Insect Molecular Biology (2004)

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(2), 165–177

© 2004 The Royal Entomological Society

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Blackwell Publishing, Ltd.

Cloning and characterization of a gut-specific cathepsin L from the aphid

Aphis gossypii

C. Deraison*†, I. Darboux*, L. Duportets*, T. Gorojankina*, Y. Rahbé† and L. Jouanin*

*

Laboratoire de Biologie Cellulaire, INRA de Versailles, Versailles, France; and

†

Laboratoire Biologie Fonctionnelle, Insectes et Interactions, UMR INRA-INSA de Lyon BF2I, Villeurbanne, France

Abstract

We have characterized proteinase activities in gutextracts from the cotton-melon aphid (

Aphis gossypii

Glover), an insect feeding strictly on protein-poorphloem. The major, if not exclusive, intestinal protein-ases of this aphid are of the cysteine type. A cDNA hasbeen cloned from a gut library and codes for thecysteine proteinase AgCatL, a cathepsin L-likecysteine proteinase. The

AgCatL

protein shows highsequence similarity with mammalian and some arthro-pod cathepsin L-like proteinases, but can be reliablydistinguished from the secreted (digestive) protein-ases identified in other arthropods.

AgCatL

is widelyexpressed in aphid intestinal cells. Immunolocaliza-tion of AgCatL showed an intense signal at the level ofthe anterior ‘stomach’ part of the midgut, and espe-cially at intracellular localization. Although the preciserole of AgCatL in aphid midgut physiology is stillunclear, this enzyme could be involved in the process-ing of exogenous ingested polypeptides.

Keywords: Hemiptera, Aphididae, cysteine proteinase,digestion, phylogeny.

Introduction

Together with moths (Lepidoptera) and beetles (Coleoptera),aphids (Hemiptera, Aphididae) belong to one of the threemajor orders of insect agricultural pests. Damage resultingfrom direct aphid feeding and from the cost of controlling

this pest family account for one of the largest sources ofeconomic losses in agriculture. Currently, the most widelyavailable method used to control aphid populations is theapplication of chemical insecticides. This approach leads toserious drawbacks, such as environmental and food-chaincontamination. Moreover, many aphid species are becomingmore and more resistant to insecticides. The use of plant-resistance genes is a common alternative to chemicals, butvery few natural aphid-resistance genes have been charac-terized (Pitrat & Lecoq, 1982; Khush & Brar, 1991; Porter

et al

., 2000), and only two have been cloned to date, the

Mi

(Rossi

et al

., 1998) and

Vat

(Brotman

et al

., 2002) genes.Furthermore, most of these genes are specific to singleaphid species, or even populations, suggesting an analogyto the race/host plant model initially described for plantpathogens (Flor, 1987).

Plant genetic engineering offers new possibilities in thecontrol of aphid populations (Gatehouse

et al

., 1993; Rahbé& Febvay, 1993; Thomas

et al

., 1995; Jouanin

et al

., 2000).Among the genes enhancing plant resistance to aphids,proteinase inhibitors (PIs) have been reported to promotetoxic effects when ingested by different aphid species (Rahbé& Febvay, 1993; Lee

et al

., 1999; Ceci

et al

., 2003; Rahbé

et al

.,2003a,b). However, owing to their peculiar digestive phys-iology, the mode of PI toxicity towards aphids is completelyunknown and the success of this control strategy dependson a better identification of PI targets in these insects.

Hemiptera is an insect order quite different and phyloge-netically distant from most other pest or model insects(

Drosophila

,

Anopheles

,

Bombyx

). In addition to lacking fullmetamorphosis, aphids share with scales and whitefliesthe peculiarity of being very strict phloem feeders. Theypenetrate plant tissues through an exclusive intercellularroute between epidermal and mesophyll cells, and feedsolely on photoassimilates translocated in phloem sieveelements. Therefore, their diet consists mainly of phloemsap, which is generally poor in proteins. Values reported fortotal proteins in the sieve tube sap of various plants rangefrom 0.2 to 2 mg/ml (Ziegler, 1975). This is why aphids arethought to lack proteolytic activity in their digestive tract(Terra & Ferreira, 1994; Mochizuki, 1998). However, someplants, such as the cucurbitacea, have a protein-rich phloem

Received 30 September 2003; accepted after revision 5 December 2003.Correspondence Dr Yvan Rahbé, UMR BF2I INRA-INSA de Lyon, Bat.Louis-Pasteur, 20 av. A. Einstein, 69621 Villeurbanne cedex, France. Tel.:+33 4 72 43 84 76; fax: +33 4 72 43 85 34; e-mail: rahbe@jouy.inra.fr

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sap, in which the protein content extends up to 60 mg/ml(Cronshaw & Sabnis, 1989). Protein concentration in excessof about 5 mg/ml could provide an adequate source ofamino acids and nitrogen for phloem-feeding insects.

Information on protein digestion in aphids is extremelyscarce. In order to examine the physiology and to under-stand the toxicity of some PIs to these insects, we analysedthe putative proteolytic activity in the gut of an aphid natu-rally feeding on a protein-rich diet.

Aphis gossypii

Glover isknown as the cotton-melon aphid and is a very efficient vec-tor of various plant viruses. It is one of the most widespreadspecies of aphids and is a major pest for many crop plants.In the present study, we demonstrate that the main diges-tive tract proteinase of this aphid is of the cysteine type, anda cDNA related to this family has been cloned. The encodedsequence, termed

AgCatL

, shows a coding sequence (cds)with significant amino acid sequence similarity to cathepsinL-like cysteine proteinases (SCPs) from the weevil

Sitophiluszeamais

(Matsumoto

et al

., 1997), and to other cathepsin Lcysteine proteinases, including mammalian ones. Molecu-lar and immunohistochemical studies allowed us to analysethe tissue distribution and expression of AgCatL, and ourresults suggest that it could play a role in digestion orprocessing of exogenous polypeptides, even though it isnot secreted in the digestive fluids, as occurs in some otherinsect species that have already been shown to rely oncysteine proteinase digestion.

