Immunity or DigestionGLUCANASE ACTIVITY IN A GLUCAN-BINDING PROTEIN FAMILY FROM LEPIDOPTERA*□S

Received for publication, August 11, 2008, and in revised form, November 20, 2008 Published, JBC Papers in Press, November 25, 2008, DOI 10.1074/jbc.M806204200

Yannick Pauchet1, Dalial Freitak2, Hanna M. Heidel-Fischer, David G. Heckel, and Heiko VogelFrom the Department of Entomology, Max Planck Institute for Chemical Ecology, Beutenberg Campus, Hans-Knoell-Str. 8,07745 Jena, Germany

The cell surfaces of microorganisms display distinct molecu-lar patterns formed from lipopolysaccharides, peptidoglycans,or �1,3-glucans. Binding of these surfaces by pattern recogni-tion proteins such as �1,3-glucan recognition proteins (�GRPs)activates the immune response in arthropods. We identified a40-kDa�1,3-glucan-bindingproteinwith sequence similarity topreviously characterized lepidopteran �GRPs from hemo-lymph, but unlike these it is secreted into the larval gut lumenand is an active�1,3-glucanase. This glucanasewas not detectedin hemolymph. Its mRNA is constitutively and predominantlyexpressed in the midgut and is induced there when larvae feedon a diet containing bacteria. Homologs of this predominantlymidgut-expressed gene frommany Lepidoptera possess key res-idues shown to be part of the active site of other glucanases, andform a cluster that is distinct from previously described �GRPs.In addition, this group includes proteins from insects such as theAnopheles gambiaeGNBP subgroup B for which a catalytic rolehas not been previously suspected. The current domain classifi-cation does not distinguish between the catalytic and noncata-lytic clades, and should be revised. The noncatalytic�GRPsmaybe evolutionarily derived from this newly described enzymefamily that continues to function catalytically in digestionand/or pathogen defense.

Recognition of invading organisms as non-self is a crucialpre-requisite for an immune response. In arthropods, immuneresponse is triggered in the hemolymph by so-called patternrecognition proteins or receptors, which bind to classes of bac-teria- and fungi-specific polysaccharides such as peptidogly-cans, �1,3-glucans, lipopolysaccharides (LPS),3 and lipotechoicacid (LTA) (1, 2). Little detail is known for Lepidoptera but inDrosophila these binding interactions lead to activation of the

innate immune response, largely regulated by two main path-ways, the Toll and Imd pathways. The innate immune systemcontains of three main effector mechanisms: the cellularresponse, the humoral response, and melanization. The activa-tion of the signaling pathways leads to a cascade of events thatresult in the induction of defensive genes in hemocytes and thefat body, including antimicrobial peptide genes (3), and also tothe activation of the prophenoloxidase (proPO) cascade. Phe-noloxidase (PO) is an essential enzyme for the cellular immuneresponses (4) but is also involved in other developmental anddefensive processes such as wound healing and sclerotization(1).Gram-negative bacteria-binding proteins (GNBPs) and�1,3-

glucan recognition proteins (�GRPs) have been extensivelystudied as pattern recognition proteins in Lepidoptera (5–10).These proteins are produced in the fat body and secreted intothe hemolymph of the caterpillar. Some are constitutively pres-ent (5, 8), whereas others are induced upon microbial infection(6). Recognition of�1,3-glucans by these proteins appears to bedue to two distinct �1,3-glucan-binding domains: a stronglybinding N-terminal glucan recognition domain and a weaklybinding C-terminal glucanase-like domain. Neither domainpossesses �1,3-glucanase activity (11).The immune response has been extensively studied in

Lepidoptera by introducing microbes into the hemocoel oflaboratory-reared insects by mechanical wounding. However,because herbivorous lepidopteran larvae consume largeamounts of plant material during their development, infectionvia food is very likely. The potential role of the midgut in trig-gering the immune response of herbivorous larvae has beenpoorly studied, but there is increasing evidence that it partici-pates in this process. Freitak et al. (12) showed that in larvae ofTrichoplusia ni that were fed an artificial diet containing bac-teria, several immune-related genes were up-regulated andimmune-related proteins increased in concentration in thehemolymph.If the midgut were to play a role in sensing potentially inva-

sive bacteria, one would expect to find pattern recognition pro-teins expressed in that tissue. Bacteria present in theDrosophilagut are thought to release peptidoglycan fragments that bind tospecific PGRP (peptidoglycan recognition protein) receptorsand induce the imd pathway (13–15). Simpson et al. (16)described a sequence (EpGRP1) with similarity to �1,3-glucan-binding proteins from Anopheles gambiae and Bombyx mori inESTs derived from the larvalmidgut ofEpiphyas postvittana. Ina proteomic study of the lumen contents of larval midgut ofHelicoverpa armigera, we (17) discovered a similar protein that

* This work was supported by the Max Planck Society. The costs of publicationof this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. S1–S3.

1 To whom correspondence should be addressed: School of Biosciences, Uni-versity of Exeter in Cornwall, Penryn, Cornwall TR10 9EZ, United Kingdom.Tel.: 441326371852; Fax: 441326253638; E-mail: Y.Pauchet@exeter.ac.uk.

2 Supported by an International Max Planck Research School (IMPRS) Ph.D.fellowship.

3 The abbreviations used are: LPS, lipopolysaccharide; LTA, lipotechoic acid;PGRP, peptidoglycan recognition protein; proPO, prophenoloxidase;GNBP, Gram-negative bacteria-binding protein; �GRP, �1,3-glucan recog-nition protein; PBS, phosphate-buffered saline; MALDI-TOF, matrix-as-sisted laser desorption ionization time-of-flight; MS, mass spectrometry;RT, reverse transcription.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 4, pp. 2214 –2224, January 23, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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we named GH16betaGRP-1 (glycosyl hydrolase family 16,�-glucan recognition protein 1) secreted into the lumen. Thefull-length cDNA showed similarities to �1,3-glucan recogni-tion proteins from the hemolymph of B. mori and Manducasexta. Because �1,3-glucanase activity had not been previouslyreported from lepidopteranmidguts, we suggested the possibil-ity of an immune-related function.Here we describe the purification and characterization of

GH16betaGRP-1 from the midgut lumen of H. armigera. Theprotein does indeed possess �1,3-glucanase activity and canaccount for the majority of such activity in the lumen. Thus wehave renamed this protein as H. armigera �1,3-glucanase-1.The mRNA is predominantly expressed in the midgut, and theprotein product is secreted into the lumen and persists stablythere. Expression is increased when the insect feeds on a dietcontaining bacteria. We found similar sequences from a num-ber of lepidopteran species derived from cDNAs generatedfrommidgut tissue samples. These form a novel protein familythat is characterized by a highly conserved signature sequenceincluding two glutamate residues previously shown to be nec-essary for catalytic activity in other �1,3-glucanases (18). Thisfamily is related to but distinct from a family of previouslydescribed �GRP/GNBP proteins primarily found in lepidopt-eran hemolymph, which do not possess this conserved site.Certain proteins from insects for which a catalytic role has notbeen suspected, including the A. gambiae GNBP subgroup B,belong to the catalytic clade and we suggest that the domainnomenclature be revised accordingly. These features suggestthat lepidopteran larvae secrete an active �1,3-glucanase intothe midgut lumen that may function in digestion of �1,3-glu-cans released by commensal or invading bacteria.

