE.

2.5. Expression and preparation of the Cry4Ba protein

Escherichia coli clone harboring the pMU388 plasmid encodingthe cry4Ba gene (Angsuthanasombat et al., 1987) was grown at37 �C in LuriaeBertani medium containing 100 ug/ml ampicillinuntil OD600 of the culture reached 0.3e0.6. Protein expression wasinduced with 0.1 mM isopropyl-b-D-1-thiogalactopyranoside(IPTG) for 4 h and harvested by centrifugation at 6000�g, 4 �C for10 min. E. coli cells, expressing the 130-kDa Cry4Ba protoxin asa cytoplasmic inclusion, were resuspended in 100 mM KH2PO4 (pH5.0) containing 0.1% Triton X-100 and 0.5% NaCl. Cells were dis-rupted in a French Pressure Cell at 10,000 psi and protein inclusionswere collected by centrifugation at 6000�g, 4 �C for 10 min. Theinclusion bodies were subsequently resuspended in distilled water.Protein concentration was determined by using a protein micro-assay (Bio-Rad, USA), with bovine serum albumin (Sigma, USA) asa standard. Protoxin inclusions (w3 mg/ml) were solubilized ina 50 mM Na2CO3 (pH 9.0) at 37 �C for 1 h. The solubilized Cry4Baprotoxin (w1e2 mg/ml) was further digested with trypsin (L-1-tosylamide-2-phenylethylchloromethyl ketone-treated, Sigma) atan enzyme/toxin ratio of 1/20 (w/w) in 50 mM Na2CO3 (pH 9.0) at37 �C for 16 h. Trypsin-treated fractions were subsequentlyanalyzed by SDS-PAGE.


Fig. 1. Multiple sequence alignment of the deduced Aa-mALP amino acid sequence against ALPs from other mosquitoes and insects: Ag_EAA10738 (XP_316433), Aa-ALP1(Fernandez et al., 2009), Ag-ALP (Hua et al., 2009), Cq-ALP (CPIJ018121-RA), Bm-mALP (Itoh et al., 1991), Hv-mALP (Perera et al., 2009) using ClustalX. Human-PLAP is used as thereference sequence denoting protein secondary structure (Le Du et al., 2001). The 13 conserved residues involved in metal binding or substrate-binding are boxed; conservativechanges for Aa-mALP are shaded light blue. The predicted N-terminal signal peptide is underlined; the putative GPI-anchor site is bolded and underlined; potential N-linkedglycosylation residues are italicized and shaded black. Amino acids are shaded dark blue, pink, red and yellow to denote degrees of identity (8/8), (7/8), (6/8), (5/8), respectively. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Dechklar, M., et al., Functional expression in insect cells of glycosylphosphatidylinositol-linked alkaline phos...,Insect Biochemistry and Molecular Biology (2010), doi:10.1016/j.ibmb.2010.11.006

Table 1Pairwise comparison of Aa-mALP sequence against twelve other A. aegypti ALPsidentified in the A. aegypti genome database, a reported Cry11A ALP receptor (Aa-ALP1) and also against other membrane-bound ALPs from other insect species.Percent identity was calculated using LALIGN program (http://www.ch.embnet.org/software/LALIGN_form.html).

Species Accession no. Identity (%)

Aedes aegypti (Aa-sALP1) AAEL000931-RA 33.7Aedes aegypti (Aa-sALP2) AAEL003317-RA 35.5Aedes aegypti (Aa-sALP3) AAEL013330-RA 36.8Aedes aegypti (Aa-sALP4) AAEL014601-RA 36.8Aedes aegypti (Aa-sALP5) AAEL003297-RA 37.2Aedes aegypti (Aa-sALP6) AAEL003313-RA 37.3Aedes aegypti (Aa-sALP7) AAEL003309-RA 37.7Aedes aegypti (Aa-sALP8) AAEL003298-RA 38.2Aedes aegypti (Aa-sALP9) AAEL003905-RA 38.2Aedes aegypti (Aa-sALP10) AAEL003289-RA 38.7Aedes aegypti (Aa-sALP11) AAEL009077-RA 39.3Aedes aegypti (Aa-sALP12) AAEL003286-RA 42.1Aedes aegypti (Aa-ALP1) EAT39089 39.3Anopheles gambiae (Ag-ALP) XP_316433 36.6Heliothis virescens (Hv-mALP) ACP39712 36.7Bombyx mori (Bm-mALP) NP_001037536 37.0Culex quinquefasciatus (Cq-ALP) XP_001842932-RA 38.1