Results

The physiological pH along the

A. gossypii

gut was meas-ured by dipping the dissected organ in pH indicator. Thisexperiment revealed the presence of a pH gradient in thegut, with an acidic segment in the anterior ‘stomach’ part(down to pH 5–6), a smooth gradient towards alkaline pH in

the mid and posterior midgut (pH 8 around the mid/hindgutjunction) and a last acidification (pH 6) occurring again atthe level of the hindgut (Fig. 1). These values estimatedvisually were confirmed when the gut was dissected andhomogenized according to its four cited anatomical parts.

The pH dependence of proteolytic activity in midgutextracts of

A. gossypii

was examined using the

125

I-globulinsubstrate in a mixed buffer system. Midgut extracts fromadult aphids showed substantial activity between pH 4.5and 6. The optimum for proteolysis was close to pH 4.5(Fig. 2), with activity falling severely at lower pH values.Assays were performed in the presence of inhibitors andactivators specific to different proteinase types to discrimi-nate this global activity into the four major mechanisticclasses described for peptidases (Dunn, 1989). Proteolyticactivity was substantially enhanced (+70%) in the presenceof DTT, a cysteine proteinase activator, and E-64 (a highlyspecific cysteine proteinase inhibitor) inhibited more than95% of this activity. The other specific inhibitors did not

Figure 2. pH dependence of proteolytic activity of A. gossypii guts. Whole extracts from the digestive tract were incubated with 125I-globulin solution (pH 4–9) and different diagnostic inhibitors or an activator at 37 °C for 24 h. Hydrolysis was measured by the resulting radioactivity in supernatants from TCA precipitation.

Figure 1. Physiological pH in A. gossypii digestive tract. After dissection, the gut was dipped in universal indicator solution. Colour change was observed under a binocular microscope and compared with standards made from buffers of known pH.

Aphid gut cathepsin L

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affect the activity, indicating that cysteine proteinases wereresponsible for most of the detectable proteinase activity.Inhibition by chymostatin is also a feature of papain-likecysteine proteinases.

Zymogram analysis of aphid gut extracts, performed onpolyacrylamide gels transferred on to a 12% gel copoly-merized with gelatin, revealed multiple zones of proteolyticactivity. Zymograms were incubated at pH 5 in order toassess aphid gut proteolytic activity at a physiologically rel-evant pH. Three major bands of activity were resolved, theestimated molecular weights of which were approximately40 (doublet) and 55 kDa (Fig. 3); all of these activities wereE-64 sensitive, in agreement with the results of the

125

I-globulin assays (Fig. 2).

Cloning a full cDNA corresponding to a cysteine proteinase

Gut RNA of

A. gossypii

was prepared for cDNA library con-struction and used in RT-PCR with two sets of degeneratedprimers designed using insect cysteine proteinases thathave been previously published (Yamamoto

et al

., 1994;Matsumoto

et al

., 1997; Tryselius & Hultmark, 1997; Girard& Jouanin, 1999). Several amplimers of 480 bp were obtainedand cloned. Analysis of all the sequences obtained revealedthat these RT-PCR fragments belong to a single sequenceof a thiol proteinase. In order to isolate the entire cDNAencoding this cathepsin, specific primers were designedand used to screen the gut library by PCR. The librarycontained more than 6

×

10

6

independent clones. The full-length cDNA (1504 bp) was obtained. The sequence hasbeen deposited in the EMBL/GenBank/DBJJ database underaccession number AJ489298 and is referred to as

AgCatL

.The deduced protein AgCatL is Swissprot Q8MN27.

Ag cathepsin L sequence analysis

Comparing the deduced amino acid sequence (Fig. 4) withsequences of the GenBank™ database demonstrated that

AgCatL belongs to the papain-family of cysteine proteinases,and more precisely to the cathepsin L subfamily, as hasbeen determined by the phylogenetic analysis (Fig. 5). ThecDNA encodes a putative preproprotein, the preregionbeing composed of an N-terminal sequence of nineteenamino acids, characteristic of a signal peptide as predictedby several algorithms, such as SignalP or TargetP (Nielsen

et al

., 1997). Canonical cleavage of the leader peptide afterthe nineteenth residue (Ser

−

105

) would leave a proenzymeof 322 amino acids. The amino acid sequence presents an‘E

−

78

R

−

74

[FY]

−

70

N

−

67

I

−

63

N

−

59

’ motif and a ‘G

−

53

N

−

44

F

−

42

D

−

40

’motif (Fig. 4), both highly conserved in the propeptide seg-ment of all cysteine proteinases except the cathepsin Bgroup (Karrer

et al

., 1993). The ‘ERFNIN’ motif is consid-ered to be a functional unit probably involved in the inhibi-tion of the enzyme activity by the propeptide (Karrer

et al

.,1993). A single potential N-glycosylation site was observedin the propeptide region (N

−

17

FTN). The potential cleavagesite of the proregion was estimated, by homology, to bebetween residues Val

−

1

and Ile

+1

, fitting the scheme of aconserved proline residue at position 2 in the matureenzyme, as for many members of the papain family. Thisproline may serve to prevent unwanted N-terminal proteol-ysis (Rawlings & Barrett, 1994). The proenzyme can beautocatalytically cleaved to produce the mature enzymeconsisting of 218 amino acid residues, with a predictedmolecular mass of 24 kDa and a pI of 6.98. The catalytictriad Cys

+25

, His

+164

and Asn

+185

is fully conserved inAgCatL, and the six cysteine residues that form disulphidebonds in papain between positions 22–65, 56–98 and 157–207, conserved in other cysteine-proteinases, are alsopresent in the

A. gossypii

sequence. Overall, the deducedamino acid sequence of

A. gossypii

cathepsin L is very sim-ilar to some other insect cysteine-proteinases (75% similar-ity to

Drosophila

) and to mammalian cathepsin L (70%similarity with human, Fig. 4).