EXPERIMENTAL PROCEDURES

Insects—The TWB strain of H. armigera Hubner (Lepidop-tera:Noctuidae)was collected from the vicinity ofToowoomba,Queensland, Australia. Neonates were reared on a chemicallydefined diet containing only casein as the protein source and noplant-derived material, as described by Vanderzant (39) at26 °C with a 16:8 (light:dark) photoperiod.Sample Preparation—Gut lumen sample was prepared

essentially as described by Pauchet et al. (17). Briefly, midgutswere dissected from actively feeding second day fifth-instar lar-vae in ice-cold phosphate-buffered saline (PBS). Peritrophicmatrix containing the food bolus was pulled out of the midgutwith forceps and gently homogenized by 10 strokes in a Potter-Elvehjem homogenizer in PBS, pH 7.5, containing a mixture ofprotease inhibitors (Complete EDTA-free, Roche Applied Sci-ence) to release soluble proteins. After centrifugation (30,000�g, 30 min, 4 °C), the supernatant containing the gut lumen-soluble proteins was kept and protein concentration was deter-mined using the Protein Dye reagent (Bio-Rad) and bovineserum albumin as standard. Hemolymph was collected via lat-eral punctuation of abdominal part of larvae withmicropipette.Collected hemolymph was immediately diluted with ice-coldN-phenylthiourea-saturated PBS containing protease inhibi-tors and hemocytes were removed by centrifugation (3,000� g,15 min, 4 °C).

CurdlanPull-downAssay—Gut lumen andhemolymph sam-ples (1 ml each) were incubated 30 min at 22 °C with 2.5 mg ofcurdlan (an insoluble�1,3-glucan isolated fromAlcaligenes fae-calis, Sigma). After centrifugation of the sample/curdlan mix-ture (16,000� g, 5min, 4 °C), the supernatant corresponding tothe unbound fractionwas saved. The curdlan pellet was washed5 times with PBS followed by 2 washes with PBS containing 1 MNaCl and finally 3 more times with PBS. Bound proteins wereeluted from curdlan by boiling 10 min in SDS-PAGE samplebuffer. After centrifugation (16,000� g, 5 min, 4 °C), the super-natant was analyzed by SDS-PAGE.Protein Analysis by Mass Spectrometry—This was done

essentially as described by Pauchet et al. (17). Protein bandswere excised manually from the SDS-PAGE gels and weredestained, trypsinized, and extracted using an Ettan TADigester running the Digester Version 1.10 software (GEHealthcare Bio-SciencesAB). Trypsin digestionwas carried outovernight with 50 ng of porcine trypsin (Promega) at 37 °C.First step analysis of the tryptic peptides was done using aMALDImicro MX mass spectrometer (Waters) used in reflec-tron mode and was calibrated using a tryptic digest of bo-vine serum albumin (MPrep, Waters). The MALDI-TOF spec-tra searches were performed with the Protein Lynx GlobalServer software, version 2.2 (PLGS 2.2, Waters), against theNCBI_insecta data base (downloaded on 15 March 2008 fromncbi.nlm.nih.gov/data base, 372,057 entries). The searchparameters were as follows: peptide tolerance of 80 ppm, onemissed cleavage, carbamidomethyl modification of cysteines,and possible Met oxidation. An estimated calibration error of0.05Dand aminimumof four peptidematcheswere the criteriafor obtaining positive data base hits.The MALDI-TOF peptide signal intensities were used to

estimate the volume of the remaining sample to be used for thesubsequent nanoLC-MS/MS de novo sequence analysis. Liquidchromatography-tandemmass spectrometry was performed toacquire fragmentation data from selected peptides. Aliquots oftryptic peptides were injected on a CapLC XE 2D nanoLC sys-tem (Waters). After concentration and desalting, eluted pep-tides were transferred to the NanoElectroSpray source of aQ-TOF Ultima tandem mass spectrometer (Waters).MS/MS spectra were collected byMassLynx version 4.0 soft-ware (Waters). ProteinLynx Global Server Browser version2.2 software (PLGS 2.2, Waters) was used for baseline sub-traction and smoothing, deisotoping, and de novo peptidesequence identification.Using PLGS 2.2, CID spectra were interpreted de novo to

yield peptide sequences. Sequences with a ladder score (per-centage of expected y- and b-ions) exceeding 30 were then usedin a homology-based search strategy using theMS BLAST pro-gram (19). MS BLAST was developed to utilize redundant,degenerate, and partly inaccurate peptide sequences in similar-ity searches of protein databases that may be derived from or-ganisms phylogenetically distant from the study species. TheWU-BLAST2 BLASTP search engine is employed with param-eter values that disallow gaps within a peptide, and that scoreonly the most significant match in the case of several peptidecandidates covering the same region in the target sequence. Inaddition, the PAM30MS matrix that accounts for the inability

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to distinguish Ile and Leu residues, and allows for unknownresiduesX, is used in the blastp similarity search (19). Scoring ofthe significance of peptidematches is not based on E or p valuesof the individual HSPs (high-scoring segment pairs) but insteadon pre-computed threshold scores conditional on the numberof query peptides and HSP hits. The color-coded output pro-duced by the MS BLAST script identifies in red those targetsequences with scores exceeding 99% of queries utilizing ran-domized peptide sequences by chance. Computational studieshave estimated a false positive rate of�3% (20).We installed anin-house MS BLAST server for searching NCBI_insecta and alocally generated EST data base from H. armigera midgut andfat body cDNA libraries (5,685 protein sequences). Additionalinformation on MS BLAST statistics and scoring can be foundin Shevchenko et al. (19).

�-Glucanase Activity Assays—Lichenan, barley �-glucan,and carboxymethyl (CM)-cellulose were from Sigma. CM-curdlan, endo-�1,3-glucanase from Trichoderma sp. and cellu-lase from Trichoderma sp. were both fromMegazyme.Plate assays were performed on 1.5% agar Petri dishes con-

taining 0.25% of each substrate in 50 mM citrate-phosphatebuffer, pH6.0.Wellswithin platesweremade by puncturing theagar with a plastic pipette and removing the agar plug by suc-tion. Two and a half-micrograms of gut lumen proteins in 2.5�lof PBS were tested together with 2.5 �l of PBS as negative con-trol, and 1 �l of a 100 times diluted solution of each endo-�1,3-glucanase and cellulase as positive controls. After 16 h incuba-tion at 37 °C, activity was revealed by staining with Congo Red(0.1% (w/v) in water) for 30 min and destaining with 1 M NaClalso for 30 min. Plates were scanned using a GS800 densitom-eter (Bio-Rad).For zymograms, sampleswere resolved bynative PAGEusing

4–15% polyacrylamide gels (Ready Gels, Bio-Rad) and Tris gly-cine, pH 8.3, as running buffer. Electrophoresis was performedat 150 V for 60 min at 4 °C. After the run, gels were washed 3times for 15 min in 50 mM citrate-phosphate buffer, pH 6.0,before being overlaid on a CM-curdlan plate prepared asdescribed above. Incubation and Congo Red staining/destain-ing were performed as described above.Cell Culture and Transfection—The Ha_GH16betaGRP-1

cDNA was amplified from a H. armigera larval midgut cDNAlibrary clone using primers GRP1-F (5�-GCCACCATGTGGT-CGGTGTTAGCGGGCGTG-3�) and GRP1-R (5�-CAAAGC-CCAAATGCGAACGTAGTC-3�) and was inserted in pIB/V5-His TOPO (Invitrogen) by TA cloning according to thesupplier’s instructions. Positive clones were selected and cor-rect insertion was confirmed by sequencing.T. ni High Five cells (Invitrogen) were cultured at 27 °C in