2.6. Immunofluorescence localization of Cry4Ba toxin againstthe recombinant Aa-mALP

The specific interaction between Aa-mALP and Cry4Ba toxinwasdetermined by immunolocalization. Aa-mALP-expressed Sf9 cellswere incubated with the trypsin-activated Cry4Ba toxin (100 mg/ml) at 26 �C. After 2 h, the cells were washed 3� with PBS (pH 7.4)and then collected by centrifugation at 2000�g for 5 min, at roomtemperature. The cell pellet was then fixed in ice-cold 4% para-formaldehyde solution for 30 min. Next, the fixed cells werewashed 3�with PBS and then blocked with 5% BSA for 1 h, at roomtemperature. After blocking, they were washed with PBS and thenincubated with primary monoclonal antibodies (clone 2F(1H2))against Cry4Ba toxin (Moonsom et al., 2007) at 1:100 dilution for1 h. After an hour, the cells were rinsed 3� with PBS, followed byincubation with FITC-conjugated mouse IgG secondary antibodies(GE Healthcare, USA) at 1:100 dilution for 1 h. The unboundconjugate was removed by washing with PBS. The cells werecarefully pipetted onto the glass slide, mounted with a cover slipand examined using an Olympus FV 1000, a confocal laser scanningmicroscope (Center of Nanoimaging, Mahidol University, Thailand).

2.7. Cytotoxicity assays on Sf9 cells-expressing Aa-mALP

Aa-mALP-expressing Sf9 cells susceptibility to Cry4Ba toxin wereobserved for changes in cell morphology using light microscope(Nikon Eclipse TS100) as well as by trypan blue cell viability assay.Briefly, w8 � 105 Sf9 cells were seeded on a six-well culture plate.After 3-day post-infection, cells were washed once with PBS, and100 mg/ml activated Cry4Ba toxin (mixed with 1 ml Sf-900 medium(Invitrogen, USA)) was added and further incubated for 3 h. Anychanges in cell morphologywere observed at this point. To determinecellmortality, trypanblueexclusion assaywasperformed. Briefly, cellswere washed once with 1 ml PBS, mixed with 1 ml 0.4% trypan blueand then quantitated under light microscope using hemocytometer.

3. Results

3.1. Identification and cloning of Aa-mALP

Even though a complete A. aegypti genome is available, its anno-tation is far from completion. To obtain the closest insect relative ofA. aegypti mALP gene, a literature survey was performed and a GPI-ALP was identified from B. mori (Bm-mALP) (Itoh et al., 1991).Therefore, the A. aegypti genome was next searched for geneencoding Aa-mALP using the deduced amino acid sequence of Bm-mALP. The result showed a clone (AAEL015070-RA) with the highestsequence identity (37%) to Bm-mALP. Then, RT-PCR was performedusing total RNAs extracted from A. aegypti larval midgut and twogene-specific primers, Topo-ALP-forward and Topo-ALP-reverse. Theresulting w1600-bp PCR product (data not shown) was cloned andsequenced. Sequence analysis showed that it is identical to the one inthe A. aegypti database. This Aa-mALP cDNA is 1608-bp long,encoding 535 amino acids with a calculated molecular mass of58.7 kDa. FromBLASTPanalysis, it showedhighest identity of 67%and65% toCulex quinquefasciatus (XP_001865437) andAnopheles gambiae(EAA10738) ALPs, respectively. However, it should be noted that theALP from this Culex species is much smaller, only 402 amino acids asdeduced from the genomedatabase.Moderate sequence identitywasfound against ALPs fromothermosquitoes and insects. For instance, itshowed only 33e35% identity to a putative Cry11Aa receptor from A.aegypti (Fernandez et al., 2009), a Cry11Ba receptor from A. gambiae(Hua et al., 2009), another ALP from C. quinquefasciatus (CPIJ018121-RA), a Cry1Ac receptor from Heliothis virescens (Perera et al., 2009),and an ALP from B. mori (Itoh et al., 1991) (Fig. 1). Using SignalP


program (www.cbs.dtu.dk/services/SignalP/), thefirst 36 amino acidsare predicted to be the signal peptide. The predicted GPI-anchoringsite is at positionCys512 as calculatedby threedifferentGPI-predictionprograms (PredGPI, http://gpcr2.biocomp.unibo.it/gpipe/pred.htm;big-PI Predictor, http://mendel.imp.univie.ac.at/sat/gpi/gpi_server.html; FragAnchor, http://navet.ics.hawaii.edu/wfraganchor/NNHMM/NNHMM.html). This Aa-mALP contains 9 out of the 13conserved amino acids (or conservative substitutions) that areproposed to be either involved in the hydrolysis of alkaline substrateor in metal binding (Sowadski et al., 1985; Itoh et al., 1999). Eventhough thisAa-mALP lacks 4 conserved residues, homologymodelingwas performed using SWISS-MODEL showing similar 3D structure toother membrane-bound ALPs (data not shown). Moreover, threepotential N-linked (Asn94, Asn210 and Asn287) and one O-linked(Thr395) glycosylation sites are foundaspredictedusingNetNGlyc andNetOGlc, respectively (http://www.cbs.dtu.dk/services).