Structural inferences

The tentative structure produced for AgCatL by homologymodelling (not shown) differs significantly from its humantemplate by three ‘junctional’ regions easily identifiable bythe gaps in the alignment: the

α

1/

α

2 helix boundary of theprosegment (

−

80 region), the prosegment C-terminus (

−

15region) and the putative light/heavy chain junction (+175region). As they all occur at the periphery of the molecule,the catalytic consequences of such large differences mightbe reduced. A few questions were considered further inorder to interpret the functional characteristics of the aphidenzyme better. Catalytic activity and its regulation by thepropeptide region were thoroughly investigated in thehuman enzyme (Coulombe

et al

., 1996; Fujishima

et al

.,1997; Groves

et al

., 1998). When exploring the importantresidues involved in the structural interactions between theenzyme and its N-terminal proregion, most of which are

Figure 3. Proteinase activity gel. Two midgut equivalents were loaded in each lane (1, standard conditions; 2, 3, E-64 10 µM or PMSF 10 µM, respectively, were added to the sample 15 min before loading).

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supported by aromatic and hydrophobic interactions (Cou-lombe

et al

., 1996), the result is an extreme conservationbetween the human, aphid and other orthologous arthro-pod sequences (fourteen conserved on fifteen critical resi-dues, highlighted in grey in Fig. 4). The only missinghydrophobic interaction concerned residue K

−

66

in the ERF-NIN motif (M in human; variant in the four lower sequencesof Fig. 4), which supports

α

1/

α

2 stabilizing interactions inthe propeptide (Coulombe

et al

., 1996). When looking atthe catalytic activity, through residues interacting with theE-64 inhibitor (Fujishima

et al

., 1997), a similar pattern ofconservation is revealed (twenty-four conserved residuesover twenty-six critical; boxed area in Fig. 4). In this case,

the two variant positions in the aphid sequence (Q

145

andT

190

from L and E, respectively, in human) share a commontrend of variation among arthropod sequences, and bothpositions concern the so-called S

′

region of the catalyticcleft, which seems important for the selectivity of inhibitorsbetween cathepins of the B and L groups (Fujishima

et al

.,1997). One consequence could be that insect cathepsinsdisplay nonstandard behaviour towards specific inhibitorsof mammalian cathepsin families. Finally, two acidic resi-dues crucial for the pH dependency of the activation ofcathepsin L are also conserved in the insect sequences,highlighting probable common activation features for allthese enzymes (except the crustacean, see D

−

40

and E

−

35

Figure 4. Deduced amino acid sequence of AgCatL (predicted mature sequence numbering) and comparison with selected cysteine proteinase sequences from the cathepsin L subgroup (Swissprot accession nos. Homo sapiens P07711; Homarus americanus P13277; Pheadon cochleariae O97397; Drosophila melanogaster Q95029; Sitophilus zeamais O46030; Bombyx mori Q26425). Gaps were introduced by CLUSTALW to maximize the homology between the prosequences, and are indicated by dots. Active site residues C25, H164 and N185 (AgCatL numbering) are highlighted in yellow, and Prosite consensus patterns are reported below. Yellow also highlights the conserved carboxylic side chains important for acid autocatalytic processing of L cathepsins. In grey are the residues potentially implicated in tight intramolecular interactions, either of the covalent–disulphide bridges, aromatic or hydrophobic types. When present, the potential lysosomal-addressing/N-glycosylation site is highlighted in blue and its pattern reported; likewise, lysine residues involved in the mammalian lysosomal mannose-phosphate processing signal are highlighted in green. The site of predicted signal peptide cleavage is shown as pattern x1, that of the activation peptide as y1 (by homology to human sequence), and the putative site of cleavage between eventual light and heavy chains as z1 (1 marking the potential downward-leaving chain N-terminus). Residues involved in the active site cleft are boxed in the human sequence, as well as their variants in the aphid sequence.

Figure 5.

Phylogenetic tree based on cysteine proteinase sequences, constructed from a PAM matrix alignment and the neighbour-joining algorithm. The tree was rooted from the CatB outgroup. Numbers near the nodes indicate percentage of bootstraps supporting a given node (500 replicates). The scale bar corresponds to 0.1 amino acid substitution per site. Insect sequences are underlined;

Drosophila

sequences are in bold type and numbered for the different loci in the genomic sequence (* for sequences found in expressed sequence tags but lacking one of the catalytic domain signatures); human sequences are italicized. Cysteine proteinase classes are indicated on the right. Sequences used are reported with their originating species followed by cathepsin class and accession numbers (gb, GenBank; sp, Swissprot).

Aphid gut cathepsin L

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in the α3 helix of prosegment, Fig. 4). The last functionalinference to be drawn from the AgCatL structure lies in theidentification of potential lysosomal targeting signals, whichassociate, in the human sequence, an N-glycosylation site(N108, blue in Fig. 4) to a lysine tandem responsible for rec-ognition by the phosphotransferase specific to the man-nose-phosphate targeting pathway (K, in green in Fig. 4). InAgCatL, one potential glycosylation site exists in the pro-region and it is conserved in four of the six arthropodsequences presented (Fig. 4). This site is located at thesame pole of the cathepsin L molecule, when comparedwith the human N-glycosylation site (only 20 Å separatethem in the model structures), despite being quite far apartin the primary sequence. In addition, lysine tandems maybe identified in the aphid prosequence, which could fit thestructural constraints set-up for the human recognition sig-nal: K-72 and K-26 in AgCatL are 33 Å apart, close to the34 Å rule established for the human Cat D/Cat L signal(Cuozzo et al., 1998). In contrast to this scheme, no poten-tial lysosomal targeting signal may be identified in thePhaedon or Homarus sequences (Fig. 4), which were iden-tified as secreted enzymes.