Express Five serum-free medium (Invitrogen) supplementedwith 2 mM L-glutamine and 10 �g/ml gentamycin. Cells weretransfected in 90-mm diameter Petri dishes with 12 �g of plas-mid DNA using Insect Gene Juice (Novagen) as transfectionreagent. Forty-eight hours post-transfection, culture mediumwas saved and the cells were detached from the plate and pel-leted by centrifugation. After two washes with PBS, cell pelletswere resuspended in PBS supplemented with Complete prote-ase inhibitor mixture (Roche Applied Science). Six cycles offreezing and thawing were performed to lyze the cells. Crude

membrane fraction was recovered by centrifugation (13,000 �g, 20 min, 4 °C) and resuspended in cold PBS/Complete.Expression was analyzed by Western blot using the anti-V5-horseradish peroxidase antibody (Invitrogen).Preparation of cDNA Libraries—TRIzol reagent (Invitrogen)

was used to isolate total RNA from whole larvae or dissectedtissues according to the manufacturer’s protocol. After DNasetreatment, total RNAwere further purified by using the RNeasyMinElute Clean up Kit (Qiagen) following the manufacturer’sprotocol. Poly(A)� mRNA were purified by binding to an oli-go(dT) column (RNA Purist, Ambion). The generation of T. niwhole larvae cDNA library has been described in Freitak et al.(12). Generation of Plutella xylostella, Pieris rapae,Colias eury-theme, Anthoracharis cardamines, and Delias nigrina has beendescribed in Fischer et al. (21). For H. armigeramidgut and fatbody and Spodoptera littoralis whole larvae, normalized, full-length enriched cDNA libraries were generated using a combi-nation of the SMARTcDNA library construction kit (Clontech)and the Trimmer-Direct cDNA normalization kit (Evrogen)generally following the manufacturer’s protocol but with sev-eral important modifications. In brief, 2 �g of poly(A)� mRNAwas used for each cDNA library generated. Reverse transcrip-tion was performed with a mixture of several reverse transcrip-tion enzymes for 1 h at 42 °C and 90 min at 50 °C. cDNA sizefractionation was performed with SizeSep 400 spun columns(GE Healthcare) that resulted in a cutoff at �200 bp. The full-length enriched cDNAswere cutwith SfiI and ligated to pDNR-Lib plasmid (Clontech). Ligations were transformed into Esch-erichia coli ELECTROMAX DH5�-E electrocompetent cells(Invitrogen).Generation of EST Databases—Plasmid minipreparation

from bacterial colonies grown in 96 deep-well plates was per-formed using the 96-robot plasmid isolation kit (Eppendorf) onaTecanEvoFreedom150 robotic platform (Tecan). Single-passsequencing of the 5� termini of cDNA libraries was carried outon an ABI 3730 xl automatic DNA sequencer (PE Applied Bio-systems). Vector clipping, quality trimming, and sequenceassembly was done with the Lasergene software package(DNAStar Inc.). Blast searcheswere conducted on a local serverusing the National Center for Biotechnology Information(NCBI) blastall program. Protein domains were determined bysearching the NCBI Conserved Domain Data base (CDD).Sequences were aligned using ClustalW software (22).BAC Library Screening and Sequencing—H. armigera BAC

library nylon filters were washed, blocked, and hybridized withhorseradish peroxidase-labeled DNA fragments containingpart of the GH16betaGRP-1 gene. Labeling, hybridization, andprobe detection were done according to specifications in theECL DNA labeling and detection kit (GE Healthcare). Positiveclones were isolated from glycerol stocks, grown in TerrificBroth, and BAC DNA was isolated with the Nucleobond XtraMidi Kit according to the manufacturer’s instructions (Mach-erey-Nagel). BACGenomic DNA quantity was measured spec-trophotometrically on a Nanodrop ND1000 and all of the pos-itive clones were digested with EcoRI and HindIII to identifyclone diversity and redundant inserts, blotted, and re-hybrid-ized to verify positive inserts. The BAC DNA was sheared intotwo different size ranges (1–1.5 kb and 4–5 kb) with a Hydro-

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shear device (Molecular Devices), blunted with the QuickBlunting Kit (New England Biolabs), isolated from an agarosegel, column purified, and ligated into the pUC19-SmaI vector(Fermentas). Ligations were transformed into E. coli ELEC-TROMAX DH5�-E electrocompetent cells (Invitrogen). Plas-mid preparation, sequencing, and assembly were performed asmentioned above.Feeding Assays—Fifteen early fifth-instar H. armigera larvae

were fed for 24 h on an artificial pinto bean-based diet soakedwith overnight liquid culture of Saccharomyces cerevisiae,Micrococcus luteus, or E. coli. Alternatively, larvae were fed onan artificial diet soaked in solutions containing either�-glucans(0.25% w/v) or bacterial cell wall polysaccharides (LTA or LPS,0.05% w/v). Larvae fed on a sterile artificial diet were used as acontrol group. Three times 5 larvae per diet were used fordetecting diet-induced changes inGH16betaGRP-1 expressionin midgut tissue. Experiments were repeated 3 times with dif-ferent patches of eggs.Quantitative Real-time PCR—Total RNAwas prepared from

dissected insect midguts, salivary glands, fat bodies, and theremainder of bodies (except head capsules) according to themethod described above. Five hundred nanograms of DNA-free total RNAwas converted into single-stranded cDNA usinga mixture of random and oligo(dT)20 primers according to theABgene protocol (ABgene). As an endogenous control gene,large ribosomal protein 1 (Lrp1) was used (forward primer:5�-CACATCAGCAAACACATCACC and reverse primer:5�-AGAAGTGAGAGCCGTGTGAAA). Gene-specific prim-ers were designed on the basis of sequence obtained for theGH16betaGRP-1 gene (forward primer: 5�-CTGGATCA-GAAGGAACACTGC and reverse primer: 5�-TACACGTC-CCAGTTCCAAGTC).Quantitative RT-PCRwas done in opti-cal 96-well plates on a MX3000P Real-time PCR DetectionSystem (Stratagene) using the AbsoluteQPCR SYBR greenMix(ABgene) to monitor double-stranded DNA synthesis in com-bination with ROX as a passive reference dye included in thePCR master mix.Phylogenetic Analysis—Genes were aligned by their amino

acid sequences using theMAFFTmultiple sequence alignmentprogram (23). The amino acid alignment was then used forphylogenetic analysis.The phylogenetic reconstructionwas done byBayesian infer-

ence using Mr. Bayes 3.1 (24). The prior was set for the aminoacid models to mix, thereby allowing model jumping betweenfixed-rate amino acidmodels.Markov ChainMonte Carlo runswere carried out for 1,000,000 generations after which log like-lihood values showed that equilibrium had been reached afterthe first 400 generations in all cases, and those data were dis-carded from each run and considered as “burnin.” Two runswere conducted for the dataset showing agreement in topologyand likelihood scores.Phylogenetic analysis was furthermore, performed using the

Neighbor Joining (NJ) method (TREECON) based on theMAFFT alignments. Distance calculations were performedafter Tajima & Nei and bootstrap analysis, running 1000 boot-strap samples. The Neighbor joining and the Bayesian treetopologies including their general subfamily relationships andnode supports were in agreement.