Further analysis of the A. aegypti genomic database using ourAa-mALP as query sequence, 13 other ALP sequences are found(Table 1). However, only this Aa-mALP showed a consensusprediction by all three GPI-prediction algorithms to contain theGPI-anchoring signal.

3.2. Expression of the recombinant Aa-mALP protein ininsect Sf9 cells

The Aa-mALP cDNAwas cloned into a transfer vector, pENTR/SD-TOPO, to generate a pENTR/Aa-mALP recombinant plasmid. Then,the Aa-mALP fragment was transferred to Baculodirect C-termLinear DNA via LR recombination method. And, finally, therecombinant Aa-mALP baculovirus DNAwas integrated into the Sf9cell genome via ganciclovir selection as verified by PCR (data notshown). Recombinant Aa-mALP expression was analyzed usingalkaline phosphatase assay and SDS-PAGE. ALP activity wasmeasured for the ability to hydrolyze PNPP substrate to p-nitro-phenol. The results showed that Aa-mALP-expressing cellsproduced significantly higher ALP activity than non-Aa-mALP-expressing cells (Fig. 2). Of course, as expected, endogenous ALPactivity was detected as well. For SDS-PAGE, the results showeda prominent 58-kDa protein of the predicted size collected from celllysate (Fig. 3A, lane 3). For the control CAT-expression, a 30-kDaprotein band was observed as expected (Fig. 3A, lane 2). To confirmthat the 58-kDa protein band indeed represents the Aa-mALP, it was


http://www.cbs.dtu.dk/services/SignalP/

http://gpcr2.biocomp.unibo.it/gpipe/pred.htm

http://mendel.imp.univie.ac.at/sat/gpi/gpi_server.html

http://mendel.imp.univie.ac.at/sat/gpi/gpi_server.html

http://navet.ics.hawaii.edu/%7Efraganchor/NNHMM/NNHMM.html



http://www.cbs.dtu.dk/services

http://www.ch.embnet.org/software/LALIGN_form.html

http://www.ch.embnet.org/software/LALIGN_form.html

Fig. 2. Alkaline phosphatase activity of Aa-mALP-expressed Sf9 cells. Non-infected,CAT-infected and Aa-mALP-infected cells were tested for ALP activity by measuring theformation of chromogenic product, free p-nitrophenol absorbance at 405 nm. Thegraph was created by SigmaPlot. Bars denote the SEM value from three independentexperiments.

M. Dechklar et al. / Insect Biochemistry and Molecular Biology xxx (2010) 1e8 5

excised and submitted for mass spectrometry analysis. The resultsshowed multiple predicted peptide sequences belonging to Aa-mALP (302RLPYAADMGEEPNKE316, 322RMVHYSLEMLQKK334,333KKEHSGGFLLFVEDGNIQEAHKENKPIKA361,444RVNPLAVLQGTPTQKE459). Furthermore, to confirm that thisrecombinant Aa-mALP is indeed GPI-linked, we performed a phos-pholipase assay using a phosphatidylinositol-specific phospholi-pase C (PI-PLC) on recombinant Sf9 cells. The result showed that inthe presence of PI-PLC, a 58-kDa Aa-mALP proteinwas cleaved fromrecombinant Sf9 cell surface and released into the supernatantfraction (Fig. 3B).