Phylogenetic affiliation

To investigate further the relationships between the clonedcathepsin sequence and those from other insect andanimal organisms, a protein phylogenetic tree was con-structed (Fig. 5). The alignment displayed high similaritiesonly in the immediate vicinity of the active site residuesCys+25, His+164 and Asn+185, as described earlier (Berti &Storer, 1995). The phylogenetic analysis was extendedto include forty representative vertebrate cathepsinsequences as well as all the arthropod cysteine proteinasesequences known at that time (including twenty-four insectsequences and all the ‘relevant’ cysteine proteinasegenomic sequences from Drosophila; a couple of relatedsequences from this species, at the cathepsin B boundaryand all those lacking active site residues from the canonicalcatalytic triad, were excluded from the analysis to ensurecorrect alignment and sufficient informative sites). Theselected alignment runs over the 311 residues delineatingthe proenzyme, resulting in a data set of eighty-six inform-ative sites (among 103 complete and 100 variable sites,most of which were in the mature enzyme). Four majorclusters were identified (Fig. 5). The first cluster containedan assortment of vertebrate and insect cathepsin L-relatedenzymes (s.l.). Being located very close to the Sitophiluszeamais (weevil) sequences, AgCatL belonged to thisgroup, and more precisely to a well-defined subgroupcontaining several insect and arthropod sequences (fromthe shrimp Artemia to the fruit fly Drosophila). This sub-group is likely to represent the orthologous group to thehuman cathepsin L gene (s.s.). In two of the other followingclusters (cathepsin H and C), no insect representative was

included. The last cluster, corresponding to cathepsin B-like enzymes, included five other insect sequences, two ofwhich belonged to the Drosophila genome. The cathepsinB family having diverged from the cathepsin L family, evenbefore the divergence of cysteine proteinases from plantand from lower organisms (Berti & Storer, 1995), thecathepsin B cluster was chosen as the outgroup for ourphylogenetic analysis.

Analysis of cysteine proteinase gene family

The results of Southern hybridization experiments suggestthe existence of a gene family encoding cysteine protein-ases in A. gossypii (Fig. 6a). Although only one stronglyhybridizing band was detected at high stringency, pro-longed exposure (17 days at −80 °C) revealed two otherweakly hybridizing signals for each digestion.

Oligonucleotide primers, corresponding to 5′ and 3′ seg-ments of the AgCatL cDNA sequence, were used for PCRamplification of A. gossypii genomic DNA. The fragmentamplified from genomic DNA was found to be similar in sizeto the cDNA product, indicating that the genomic locusdoes not harbour introns anywhere in the entire regionencoding the preproprotein.

Pattern of tissue expression

Northern-blot experiments were performed to determinethe expression profile of the AgCatL. Total RNA isolatedfrom whole insect, gut and carcass (whole body minus gutand embryos) were hybridized with a radiolabelled AgCatLprobe. Two transcripts of 1.6 and 2.0 kb were expressed ata high level in the gut, and at least ten-fold higher than thelevels detected in the carcasses (Fig. 6b).

Expression in E. coli

The fusion protein of 70 kDa (corresponding to theexpected molecular weight) was observed on SDS PAGEand Western blots from E. coli cells after induction (Fig. 6c).The polypeptide was accumulated in inclusion bodies.Recovered solubilized fusion protein was purified by affinitychromatography in denaturing conditions. The pH of theeluted fraction was lowered to 5.5 to promote potentialautocatalytic processing (Jerala et al., 1998). The matura-tion of cathepsin proteinases needs propeptide processingand it is generally achieved by autocatalytic activation atacidic pH, or by proteolytic activation. Anti-SCP antibodiesdetected two bands in this acidic extract, one correspond-ing to the fusion protein and the other to the putative matureenzyme (about 30 kDa), because it migrated at the samelevel as the lower band observed in aphid and S. zeamaisguts (Fig. 6c). However, no enzymatic activity could bedetected in these extracts.

In total extracts from S. zeamais larvae, as in the totalextract from A. gossypii midgut, the antibody recognizedtwo bands in the immunoblot analyses (Fig. 6c). The upper

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band (about 49 kDa) is likely to represent the proenzymeform of AgCatL and the other, approximately 30 kDa in size,should correspond to the mature AgCatL enzyme, by ana-logy with SCP (Matsumoto et al., 1997). Both forms aretherefore detectable in aphid midguts. However, in contrastto S. zeamais gut extract, A. gossypii guts did not containequal amounts of the 49 and 30 kDa forms but, instead,they contained significantly more of the large AgCatL49 kDa (pro-) form.

Tissue distribution of AgCatL

In order to localize AgCatL in the aphid, adult A. gossypiireared on their host plant (melon) were fixed with para-formaldehyde, embedded in paraffin wax and sectioned.Fixed sections were immunoreacted with anti-SCP anti-bodies. An intense and specific signal was detected intra-cellularly in the aphid digestive tissues, with a clear gradientdecreasing along the digestive tract (Fig. 7); the anterior

Figure 6. Molecular analysis of AgCatL. Southern-blot hybridization with radiolabelled AgCatL cDNA probe (1 and 2, 10 µg of genomic DNA digested with EcoRI and EcoRV). Northern blot hybridization with radiolabelled AgCatL cDNA probe (3 and 4, total RNA isolated from carcass and guts, respectively). Western-blot hybridization with affinity-purified antibodies against SCP (5, inclusion bodies solubilized in denaturation buffer; 6, eluted fraction from nickel chromatography and dialysed under acidic conditions; 7, total protein extract from one A. gossypii gut equivalent; 8, total protein extract from one larva of S. zeamais).