RESULTS

A �1,3-Glucan-binding Protein from H. armigera LarvalMidgut—Midgut lumen contents of unchallenged H. armigeralarvae were screened for glucan-binding proteins using curd-lan, an insoluble �1,3-glucan isolated from the Gram-negativebacterium A. faecalis, as an affinity matrix in pull-down assays.The bound fraction was freed from the matrix by boiling andafter SDS-PAGE, a single main protein band (apparent molec-ular mass 40 kDa) was detected along with trace amounts ofother proteins (Fig. 1A). The peptide mass fingerprint of thismain protein band by MALDI-TOF/MS analysis exactlymatched the cDNA sequence of H. armigera GH16betaGRP-1(ABU98621.1, supplementary Fig. S1) showing that this previ-ously identified protein, which had been named on the basis ofsequence similarity to other Lepidopteran proteins, was themain �1,3-glucan-binding protein in the larval midgut lumen.Expression patterns of this gene in different tissues were fur-

ther investigated by quantitative RT-PCR (Fig. 1B). The expres-

MW 1 2 3

15

202530

40506070100200

1

B

FIGURE 1. Purification and tissue expression of H. armigera glucanase-1.A, curdlan pull-down assay on gut larval lumen sample. Proteins were sepa-rated by SDS-PAGE using a 12% polyacrylamide resolving gel and stainedwith Coomassie. Lane 1, 10 �g of gut lumen proteins; lane 2, 10 �g of gutlumen flow-through; lane 3, 15 �l of elution fraction. Tryptic peptidesobtained from band 1 were analyzed by MALDI-TOF/MS (see supplementalFig. S1). B, tissue expression analysis of glucanase-1 by real-time quantitativeRT-PCR, the different tissues investigated were midgut, salivary glands,hemocytes, fat body, and rest of body. Relative -fold differences were calcu-lated in comparison to glucanase-1 expression in a pooled sample of adultsand pupae for which relative -fold expression was set to 1 (whole animals).

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sion level was very high in larval midgut; much lower butdetectable in salivary glands, hemocytes, and the fat body; andundetectable in the rest of the larval body tissue.Glucan-binding protein has �1,3-Glucanase Activity—We

screened for glucanase activity in H. armigera larval midgutlumen contents by plate assays on carbohydrate polymers fol-lowed by staining of any remaining polymer with Congo Red(Fig. 2A). The only tested substrate that was degraded by thelumen sample was CM-curdlan, which contains exclusively�1,3-glucan linkages. Neither cellulose (exclusively �1,4-link-ages) nor lichenan and barley �-glucan (with mixed �1,3- and�1,4-linkages) were degraded by the lumen sample. The lattertwo substrates could be partially degraded by fungal enzymesspecific for �1,3-glucans or �1,4-glucans. However, the lumensample was incapable of degrading the �1,3-glucan linkages inthe lichenan or barley �-glucan (Fig. 2A).To investigate the correlation between �1,3-glucan binding

and glucanase activity, an untreated gut lumen sample wascompared with the flow-through from the curdlan pull-downassay by native PAGE followed by an overlay with CM-curdlanand staining with Congo Red (Fig. 2B). Although the resolutionof the zymogram was poor, a broad band indicating activitycould be detected only in the untreated sample, whereas noactivity was detected in the flow-through. The broad activityband is likely due to the major band seen in Fig. 1A after thecurdlan pull-down, which is poorly resolved on the native gel;although a contribution of some of the fainter bands in Fig. 1Acannot be completely ruled out.To further test whether the glucan-binding protein itself

could be responsible for this �1,3-glucanase activity, its codingsequence fused to a C-terminal V5/(His)6 tag was transientlyexpressed in High Five insect cells (Fig. 2C). Using the anti-V5antibody in a Western blot, a protein with an apparent molec-ular mass of 44 kDa was detected in the culture medium oftransfected High Five cells, and a lesser amount was associatedwith the crude membrane fraction. The increase in apparentmolecular mass of about 4 kDa between the gut lumen form ofthe protein and its recombinant form expressed in insect cells isdue to the V5/(His)6 tag. A zymogram using CM-curdlan assubstrate revealed a single activity band in the culture mediumof cells transfected with the protein construct but not withempty vector (Fig. 2D). These results show that the protein thatwe had namedGH16betaGRP-1 is an active�1,3-glucanase andsuggest that the activity detected in the gut lumen ismainly dueto this protein. We thus rename this protein as �1,3-glu-canase-1 from H. armigera.Induction of Glucanase-1 after Ingestion of Bacteria—H.

armigera larvae were fed for 24 h on an artificial diet containingone of three types of non-pathogenic microbes: Gram-negative(E. coli) and Gram-positive (M. luteus) bacteria and yeast (S.cerevisiae). Expression of glucanase-1 was investigated byquantitative RT-PCR (Fig. 3). A �2.5-fold increase in mRNAlevels was observed for larvae fed on Gram-negative bacteria aswell as a �4.5-fold increase for larvae fed on Gram-positivebacteria. No significant induction was observed for larvae fedon yeast.Alternatively, larvae were fed on a diet containing bacterial

cell wall polysaccharides: LTA for Gram-positive or LPS for

Gram-negative bacteria. A �2-fold increase in mRNA levelswas observed for larvae fed on LTA as well as a �1.8-foldincrease for larvae fed on LPS. In addition, no significant induc-tion was observed for larvae fed on an artificial diet containingone of three types of �-glucans (Curdlan, Lichenan, and barley�-glucan).Glucanase-1 Is Not Detected in the Hemolymph—To further

confirm the expression specificity of glucanase-1 in themidgut,

1 2 3 4

CM-curdlan

Lichenan

CM-cellulose

1 2

Barley β-glucan

1 2 3 4 1 2

96-72-56-

36-28-

17-

MW

FIGURE 2. Glucanase activity assays on H. armigera larval gut lumen.A, assays were performed on 1.5% agar plates containing 0.25% of severaltypes of �-glucans: CM-curdlan (�1,3-glucan), lichenan and barley �-glucan(mixed �1,3:1,4-glucans), and CM-cellulose (�1,4-glucan). Activity wasrevealed by Congo Red staining. Lane 1, larval gut lumen; lane 2, PBS (negativecontrol); lane 3, endo-�1,3-glucanase from Trichoderma sp.; and lane 4, cellu-lase from Trichoderma sp. (both positive controls). B, �1,3-glucan zymogramof H. armigera larval gut lumen. Ten micrograms of proteins were separatedby native PAGE and the gel overlaid on a CM-curdlan-containing agar plate.Activity was revealed by Congo red staining. Lane 1, gut lumen proteins; lane2, gut lumen proteins after curdlan-pull down assay (flow-through). C, analy-sis of the expression of H. armigera glucanase-1 in transfected T. ni High Fivecells. Ten micrograms of proteins from culture medium and crude membranefraction of both glucanase-1- and empty vector-transfected cells wereresolved by SDS-PAGE using a 12% polyacrylamide resolving gel, then trans-ferred to polyvinylidene difluoride and revealed by Western blot using theanti-V5-HRP antibody. Lane 1, culture medium from control cells; line 2, cul-ture medium from glucanase-1-transfected cells; lane 3, crude membranefraction from control cells; lane 4, crude membrane fraction from glucanase-1-transfected cells. D, �1,3-glucanase activity in culture medium from controland glucanase-1-transfected cells was investigated by zymogram. Ten micro-grams of proteins were resolved by native PAGE and the gel overlaid on aCM-curdlan-containing agar plate. Activity was revealed by Congo red stain-ing. Lane 1, culture medium from glucanase-1-transfected cells; lane 2, culturemedium from control cells.