3.3. Binding of Cry4Ba to Sf9 cells-expressing recombinantAa-mALP

To investigate the interaction between Cry4Ba and the recombi-nant Aa-mALP, Sf9 cells-expressing Aa-mALP were incubated with

A B

kDa

166

66

200

45

31

97

21

58 kDa

30 kDa

M 1 2 3

97

kD

200

45

31

21

166

66

Fig. 3. Expression and PI-PLC analysis of recombinant Aa-mALP protein in Sf9 cells. (A) Sf9 cpre-stained protein markers; lane 1 is non-infected cells; lane 2 is recombinant CAT-infectedAa-mALP is observed in the cell lysate fraction (lane 3, arrow head). The expression of the 30PLC and both the pellet and supernatant fractions were collected and analyzed on 10% SDS-and supernatant fractions of recombinant Aa-mALP without PI-PLC treatment; lanes 3 andcleavage product of Aa-mALP is detected only in the supernatant fraction of PI-PLC treated


the 65-kDa trypsin-activated Cry4Ba toxin and visualized using thepreviously characterizedmonoclonal antibody against the domain IIIof the toxin (Moonsom et al., 2007). The results showed that only Aa-mALP-expressing cells are recognized by Cry4Ba toxin (Fig. 4); therewas no Cry4Ba-binding signal in either non-infected or in CAT-infected cells (Fig. 4D and E). Furthermore, upon closer examination,the results of Cry4Ba-immunoreactivity indicated that Aa-mALPs arefound mainly on the surface membrane of Sf9 cells.

3.4. Cytotoxic effect of Cry4Ba toxin on Sf9 cells-expressingrecombinant Aa-mALP

To determine if indeed, like other lepidopteran mALPs, themosquitomALP canalso function as aCry toxin receptor, the trypsin-activated Cry4Ba toxin (Fig. 5, insert) was applied to the Sf9 cells-expressing Aa-mALP. The results, after 3-h incubationwith the toxin(100 mg/ml), showed that only Aa-mALP-expressing cells incubatedwith Cry4Ba underwent cell lysis as this can be seen by the swellingof the cells (Fig. 5F). In contrast, controls CAT-infected cells and non-infected cells were insensitive to Cry4Ba (Fig. 5D and E).

Finally, to quantitate the number of cell deaths, trypan blueexclusion assay was performed. Cell mortality of Aa-mALP-infectedcells was shown to increase significantly in the presence of Cry4Ba(Fig. 6) e 61% (with Cry4Ba) vs 34% (without Cry4Ba). As expected,cell mortality of non-infected (18% vs 22%) or CAT-infected cells(32% vs 37%) was not significantly different in the presence ofCry4Ba.

4. Discussion

Membrane-bound alkaline phosphatases are thought to act as BtCry binding protein in both lepidopteran and dipteran insect larvae(Jurat-Fuentes and Adang, 2004; Fernandez et al., 2006). In thiswork, a cDNA encoding a novel membrane-bound alkaline phos-phatase was isolated from A. aegypti larval midgut (Aa-mALP),comprises 1608 nucleotides, encoding 535 amino acids. This Aa-mALP is believed to be a membrane-bound GPI-anchored proteinand is predicted to contain possible N-linked and O-linked glyco-sylation sites. However, since the size of the protein on the SDS-

M 1 2 3 4a

58 kDa

ell lysates were harvested and analyzed on SDS-PAGE (12% gel). Lane M is broad-rangecells; and lane 3 is recombinant Aa-mALP-infected cells. The expression of the 58-kDa-kDa CAT protein is detected in lane 2. (B) Recombinant Aa-mALP was treated with PI-PAGE. Lane M is broad-range pre-stained protein markers; Lanes 1 and 2 are cell lysate4 are cell lysate and supernatant of recombinant Aa-mALP treated with PI-PLC. Therecombinants (lane 4, arrow head).


Fig. 4. Immunolocalization of Cry4Ba toxin against Aa-mALP-expressed Sf9 cells. A, B and C are Nomarski interference contrast view of non-infected, CAT-infected and Aa-mALP-infected cells, respectively. The cells were treated with Cry4Ba (100 mg/ml) toxin for 2 h before they were fixed with 4% paraformaldehyde, blocked, and incubated with antibodyagainst Cry4Ba toxin followed by secondary antibody conjugated with FITC. D, E and F are fluorescent signal of non-infected, CAT-infected and Aa-mALP-infected cells, respectively.Similar results were obtained from three independent experiments. Scale bar is 10 mm.


PAGE is of the predicted 58 kDa, this suggested that it was notglycosylated in the Sf9 cells. This indicated that Aa-mALP, unlikeAPNs (Knight et al., 2004) or other lepidopteran ALPs (Jurat-Fuentesand Adang, 2004; Perera et al., 2009), does not need glycosylationto be a functional receptor. Our finding is in agreement with that ofBuzdin et al. (2002) which showed that N-acetylgalactos aminedoes not inhibit the binding between the Cry4Ba and ALPs. The high

Fig. 5. Effects of Cry4Ba toxin on Aa-mALP-expressed Sf9 cells. A, B and C are non-infected,and F are non-infected, CAT-infected and Aa-mALP-infected cells, respectively, with 100 mg/the two trypsin-resistant bands of 47 and 20 kDa.


expression of the 58-kDa Aa-mALP was obtained in the very latestage of viral infection (3e5 days of post-infection) and its role inCry4Ba toxicity was investigated using the recombinant Sf9 cells.