Figure 7. Immunohistological localization of AgCatL in Aphis gossypii. Detection of section-bound primary antibodies was accomplished using secondary immunohistochemical reagents labelled with fluorochrome (FITC-conjugated (green) antirabbit IgG), as described in the Experimental procedures section. (A) Transverse sections of adult aphid. (B) Magnification of the stomach. Stomach (S), midgut (M), hindgut (H), bacteriocytes (bct), embryos (E), haemolymph (hae) and modified perimicrovillar material (mpm).

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‘stomach’ part was always strongly labelled. Homogeneityof cell staining indicated an intracytoplasmic localizationof AgCatL. AgCatL was not clearly detected in the reddish-coloured apical structures and seems not to be secreted inthe gut lumen.

A strong signal was also observed at the level of thebacteriocytes, which contained A. gossypii endosymbionts(Buchnera aphidicola, an intracellular obligate enterobac-terial symbiont of aphids (Charles et al., 2001). However,because antibodies were raised against SCPs expressedin E. coli cells, the staining could result from artefactualcross-detection of contaminants. A control using a batchof affinity-purified antibody was therefore performed, andalthough the signal was lower in these slides, it remainedstrong enough to confirm the presence of CatL at bacterio-cyte level. CatL activity was also directly detected in this tis-sue by enzymatic assay on dissected bacteriocytes with aZ-Phe–Arg pNA substrate, specific to cysteine proteinase(Rahbé et al., 2003a). By contrast, the presence of AgCatLwas not clearly identified in fat body or in other tissues(Fig. 7), which may differ from the situation describedfor Sarcophaga (Fujii-Taira et al., 2000), Drosophila (Mat-sumoto et al., 1995) or Sitophilus (Matsumoto et al., 1997).

Discussion

In spite of the relative abundance of proteins in the phloemof some plant species, which include preferred hosts ofthe cotton-melon aphid A. gossypii, polypeptides are notusually considered to be a major source of dietary nitrogenfor most phloem-feeding insects (Terra & Ferreira, 1994;Rahbé et al., 1995; Mochizuki, 1998). However, a whiteflyspecies, although being also a strict phloem-feedingHomoptera, was shown by radiotracer experiments toingest plant proteins, to degrade part of them to free aminoacids and to use these for de novo protein synthesis (Sal-vucci et al., 1998). Therefore, the previous inability to detectany proteinase activity in the digestive tracts of Homopteraseems to be clearly artefactual. We showed that the mainreason for this, at least for aphids, was probably the insol-ubility, together with the dependence on a single catalyticclass, of their digestive tract endoproteinases.

We have found that midgut extracts of A. gossypii adultsdo possess endoproteolytic activity on both protein andsynthetic substrates. An acidic pH optimum, enhancement byreducing agents and sensitivity to a wide range of inhibitorsclearly suggest a strong dominance of aphid proteinasesby the cysteine class. Although in-gel assays may restrictdetection to those proteinases able to renature after SDS/electrophoretic treatments, all our enzymatic and molecularresults are consistent with the occurrence of this singlemajor class in aphid midguts. These results are confirmedby a more extensive study of the hydrolytic enzymes of themodel and of the larger pea aphid, from which a cysteine

proteinase has been purified and is now available for detailedenzymological characterization (Cristofoletti et al., 2003).

One cDNA encoding a putative cysteine proteinase hasbeen cloned from A. gossypii midguts. Owing to its gut-specific expression pattern, to its identification as a regularcathepsin L enzyme, compatible with the substrate specif-icity shown by whole midgut extracts (preferential activityon Z-Phe–Arg substrate), and to the presence of a singlemajor homologous gene encoding CatL in A. gossypii, thisgene is quite probably responsible for the main activityobserved in aphid midguts. The presence of two transcriptsmay reflect the use of two consensus polyadenylation sites.A preliminary systematic sequencing of a substractedcDNA library (guts vs. carcasses) from A. gossypii con-firms the strict reliance of aphid guts on cysteine proteinasein that only three endopeptidase-related transcripts wereidentified in this library, all of the cysteine type (AgCatL plustwo additional cathepsin B-related cDNAs; C.D., unpub-lished results). Within the AgCatL cDNA sequence, a singlelong open reading frame was found that encoded a typicalCatL preproprotein of 341 amino acids. The propeptidestructure seemed to be canonical, and processing wasobserved in vivo and in vitro, although no resulting activitywas obtained from the expressed/processed AgCtL geneproduct. This may be due to an incorrect folding process inE. coli, which involves the formation of three potential dis-ulphide bridges and relies on the chaperone properties ofthe propetide (Matsumoto et al., 1998). The overall patternof the structure and expression of AgCatL is reminiscent ofwhat occurs in Drosophila, in which a single orthologousgene (CATL_DROME, or CP1) has been analysed (Mat-sumoto et al., 1995; Tryselius & Hultmark, 1997; Grayet al., 1998), also showing partial gut-specific expression.One difference may lie in the absence of introns in the aphidgene (three in the Drosophila gene), but this was also foundwith the weevil genes.

Regarding the catalytic properties of AgCatL, as derivedfrom comparative sequence and structure information, thestriking feature is the extreme conservation of all residuesthat were shown to be critical for the functioning of a canon-ical mammalian cathepsin L (except the S′ subsite residuesalready discussed, and featuring specificities common toall insect orthologous CatLs). Among various subfamilies oflysosomal cysteine proteinases (B, H or L), cathepsin Lfavours aromatic residues at P2, which distinguishes it, forexample, from the closely related enzymes cathepsin Sand K. Human Ala135 (as opposed to Gly found in cathepsinS) is largely responsible for this discrimination (Barrettet al., 1998), and this diagnostic conservation is present asAla136 in AgCatL (Fig. 4). This extreme conservation is notthe rule for all arthropod CatLs, but restricted to our so-called orthologous group, featuring Drosophila, Aphis orSitophilus sequences: the Phaedon sequence does notconserve the critical Ala136 residue, and the Homarus