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affinity to curdlan was used to pull down proteins from H.armigera hemolymph sample of unchallenged larvae (Fig. 4).Seven protein bands were recovered and tryptic peptides weresequenced de novo and identified. None of the proteinsmatched the glucanase-1 sequence although two (bands b andc) having an apparent molecular mass of around 56 kDa wereidentified as �GRPs (Table 1) by their matching ESTs from H.armigera fat body. Predicted protein sequences of �GRP-1 and-2a are very similar to previously characterized proteins puri-

fied from larval hemolymph of the lepidopteran species M.sexta and B. mori (8, 9). In addition to �GRPs, proPO subunit 2(band a), a proPO-activating factor-like protein (serine-pro-teinase like protein 1, band e), a C-type lectin (band f), and aserine-proteinase homolog (band g) were identified in thehemolymph curdlan pull-down fraction (Table 1). All hitsobtained usingMS-BLAST were of high confidence as given bythe search engine except for the C-type lectin (supplementaryinformation). This protein was identified with only a single 9-amino acid peptide (SVIPGNFDK) that was returned as a “bor-derline” hit by MS BLAST. We took this hit into considerationbecause the predictedmolecularmass of the full-length protein(36.8 kDa)was similar to the apparentmolecularmass observedon the gel (Fig. 4).Glucanase-1 Is Conserved among Lepidoptera and Other

Insect Orders—TheH. armigera glucanase-1 is 375 amino acidslong and it contains a predicted signal peptide with a cleavagesite between amino acids 17 and 18 suggesting secretion. Resi-dues 40 through 375 correspond to a conserved domain iden-tified as the glycosyl hydrolase family 16 (pfam00722 Glyco_hydro-16, E � 4e-10) and the subfamily �1,3-glucanrecognition proteins (cd02179 GH16_beta_GRP, E � 3e-103)as indicated by a search against the Conserved Domain Database. Furthermore, a conserved GH16 active site was foundbetween amino acids 188 and 199 including 2 glutamate resi-dues in positions 188 and 193 that have been shown to be cru-cial for catalytic activity in the glucanase from Bacillus licheni-formis (18) (Fig. 5A).H. armigera �GRP-1 and -2a from hemolymph also possess

the glycosyl hydrolase 16 domain at the C terminus, however,the GH16 active site with the 2 glutamate residues is not pres-ent (Fig. 5A). Both proteins have an additional domain at the Nterminus of �150 residues (Fig. 5B). This domain has nodetectable similarity to the glycosyl hydrolase 16 family, andhasnot yet been assigned to a conserved domain family, but itoccurs in a variety of �1,3-glucan-binding proteins isolatedfrom lepidopteran hemolymph. Fabrick et al. (11) denoted thisdomain as carbohydrate recognition domain and showed thattruncated proteins from Plodia interpunctella expressing onlycarbohydrate recognition domain could bind to curdlan andcould activate the prophenoloxidase cascade.Putative orthologs toH. armigeraGlucanase-1 were found in

several species including B. mori, Spodoptera frugiperda, E.postvittana, Ostrinia nubilalis, and P. interpunctella (supple-mentary Fig. S2). EST coverage was sufficient to obtain 3 full-length sequences. The O. nubilalis sequence was truncatedmissing 30 nucleotides, and the full-length sequence was kindlyprovided by Dr. Siu Fai Lee (University of Melbourne, CESARcenter). All EST accession numbers used to obtain the datapresented here are given in the supplementary information. Anorthologous protein annotated as �1,3-glucanase from the sug-arcane borer Diatraea saccharalis was found in GenBankTM(ABR28479); this protein is truncated missing a few aminoacids at the N terminus. Finally, a S. frugiperda ortholog wasalso found in GenBank (ABR28478). This protein differs by 4amino acid substitutions from the protein we obtained viaESTs. The GenBank protein is two amino acids longer than ourprotein due to a repetition of amino acids 165–166: DW (GAT-

FIGURE 3. Variation of expression of glucanase-1 in the midgut of H.armigera larvae upon feeding on microbial- or polysaccharides-contam-inated diet. Larvae were fed for 24 h on artificial diet contaminated witheither microbes (E. coli, M. luteus, and S. cerevisiae) or bacterial cell wallpolysaccharides (LTA and LPS) or various �-glucans (Lichenan, Curdlan, andbarley �-glucan). Relative -fold differences were calculated in comparison tothe data obtained for larvae fed on sterile artificial diet for which relative -foldexpression was set to 1.

MW 1 32

200

100

706050

40

30

25

abcdefg

FIGURE 4. Curdlan pull-down assay on larval hemolymph sample col-lected from unchallenged larvae. Proteins were separated by SDS-PAGEusing a 10% polyacrylamide resolving gel and stained with Coomassie. Lane 1,10 �g of cell-free hemolymph proteins; lane 2, 10 �g of cell-free hemolymphflow-through; lane 3, 15 �l of elution fraction. Tryptic peptides obtained frombands a– g were analyzed by nanoLC-MS/MS and interpreted de novo, searchresults are given in Table 1.

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TGG) in our sequence, DWDW (GATTGGGATTGG) in theABR28478 sequence. Similar to theH. armigera sequence, the 4orthologs are 375 amino acids long and harbor a conservedGH16 active site (Fig. 5A).Other non-lepidopteran insect species possess likely orthologs

of Glucanase-1 that have previously been annotated as GNBP.They are found in several termite species (25), in the genome oftwo mosquito species Aedes aegypti (GNBPs, XP_001659797.1,XP_001664289.1, XP_001659796.1, XP_001664288.1, and XP_001652521.1) and A. gambiae (GNBP subgroup B, XP_312116.3,XP_312118.3, XP_312115.1, and XP_313748.3), and in thegenome of the red flour beetle Tribolium castaneum (predicted:similar to �1,3-glucanase, XP_970010.1). All of these sequenceshave around 60–65% amino acid identity compared with lepi-dopteran Glucanase-1 and all of them possess a conserved GH16active site (Fig. 5A). Orthologous sequences were not found inDrosophila sp. or in the honeybee genome.Identification of 4Distinct�-GRPClades in Lepidoptera—To

examine the relationships among �GRP proteins in Lepidop-tera, a total of 32 sequences from 17 species (for details refer tosupplemental Fig. S3) were collected and used to construct aBayesian phylogeny (Fig. 5C). The analysis revealed that thesesequences clustered in two distinct clades. One of them fallsinto three subclades (clades 1, 2, and 3) containing many pro-teins that had previously been found in insect hemolymph:Mse_BGRP1 and 2 (6, 8), Bmo_BGRP and p50_GNBP (7, 9),Pin_BGRP (5), and Ha_BGRP1 and 2a (the present study). Thesecond cluster (clade 4) contains the H. armigera Glucanase-1protein isolated from the midgut, and sequences from cDNAlibraries made frommidgut tissue of different Lepidoptera spe-cies. This cluster is clearly separated from the other clades by aposterior probability of one and a large branch length. Thetopology of the tree is unchanged when the comparison isrestricted to the glycosyl hydrolase 16 domains of the proteins,and is similar when the neighbor-joining method is used (datanot shown). This phylogeny suggests an ancient duplicationevent leading to paralogues that have different tissue specificityas well as function.Orthologous Glucanase Genes in H. armigera and B. mori—

To obtain the intron/exon organization of the Glucanase-1gene, a probe from theH. armigera cDNA sequencewas used toscreen aBAC library, yielding a 120-kb clone.When sequenced,it was found to contain the entire Glucanase-1 genomic region