Immunofluorescence study showed that Cry4Ba was able tobind only to those Sf9 cells-expressing Aa-mALP and that thisbinding appears to be localized only on the peripheral membrane.Moreover, from the PI-PLC digestion experiment and the presence

CAT-infected and Aa-mALP-infected cells, respectively, without Cry4Ba treatment. D, Eml Cry4Ba treatment. Inset is Cry4Ba toxin after activation by trypsin (lane 1) showing


Fig. 6. Cry4Ba is toxic against Sf9 cells-expressing Aa-mALP as assayed by trypan blueexclusion activity. Non-infected, CAT-infected and Aa-mALP-infected cells are countedafter incubating with Cry4Ba (100 mg/ml) for 3 h. For controls, the cells were incubatedin the same condition except that the toxin was omitted. The number of death cellswas counted by hemocytometer. The graph was drawn using SigmaPlot. Plot values arecalculated from the mean of three independent experiments. Bars denote the SEMvalue from triplicate experiments. Statistical analysis was performed by one-wayANOVA with Tukey Test. The level of statistical significance was assigned at theP � 0.05. The asterisk represents a statistically significant difference (P < 0.01)compared to other two groups. - and denote cell mortality (%) in the absence andpresence of Cry4Ba toxin, respectively.

M. Dechklar et al. / Insect Biochemistry and Molecular Biology xxx (2010) 1e8 7

of GPI sequence signature at the protein C-terminal, this stronglysuggested that Aa-mALP in Sf9 cells to be membrane-bound. Also,from the cytotoxicity results, Aa-mALP-expressing cells showedsusceptibility to the Cry4Ba toxin; in contrast, cell mortality of non-infected cell and CAT-infected cell did not significantly increase inthe presence of Cry4Ba. Altogether, our results indicated that a GPI-anchored Aa-mALP is one of the Cry4Ba receptors.

A recent publication (Fernandez et al., 2009) demonstrated thatanother membrane-bound ALP (ALP1) from A. aegypti larva caninteract with another mosquito-larvicidal protein e Cry11Aa andthat this ALP1 can also interact weaklywith Cry4Ba asmeasured viaELISA binding assay (however, no toxicity data was reportedregarding Cry4Ba and ALP1). Of interest here is the fact that thesetwo ALPs, Aa-ALP1 and Aa-mALP, shared only 39.3% amino acidsequence identity (Fig. 1). This suggested that Cry4Ba and Cry11Aamay employ different ALPs as their midgut receptors. However, itwas reported, based on ligand blot study that Cry1Aa can bind toboth the 113-kDa and the 170-kDa APNs whose amino acidsequence identity is just only 33%; but, on the other hand, Cry1Abcan only interact with the 113-kDa APN (Budatha et al., 2007). Afuture toxicity assay using Cry11Aa will need to be performed onthe recombinant Sf9 cells to answer this question. Also recently, inthe A. gambiae mosquito larva, it is shown that an ALP with pre-dicted GPI-anchor site can act as a receptor to Cry11Ba from Btstrain jegathesan (Hua et al., 2009). Thus, like lepidopterans inwhich numerous Bt receptors studies have been conducted,multiple Bt receptors may also be needed in the dipterans for thetoxins to exert their effects. For instance a recent report suggestedboth GPI-anchored APN and ALP are employed as functionalreceptors for Cry1Ab in Manduca sexta larvae (Arenas et al., 2010).Our ongoing research is to co-express other potential Bt receptorsto answer this question.

In conclusion, our results indicated for the first time that the 58-kDa GPI-Aa-mALP protein was responsible for mediating Cry4Batoxicity against Sf9 cells, suggesting its role as a receptor for this


mosquito-active toxin in A. aegypti larvae. Further studies for moreinsights into the structural details of specific interactions betweenthe Cry4Ba toxin and its characterized receptor, i.e. Aa-mALP, wouldbe of great interest.

Acknowledgements

This research was supported in part by grants from BIOTEC andthe Thailand Research Fund in cooperationwith the Commission onHigher Education (CHE), Ministry of Education (Thailand). TheStrategic Scholarship from CHE to MD is gratefully acknowledged.

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