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sequence does not conserve residue E−35, previouslyquoted as crucial for the acid autoactivation of procathepsinL (Fig. 4). When comparing the phylogenetic grouping of allarthropod cathepsin L sequences (Fig. 5), it is striking tonote that three groups emerge, and share within theirmembers both phylogenetic and functional features thatmay indicate specific ‘physiological’ recruitments of thesesequences during the evolution of the family: (i) the crusta-cean sequences may represent a distinct ‘digestive’ recruit-ment from a basal cathepsin L lineage, having been provenexperimentally to be secreted proteins from the digestivetract of their source organism (Laycock et al., 1989); (ii) incontrast to this, none of the members of the AgCatL grouphas been shown, biochemically, to be present in the diges-tive secretions of its source organism, and although differ-ent members are preferentially expressed in the digestivetract, including AgCatL, they may be regarded overall as‘standard’, and functionally conserved, insect lysosomalenzymes (thus called the insect orthologous CatL group);(iii) finally, a group of quite divergent enzymes fromColeoptera (the Phaedon–Hypera clade), probably secreted,may have appeared from the same basal Cat L group but,alternatively, also from more distantly related CatL-likegroups, such as the CatF lineage or other genes present atthis point in the Drosophila genome (Fig. 5).

As for the subcellular expression/localization of AgCatL,the situation might be complex, as pointed out already bythe presence of two related transcripts in gut mRNAs. On theone hand, AgCatL features many very standard traits oflysosomal cathepsins:1 Structural / functional conservation indicative of strongselective pressures by structural constraints on (endog-enous, conserved?) substrates.2 Propeptide organization, including acid activation char-acteristic of the lysosomal compartment; to protect cellsfrom the potentially disastrous consequences of uncon-trolled degradative activity, essentially all known cellularproteolytic enzymes are synthesized as inactive precur-sors. Activation of the enzyme is accomplished by cis- andtrans-cleavage of the propeptide (Menard et al., 1998) invery acidic conditions (Jerala et al., 1998).3 Strict intracellular expression, as indicated by immunolo-calization and enzymatic assays.4 Presence of potential lysosomal targeting signals througha mannose-phosphate pathway.

The last trait may not be consensual, and it has beenchallenged in an insect heterologous cell expression system(Aeed & Elhammer, 1994), but it was shown here to bestrikingly correlated with the three functional /phylogeneticgroups of arthropod CatL discussed above: a conservedN-glycosylation pattern in the prosequence region −20 (anecessary, though not sufficient, condition for mammalianmannose-phosphate targeting) is present in all insectsequences of the AgCatL clade, whereas it is absent from

all sequences in both the crustacean clade and the diver-gent coleopteran group (Hypera, Phaedon, Diabrotica), allencoding putative secreted cathepsins (Fig. 5).

On the other hand, some other traits do not fit in with astrict lysosomal expression, such as:1 the striking insolubility of the CatL/Z-Phe–Arg activity inthe absence of SDS;2 the consistent gut-specific expression of many insectCatL genes, associating this molecule with (intracellular?)digestive functions;3 the detection of a major proenzyme pool in aphid andother insect cells by immunoblotting (Homma et al., 1994;Matsumoto et al., 1997); and4 the localization of cathepsin L by EM/immunogold label-ling in vesicular, multivesicular and modified perimicrovillarmembrane structures of the aphid enterocytes localized inthe anterior part of the midgut (Cristofoletti et al., 2003).

Indeed, this complex expression pattern is also a typicalfeature of mammalian CatLs, which show multiple expres-sion phenotypes (e.g. lysosomal vs. secretory) dependingon cell context, associated with potential alternative splicingand numerous post-transcriptional signals (N-/C-terminalpropeptide and mature enzyme structures necessaryfor folding and secretion, signalling for mannose-phosphatedependent and independent lysosomal targeting, etc.).Insect cathepsins from the orthologous CatL group mightshare this expression complexity, which makes them inter-esting genes and molecules to study in model (Drosophila,Bombyx) or non-model organisms (weevils, aphids), in orderto understand better their involvement in physiologicalfunctions as diverse as digestion, development or otherinteraction-related processes (defence, detoxication, etc.).The inhibition of aphid AgCatL by oryzacystatin, an inhibitorof cysteine proteinase originated from rice, delivered eitherthrough transgenic plants or through a diet, was recentlyshown to alter aphid reproductive functions (Rahbé et al.,2003a); the distribution of the ingested inhibitor in the insectbody, as revealed by immunohistochemistry (Rahbé et al.,2003a), is strikingly similar to that of AgCatL, includingin internal organs (Fig. 7), which confirms that AgCatL is asuitable target for plant-derived defence strategies againstaphids, and even against other insects that do not rely ondigestive cysteine proteinases (e.g. flies and weevils).

Experimental procedures

Materials

Universal indicator of pH 4–10 was obtained from Merck (Darmstadt,Germany). Restriction endonucleases, reverse transcriptase andTRIZol reagent were purchased from Gibco-BRL (Life Science,NY, USA). SMART cDNA library construction kit and Prime aGene™ labelling kit were obtained from Clontech (Palo Alto, CA,USA) and from Promega (Madison, WI, USA), respectively. Otherchemicals were from Sigma (St Quentin Fallavier, France).

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Insects

The ‘melon strain’ of Aphis gossypii (clone Ag-LM02) originatedfrom INRA Montfavet and has been reared on Cucumis melocv. Védrantais in the laboratory since 1991. Aphids were rearedin ventilated Plexiglas cages (21 °C, 70% relative humidty,light–dark of 16 : 8 h). The maize weevil Sitophilus zeamais,reared on wheat, was used as the positive control for cathepsinimmunodetection.

Aphid dissection

Guts from adult aphids (mean adult weight 100–150 µg) weredissected under a binocular microscope. Damaged tracts werenot used and entire midguts (‘stomach’, median and posteriormidguts, hindguts) were kept on ice until an adequate numberhad been collected, then used or stored at −80 °C.