(3407 bp) consisting of 9 exons, with the initiator methioninecodon at the start of exon 2 (Fig. 6A). For an interspecific com-parison, theB.mori cDNA sequencewas used in a blastn searchof whole genome shotgun (wgs) sequences at NCBI, identify-ing a total of 5 hits (AADK01004415, BAAB01063034,BAAB01133393, BAAB01043260, and BAAB01083370) thatclustered together in one unique contig of 18,022 bp, whichcovers the entire Glucanase-1 gene between base pairs 9395and 15120 for a total of 5725 bp. Similar to the H. armigeragene, theB.mori gene consists of 9 exonswith the transcriptioninitiation methionine codon also at the beginning of exon 2(Fig. 6A).We further identified the flanking genes of Glucanase-1 in

both species (Fig. 6B). To do so, we recovered a scaffold of theB.mori genome assembly from SilkDB (scaffold000559, silk-worm.genomics.org.cn) that covered the entire B. mori Glu-canase-1 gene as well as flanking genes. ForH. armigera, a partof the 120-kb BAC clone described abovewas used for the com-parative analysis. All of the sequenceswere blasted via theNCBIwebpage against the non-redundant database using BLASTX.A total of 10 and 7 genes were found on theH. armigera and B.mori sequences, respectively (Fig. 6B). The genomic regions ofthe 2 species contain four genes in common: Transcription fac-tor IIE, Tyrosine receptor kinase, GTPase, and Glucanase-1,with similar but not identical orientations. No additional orduplicated BGRP gene was identified within the respectivegenomic regions. These data strongly suggest that the B. moriandH. armigera Glucanase-1 genes are orthologous and that alarger genomic region is highly syntenic between these two lep-idopteran species.

DISCUSSION

Here we present themolecular characterization of amemberof a newclass of�1,3-glucan recognition protein in Lepidopterathat binds strongly to curdlan, is predominantly expressed inthe midgut, is located in the gut lumen thus in direct contactwith the food bolus, and exhibits �1,3-glucanase activity.Glucanase-1 orthologs are present inmany insect orders and

thus this �GRP/GNBP gene family is not mosquito-specific assuggested by Warr et al. (26). Within the Lepidoptera, we canfind a clear distinction between two classes of �GRPs, one pos-sessing the signature of a glucanase active site and expressedprimarily in midgut, and the other lacking this signature but

TABLE 1Protein identification from the curdlan pull-down assay on H. armigera larval hemolymph

Band Databasesearched Hita Species Accessionb De novo

sequenced peptidescPeptides matchedby MS BLAST

MS BLASTscored

a ncbi_insecta ProPO subunit 2 H. armigera AAZ52554 5 5 336b Ha_EST_db �1,3-Glucan recognition protein 2a H. armigera EU770382* 4 1 78c ncbi_insecta �1,3-Glucan recognition protein B. mori NP_001036840 5 3 182

Ha_EST_db �1,3-Glucan recognition protein 1 H. armigera EU770381* 5 1 101d Not identified 6e Ha_EST_db Serine-proteinase like protein 1 H. armigera EU770389* 3 2 145f Ha_EST_db C-type lectin H. armigera EU770390* 3 1 64g ncbi_insecta Masquerade-like serine protease B. mori NP_001037053 6 2 113

Ha_EST_db Serine-proteinase homolog-1 H. armigera EU770391* 6 4 192a For hits obtained by searching ncbi_insecta, the annotation is the one given by the database. For hits obtained by searching ourH. armigera EST database, the annotation hasbeen given according to theM. sexta homologs (1).

b GenBank accession numbers (asterisk indicates from this study).c Details on the de novo sequenced peptides are given in Supplementary Information S4.d MS BLAST scoring is detailed under “Experimental Procedures.”

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possessing an additional C-terminal domain and expressed inother tissues including hemolymph. The difference in function,however, is not clear yet. We found that the expression of thegene coding for this protein is induced in the midgut of larvaefed on Gram-negative or Gram-positive bacteria in the diet,suggesting a role in immune response.The result of our curdlan pull-down assay with the hemo-

lymph sample was complex and several unexpected proteins

were identified apart from the �GRPs 1 and 2a. Finding aC-type lectin associated with curdlan was not surprisingbecause lectins are well known polysaccharide-binding pro-teins and a previously characterized protein from H. armigerahas been shown to have some affinity for curdlan (27). Thepresence of proPO subunit 2, a proPO-activating factor protein(serine proteinase-like protein) and a serine-proteinase homo-log was surprising and is unlikely due to direct binding to curd-

signal peptide

GlucanaseMidgut β-1,3-glucanase-1

0

1

0.

1

0.

.1

1 0

.1 Dni_BGRP200

0 2

Aga_GNBPB3.0

Ncr_glucanaseTmo_BGRPDme_GNBP3Mse_BGRP2Bmo_BGRP2Pin_BGRPHar_BGRP2aSfr_BGRP2Tni_BGRP2

Har_BGRP2bSli_BGRP2

.51.00

1.00

Pra_BGRP2

1.00

0.97

1.00

1.00

Mse_BGRP1Gme_BGRPBmo_BGRPHar_BGRP1Pra_BGRP1

.500.69

1.00

1.00

1.00

0.89

Bmo_p50(GNBP)Har_BGRP3Hcu_BGRP

.001.00

Dni_BGRP3Pra_BGRP3

1.00

1.00

Ame_GNBP1

Dme_GNBP185

0.83

0.55

1.00

Nfu_GNBP1Pxy_gluc-1Pin_gluc-1Epo_gluc-1Dsa_gluc-1Ceu_gluc-1

Pra_gluc-1Aca_gluc-1

.001.00

0.61

Bmo_gluc-1Onu_gluc-1

99

Har_gluc-1Sfr_gluc-1Sli_gluc-1

001.00

0.83

0.98

0.47

0.98

1.00

0.88

1.00

clade 1

clade 2

clade 3

70 7.

C

GH16 consensus E L D L x E x x G K x P I I Q R S V V N T F F A

Har-Gluc1 186-S G E I D L V E S R G N R A-199Bmo-Gluc1 188-S G E I D L V E S R G N K N-199Aga-GNBPB4 187-S G E I D L M E S R G N L D-198Aae-GNBP 204-S G E I D L M E S R G N L D-215Nfu-GNBP 188-S G E I D L T E S R G N K Q-199

Mse-BGRP2 317-S G L M R V A F V R G N D V-330

Har-BGRP2a 324-S G M M R V A F A R G N P S-337

CRD Glucanase-like

signal peptide

Hemolymph beta-GRP

FIGURE 5. A, alignment of the predicted GH16 active site of 2 lepidopteran Glucanase-1 and GNBPs from termite and mosquitoes compared with twolepidopteran hemolymph-specific proteins. Consensus active site for GH16 family was obtained from PROSITE (expasy.ch/cgi-bin/nicedoc.pl?PDOC00794).Bmo, B. mori; Har, H. armigera; Nfu, N. fumigatus; Aae, A. aegypti; Aga, A. gambiae; and Mse, M. sexta. B, schematic structure of lepidopteran Glucanase-1 andhemolymph-specific �GRPs. CRD, carbohydrate recognition domain. C, bayesian tree (rooted) of putative �GRPs protein sequences expressed in severallepidopteran species. Sequences were obtained from GenBank, in-house EST databases and SilkDB. Posterior probability for each branch point is given.Relevant information concerning the sequences used for this Phylogenetic analysis is summarized in supplemental Fig. S3.

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lan. ProPO is responsible for melanization and has no knownaffinity for polysaccharides (28) similar to proPO-activatingfactors (1). Pattern recognition receptors (in our case �GRPsand C-type lectins), proPO, and proPO-activating factors allparticipate in the proPO activation cascade (1) and conse-quently might at some point interact together. Hence, our datasuggest that we have precipitated a complex of these proteins.This is consistentwith the suggestion of Yu et al. (29) that, inM.sexta, Immunolectin-2 (a C-type lectin), after binding to sur-face polysaccharides of microbes, forms a complex togetherwith serine-proteinase homologs and proPO-activating factors.Our results on the dietary induction of Glucanase-1 by bac-

teria as well as by LTA and LPS correlate well with observationsof one of its orthologs in the mosquito A. gambiae, GNBP-B1,for which the expression is induced upon bacterial challengewith both Gram-negative and Gram-positive bacteria with astronger effect observed with the latter (30). No induction ofGNBP-B1 is observed upon challenge with S. cerevisiae (30).Furthermore, Warr et al. (26) showed that knocking down theexpression of GNBP subgroup B genes in adult female mosqui-toes by RNA interference increased their susceptibility toimmune challenge and subsequently their death rate. Our find-ings suggest that these results should nowbe considered in lightof a possible enzymatic role of GNBP-B proteins. A similarknock-down approach with lepidopteran Glucanase-1 genescombined with bioassays testing larvae behavior feeding on ahighly microbial-contaminated diet would be a valuable tool inunderstanding the potential role in immune defense of Glu-canase-1 in caterpillars. Development of gene manipulation

techniques for Lepidoptera is a growing research field, but theiravailability is still limited although some progress has beenmade recently especially with H. armigera (31).In Lepidoptera and other insect orders, pattern recognition

proteins including �GRPs/GNBPs trigger an innate immuneresponse after recognition of pathogen-associated molecularpatterns by activating the prophenoloxidase cascade (1, 3) andthe Toll receptor pathway in hemocytes (32). These events takeplace in the hemolymph and thus it is unlikely that Glucanase-1could activate this cascade from its site of activity in themidgutlumen. Furthermore, the presence of two distinct groups of�GRPs in Lepidoptera suggests an ancient duplication eventleading to paralogous genes. Subfunctionalization and neo-functionalization could have led to different functions of bothof these paralogs. Hence, hemolymph-specific �GRPs mighttrigger an innate immune response, whereas midgut specific�GRPs (Glucanase-1) possibly fulfill a different function withinthe organism.The recognition that glucanases and non-catalytic glucan-

binding proteins are similar but distinct clades is hampered bycurrent nomenclature, including the misleading definition of aconserved domain in the NCBI CDD data base. Version 2.15lists a Conserved Domain, cd02179: G16_beta_GRP, describedas follows: “Beta-GRP (�1,3-glucan recognition protein) is oneof several pattern recognition receptors, also referred to as bio-sensor proteins, that complexes with pathogen-associated�1,3-glucans and then transduce signals necessary for activa-tion of an appropriate immune response…” This description issupported by citations (8, 11) describing studies on the non-

FIGURE 6. A, schematic representation of the intron/exon structure of H. armigera and B. mori Glucanase-1 genes. Boxes represent exons: white boxes cor-respond to the gene CDS and gray boxes represent the 5�- and 3�-untranslated region found on the transcripts. Size (in nucleotides) is shown below each exon. Linesrepresent intron insertions (drawn to scale), and the size of each intron is indicated above the line. Initiator methionine codon (ATG) and stop codon (TAA) are alsoindicated for both species. B, schematic representation of the genomic region around the Glucanase-1 gene deduced from a B. mori scaffold obtained from NCBI andsequencing of a H. armigera BAC clone. Black arrows, putative genes common between the 2 species; gray arrows, other putative genes.

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catalytic hemolymph �GRP proteins from Lepidoptera. How-ever, none of these lepidopteran proteins lacking the GH16active site with two glutamates are among the 14 sequencesused to construct the consensus sequence for this cd02179domain. Instead, all 14 sequences possess the active site, includ-ing 6 from fungi, 2 from earthworms, 4 from crustaceans, and 2mosquito sequences (one is a GNBP-B from A. gambiae, whichas far as we know has not been tested for glucanase activity).Thus biological information from lepidopteran hemolymph�GRP sequences has been associated with the definition of adomain possessing the catalytic site, yet those sequences havenot been used to represent a separate domain lacking the cata-lytic site. Recognition of an additional subfamily containingbona fide GRPs lacking the active site would better reflect theexistence of similar but distinct catalytic and non-catalyticclades that we have found.Our data show that glucanase-1 hydrolyzes specifically �1,3-

glucans. Themajor source for this type of polysaccharides is thecell wall of bacteria, yeast, and fungi. One of its orthologs in A.gambiae, GNBP-B4, binds to the surface of bothGram-negativeand Gram-positive bacteria (26), but its potential �1,3-glu-canase activity has not been tested yet. Genta et al. (33) purifieda 46-kDa endo-�-1,3-glucanase from salivary glands of thecockroach Periplaneta americana. Their purified protein wasable to lyse S. cerevisiae cells in hypotonic medium lackingnutrients, making their content available as a nutritive source,but no lysis was observed in isotonic nutritive medium. Theauthors also pointed out that although it is conceivable that thisenzyme may protect midgut cells from microbial invasion, itspresence in saliva and the large amounts of fungi usually foundin detritus, the major food source of P. americana, points morestrongly to a digestive role. We could not observe any lyticactivity by plate assay on any microbe tested using partiallypurified Glucanase-1 from culture medium of High Five trans-fected cells (data not shown) although the recombinant proteinexhibits glucanase activity. We cannot exclude that this lack ofactivity is due to too low of a concentration of recombinantGlucanase-1. A more thorough investigation of the potentialanti-microbial activity of Glucanase-1 should be undertaken.Similarly, it has been proposed that �1,3-glucanase from

plants may act synergistically with other hydrolases, such aschitinases and proteinases, to disrupt the structural integrity offungal cell wall (34). Alternatively theymay release oligosaccha-rides from �1,3-glucan substrates that may serve as chemicalsignals leading to the activation of other defense responses (35).In addition, the midgut of herbivorous caterpillars is com-

posed of a highly diverse microbial community that might con-tribute to its physiological function (36–38) and it is unclearhow Glucanase-1 could differentiate between them, as themolecular motifs activating the innate immune response arepresent in both beneficial and intruding microorganisms. Onehypothesis is that Glucanase-1 has a similar function asPGRP-LB in Drosophila in preventing local immune activationby commensal gut bacteria (15). PGRP-LB, in contrast toPGRP-LC, is an active amidase able to digest peptidoglycanmolecules released by dividing Gram-negative bacteria.PGRP-LB regulates the level of immune response by scavengingpeptidoglycan present in the Drosophila larval gut. If this scav-

enging effect is overwhelmed, by bacterial infection for exam-ple, the excess peptidoglycan is recognized by the nonenzy-matic PGRP-LC,which activates the immune response throughthe Imd pathway. In the case of peptidoglycan released by com-mensal gut flora, the scavenging effect of PGRP-LB is sufficientto prevent activation of the Imd pathway and thus to inhibit anyimmune response.Our results raise the following questions. (i) Can Glu-

canase-1 be considered as a true pattern recognition protein?(ii) What is the role of Glucanase-1 in the midgut: immunedefense protein or digestive enzyme?The present study is a firststep in understanding the physiological role of this new lepi-dopteran midgut-specific �GRP protein family. More investi-gations have to be conducted to fully address these questions.