Determination of digestive tract pH

The pH of the different gut parts was estimated using a universalindicator solution. Guts were either homogenized in four parts ordipped entirely in indicator, and the resulting tissue colour wascompared either with the pH values obtained using standardsmade from buffers or with a colour chart.

Determination of A. gossypii proteinase activities

Digestive tracts were homogenized in ice-cold 0.15 M NaCl. Gen-eral proteinase activity was determined using 125I globulin as asubstrate protein between pH 4 and 9 (Sarath et al., 1989).Labelled substrate was obtained by coupling 125I to commercialpurified γ globulin using the iodobead protocol (Pierce, France).Two gut equivalents were mixed with 5 µg (5 × 105 d.p.m.) oflabelled globulin, with 10 µl of water or inhibitors and buffer to givea final volume of 300 µl. Inhibitors (E-64, benzamidine, EDTA, pep-statine) or activator (DTT) were pre-incubated with gut extract atroom temperature for 15 min prior to the addition of substrate. Thereaction mixture was then incubated at 37 °C for 24 h. Five hun-dred micolitres of chilled 20% TCA and 250 µl of 2% casein wereadded to stop the reaction, then centrifuged at 10 000 g for 5 minto remove the precipitated proteins. The radioactivity recovered inthe supernatant was measured for each pH value, and checkedwith enzyme blanks.

Assay of proteinase activity on polyacrylamide gel electrophoresis

Tissues were homogenized in cold extraction buffer (0.15 M NaCland 1% SDS), incubated at room temperature for 2 h and cen-trifuged for 5 min at 17 000 g and 4 °C. SDS was eliminated bydialysis against 0.15 M NaCl at 4 °C overnight. Preliminaryexperiments on A. gossypii and another aphid species showedthat the use of SDS was necessary to solubilize endoproteolyticactivity, and that other tested detergents did not result in substan-tial solubilization (Cristofoletti et al., 2003).

Samples containing two gut equivalents were diluted two-fold inLaemmli buffer (62.5 mM Tris HCl, pH 6.8, 2% (w/v) SDS, 10%(v/v) glycerol, 5% (v/v) β-mercaptoethanol, 1‰ (w/v) bromophenolblue). After migration on a 0.1% SDS−10% polyacrylamide gel(Bio-Rad Mini Protean II™ system) conducted at 4 °C, the proteinswere electrotransferred on to a 10% polyacrylamide/0.1% gelatinreplica gel where in situ proteolytic visualization was carried out,as described by Michaud et al. (1993).

RNA isolation, RT-PCR and Northern-blot hybridization

Total RNA was isolated from both the 200 dissected guts and theresulting carcasses (whole aphids minus guts and minus embryos–containing gut tissues). The aphids used were young adults, andRNA was extracted using TRIzol™ reagent, as described by themanufacturer.

Five micrograms of total RNA was submitted to reverse tran-scription (RT) prior to PCR. The RT reaction was carried out at37 °C in the presence of oligo(dT) and M-MLV reverse tran-scriptase, according to standard instructions. The PCR reactionwas performed using 1 µl of the RT reaction, and 10 pmol of eachdegenerated primer (Flav1 : 5′-CARGGWSAMTGYGGVWSNT-GYTGGDSMTT-3′, Flav2 : 5′-CCGWADCCMACNACMWDMAC-MCCRTGRT-3′).

For Northern blot analysis, the total RNA from carcasses (bodyminus gut and embryos) or from guts was prepared from fifty dis-sected or whole adult aphids. Three micrograms of total RNA wasanalysed on a 1.5% agarose gel containing 2.2 M formaldehydeand then transferred to a nylon membrane Gene Screen Plus(NEN™, Life Science, Boston, MA, USA). The RNA/DNA hybridi-zation was performed overnight at 65 °C according to the Church& Gilbert (1984) standard protocol, with a radiolabelled probe cor-responding to the entire cDNA AgCatL. The same filter wasstripped and hybridized to an 18S ribosomal RNA probe to checkthe RNA loading. The autoradiograms were scanned using a den-sitometer and data points were corrected for input RNA variations.

cDNA cloning

The gut cDNA library in the pTriplEX2™ vector (SMART cDNA Library,Clontech) was constructed according to the manufacturer’s instruc-tions. About 4 × 106 plaques were differentially screened by PCRusing two specific AgCatL primers deduced from the sequenceof the RT-PCR fragment previously isolated (GossF: 5′-CTACT-GGATCGCTGGAAGGAC-3′, GossR: 5′-GGGCATCTTCATCAC-CTTCAGG-3′). The cDNA library was diluted and each quarterof the Petri dish was screened by PCR. The positive part was titredand subsequently diluted. This step was repeated until one phagemidclone was obtained. The cDNA containing phagemids were rescuedfrom the phage according to the kit’s instructions. The cDNA wascharacterized by DNA sequencing on both strands.

Genomic Southern-blot hybridization

Genomic DNA was extracted from whole mature aphids using aCTAB-based method (Doyle et al., 1990). Ten micrograms of DNAwas fully digested with EcoRI or EcoRV (restriction sites absent inthe AgCatL gene sequence) and migrated on a 0.8% agarose gel.The DNA was transferred on to a nylon membrane Gene ScreenPlus (NEN™, Life Science) and hybridized overnight at 65 °C,according to a standard protocol (Church & Gilbert, 1984), with acDNA AgCatL probe labelled with α-32PdCTP using the oligolabel-ling kit from Promega. The membrane was washed in 2× SSC,0.1% SDS for 20 min at room temperature (two washes) and twicewith the same buffer at 40 °C, then exposed to X-ray film.