Acknowledgments—We are particularly grateful to A. Muck and A.Svatos for the Mass Spectrometry support and to D. Schnabelrauchand H. Ringys-Beckstein for providing technical assistance forsequencing. We are grateful to Dr. Siu Fai Lee for providing the O.nubilalis Glucanase-1 sequence.

REFERENCES1. Kanost, M. R., Jiang, H., and Yu, X. Q. (2004) Immunol. Rev. 198, 97–1052. Medzhitov, R., and Janeway, C. A., Jr. (1997) Cell 91, 295–2983. Kim, Y. S., Ryu, J. H., Han, S. J., Choi, K. H., Nam, K. B., Jang, I. H.,

Lemaitre, B., Brey, P. T., and Lee, W. J. (2000) J. Biol. Chem. 275,32721–32727

4. Cerenius, L., and Soderhall, K. (2004) Immunol. Rev. 198, 116–1265. Fabrick, J. A., Baker, J. E., and Kanost, M. R. (2003) Insect Biochem. Mol.

Biol. 33, 579–5946. Jiang, H., Ma, C., Lu, Z. Q., and Kanost, M. R. (2004) Insect Biochem. Mol.

Biol. 34, 89–1007. Lee, W. J., Lee, J. D., Kravchenko, V. V., Ulevitch, R. J., and Brey, P. T.

(1996) Proc. Natl. Acad. Sci. U. S. A. 93, 7888–78938. Ma, C., and Kanost, M. R. (2000) J. Biol. Chem. 275, 7505–75149. Ochiai, M., and Ashida, M. (2000) J. Biol. Chem. 275, 4995–500210. Shin, S. W., Park, S. S., Park, D. S., Kim, M. G., Kim, S. C., Brey, P. T., and

Park, H. Y. (1998) Insect Biochem. Mol. Biol. 28, 827–83711. Fabrick, J. A., Baker, J. E., and Kanost, M. R. (2004) J. Biol. Chem. 279,

26605–2661112. Freitak, D.,Wheat, C.W., Heckel, D. G., andVogel, H. (2007)BMCBiol. 5,

5613. Liehl, P., Blight,M., Vodovar, N., Boccard, F., and Lemaitre, B. (2006) PLoS

Pathog. 2, e5614. Nehme, N. T., Liegeois, S., Kele, B., Giammarinaro, P., Pradel, E., Hoff-

mann, J. A., Ewbank, J. J., and Ferrandon, D. (2007) PLoS Pathog. 3, e17315. Zaidman-Remy, A., Herve, M., Poidevin, M., Pili-Floury, S., Kim, M. S.,

Blanot, D., Oh, B. H., Ueda, R., Mengin-Lecreulx, D., and Lemaitre, B.(2006) Immunity 24, 463–473

16. Simpson, R. M., Newcomb, R. D., Gatehouse, H. S., Crowhurst, R. N.,Chagne, D., Gatehouse, L. N., Markwick, N. P., Beuning, L. L., Murray, C.,Marshall, S. D., Yauk, Y. K., Nain, B., Wang, Y. Y., Gleave, A. P., andChristeller, J. T. (2007) Insect Mol. Biol. 16, 675–690

17. Pauchet, Y., Muck, A., Svatos, A., Heckel, D. G., and Preiss, S. (2008) J.Proteome Res. 7, 1629–1639

18. Juncosa, M., Pons, J., Dot, T., Querol, E., and Planas, A. (1994) J. Biol.Chem. 269, 14530–14535

19. Shevchenko, A., Sunyaev, S., Loboda, A., Bork, P., Ens, W., and Standing,K. G. (2001) Anal. Chem. 73, 1917–1926

20. Habermann, B., Oegema, J., Sunyaev, S., and Shevchenko, A. (2004)Mol.Cell. Proteomics 3, 238–249

21. Fischer,H.M.,Wheat, C.W.,Heckel, D.G., andVogel,H. (2008)Mol. Biol.Evol. 25, 809–820

22. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., and Higgins,

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JANUARY 23, 2009 • VOLUME 284 • NUMBER 4 JOURNAL OF BIOLOGICAL CHEMISTRY 2223

by guest on February 12, 2018http://w

ww

.jbc.org/D

ownloaded from

D. G. (1997) Nucleic Acids Res. 25, 4876–488223. Katoh, K., Misawa, K., Kuma, K., and Miyata, T. (2002)Nucleic Acids Res.

30, 3059–306624. Ronquist, F., and Huelsenbeck, J. P. (2003) Bioinformatics 19, 1572–157425. Bulmer, M. S., and Crozier, R. H. (2006)Mol. Biol. Evol. 23, 317–32626. Warr, E., Das, S., Dong, Y., andDimopoulos, G. (2008) InsectMol. Biol. 17,

39–5127. Chai, L. Q., Tian, Y. Y., Yang, D. T.,Wang, J. X., andZhao, X. F. (2008)Dev.

Comp. Immunol. 32, 71–8328. Hall, M., Scott, T., Sugumaran, M., Soderhall, K., and Law, J. H. (1995)

Proc. Natl. Acad. Sci. U. S. A. 92, 7764–776829. Yu, X. Q., Jiang, H., Wang, Y., and Kanost, M. R. (2003) Insect Biochem.

Mol. Biol. 33, 197–20830. Dimopoulos, G., Richman, A., Muller, H. M., and Kafatos, F. C. (1997)

Proc. Natl. Acad. Sci. U. S. A. 94, 11508–1151331. Mao, Y. B., Cai, W. J., Wang, J. W., Hong, G. J., Tao, X. Y., Wang, L. J.,

Huang, Y. P., and Chen, X. Y. (2007) Nat. Biotechnol. 25, 1307–131332. Ao, J. Q., Ling, E., and Yu, X. Q. (2008)Mol. Immunol. 45, 543–55233. Genta, F. A., Terra, W. R., and Ferreira, C. (2003) Insect Biochem. Mol.

Biol. 33, 1085–109734. Mauch, F., Mauch-Mani, B., and Boller, T. (1988) Plant Physiol. 88,

936–94235. Lamb, C. J., Lawton, M. A., Dron, M., and Dixon, R. A. (1989) Cell 56,

215–22436. Ping, L., Buchler, R., Mithofer, A., Svatos, A., Spiteller, D., Dettner, K.,

Gmeiner, S., Piel, J., Schlott, B., and Boland,W. (2007) Environ. Microbiol.9, 1572–1583

37. Spiteller, D., Dettner, K., and Boland, W. (2000) Biol. Chem. 381,755–762

38. Xiang, H., Wei, G. F., Jia, S., Huang, J., Miao, X. X., Zhou, Z., Zhao, L. P.,and Huang, Y. P. (2006) Can. J. Microbiol. 52, 1085–1092

39. Vanderzant, E. S. (1968) Ann. Entomol. Soc. Am. 61, 120–125

Lepidopteran Glucan-binding Proteins

2224 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 4 • JANUARY 23, 2009

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VogelYannick Pauchet, Dalial Freitak, Hanna M. Heidel-Fischer, David G. Heckel and Heiko

PROTEIN FAMILY FROM LEPIDOPTERAImmunity or Digestion: GLUCANASE ACTIVITY IN A GLUCAN-BINDING

doi: 10.1074/jbc.M806204200 originally published online November 25, 20082009, 284:2214-2224.J. Biol. Chem. 

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