Phylogenetic analysis

A phylogenetic analysis was performed on AgCatL to address thefollowing questions: what is the correct functional assignment ofthis protein, and how is it linked to the main classes of vertebratecysteine proteinases (on which the functional classification is

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based)? How does AgCatL compare to all other known arthropodsequences? How is AgCatL placed relative to the model set of fullysequenced insect cysteine proteinases (full set of homologuesfrom Drosophila)?

In order to select vertebrate sequences, the HOVERGEN data-base was used (Duret et al., 1995). It contains all the vertebratesequences from GenBank (excluding expressed sequence tags)and is cleared for redundancy; homologous coding sequences havebeen classified in gene families, and protein multiple alignmentsand phylogenetic trees have been computed for each family. Onerepresentative sequence was retained from every family of cysteineproteinases (B, C, H, S and L; NC-IUBMB enzyme classification).All of these protein sequences were then blasted against Gen-BankP™ to extract any homologous arthropod sequences, whichwere retained after verifying that they possess the three catalyticamino acids (included, however, if from Drosophila). Proteinsequences were aligned using the CLUSTALW program (Thompsonet al., 1994) followed by manual refinement. Gaps and regions withambiguous alignments were excluded from the analysis. Thephylogenetic tree was generated using the Phylo_win software(Galtier et al., 1996) and the neighbour-joining algorithm withthe PAM distance. Confidence limits on grouping were determinedby the CLUSTALW bootstrapping technique (500 repeats).

Molecular modelling

As a basis for a spatial localization of the critical residues, patternsand features of the aphid cathepsin L molecule, a first approachmodelling was performed through the Expasy protein resourceserver (http://www.expasy.ch), using the Swiss-Model automatedprotein modelling facility (Guex et al., 1999). The target sequenceused was AgCatL prosequence (Fig. 4), and the selected tem-plates were two PDB accessions for human cathepsin L crystalstructures, 1CS8/A-chain (Groves et al., 1998) and 1CJL (Cou-lombe et al., 1996). Sequence similarity between the target andthe template was high (52% identical and 69% positive residuesbetween AgCatL and 1CJL template), ensuring robust alignmentand global correctness of the model for the purposes required.

Expression in E. coli

The AgCatL coding region (1023 bp), excluding the N-terminal57 bp signal peptide sequence and stop codon, was inserted intothe polylinker site downstream of the lacZ promoter of the pET41bexpression vector (Novagen, Darmstadt, Germany), in the sameorientation as lacZ and in frame with several N- and C-terminaltags. Recombinant protein production was induced with isopropyl-β-D-thiogalactopyranoside. Inclusion bodies were solubilized withdenaturation buffer (6 M guanidine–HCl, 300 mM NaCl, 50 mM Naphosphate buffer, pH 8, and 10 mM DTT). Recovered solubilizedproteins were purified by affinity chromatography on a nickelcolumn in denaturing conditions with imidazole elution (150 mM,pH 8), according to the manual’s instructions (TALON, Clontech).The eluted fraction was then dialysed against renaturation buffer(25 mM Tris–HCl, pH 8, 100 mM NaCl,; 5 mM EDTA) and the pHwas lowered to 5.5 by addition of HCl to induce autocatalyticprocessing.

Western blot analysis

Total extracts of aphid guts and of S. zeamais at the larval stage(positive sample) were prepared by homogenizing tissues in one

volume of Laemmli buffer. Proteins were electrophoresed on a10% SDS polyacrylamide gel and electroblotted on to a nitrocellu-lose membrane; the membrane was immunoreacted with antibodyagainst SCP (1 : 500 dilution in PBS plus 5% milk) and the immu-noreacted proteins were visualized using peroxidase-conjugatedantirabbit IgG antibody and an ECL kit (Amersham-PharmaciaBiotech, France). Because the amino acid sequences of weevil(SCP) and aphid were highly similar (Fig. 4), the anti-SCP anti-body (Matsumoto et al., 1997) was expected to cross-react withAgCatL.

Immunohistochemical study

Adult aphids were fixed overnight in Bouin-Holland fixative (32%ethanol, 4% neutral formaldehyde, 2.6 mg picric acid, 4% ice-coldacetic acid). After dehydration in graded ethanol, the cuticle wassoftened in 100% butanol for 48 h. After rehydration in H2O for15 min, the material was embedded in 1.3% agarose, 0.04% neu-tral red in order to position the aphid correctly. Agarose blocks ofthree individuals were embedded in paraffin. Serial sections(6 µm) were cut and mounted on poly-L-lysine-coated slides, de-paraffinized, blocked with 1% bovine serum albumin in PBS andincubated at room temperature overnight with the anti-SCP anti-body diluted 1 : 100 in PBS plus 0.5% BSA. The samples wererinsed in PBS at room temperature and incubated with the second-ary antibody, fluorescein isothiocyanate (FITC)-conjugated swineantirabbit immunoglobulin, diluted 1 : 100 in PBS plus 0.5% BSA(1 h at room temperature and in the dark). After rinsing in PBS atroom temperature, the sections were mounted in Citifluor mount-ing medium (Sigma, Steïnheim, Germany) and observed usingLeica TCSNT confocal laser scanning microscopy (Leica, Heidel-berg, Germany) equipped with an argon/krypton laser (Omnichrome,Chino, Canada). To visualize the AgCatL/FITC fluorochrome weused a 530 ± 30 nm band-pass filter (green signal), and to observetissue organization, autofluorescence was collected at 590 ± 20 nm(red signal); composite images were generated using the Zeisssoftware. All sections were first scanned at low magnification (×16objective, electronic zoom ×1 or ×2.64).

Acknowledgements

We thank Marie-Gabrielle Duport for her help in aphid rear-ing and dissection, Dr Hubert Charles for his help in thephylogenetic analysis, and gratefully acknowledge theEuropean Union for a doctoral fellowship to C.D., through aFAIR collaborative programme CT98-4239, coord. M. A.Jongsma.

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