Proc. Nat!. Acad. Sci. USAVol. 83, pp. 14-18, January 1986Biochemistry

L-Canavanine and protein synthesis in the tobacco hornwormManduca sexta

(protein degradation/aberrant protein/amino add analog/plant-Insect interactions)

GERALD A. ROSENTHAL*t, AND DOUGLAS L. DAHLMANt**T. H. Morgan School of Biological Sciences, tDepartment of Entomology, and tGraduate Center for Toxicology, University of Kentucky,Lexington, KY 40506

Communicated by Robert L. Metcalf, August 16, 1985

ABSTRACT L-Canavanine, a nonprotein amino acid ofcertain leguminous plants, manifests potent Insecticidal prop-erties in a canavanine-sensitive insect such as the tobaccohornworm Manduca sexta (L.) (Sphingidae). This arginineanalog is activated and aminoacylated by arglnyl-tRNA syn-thetase and incorporated into nascent polypeptide chains tocreate structurally aberrant, canavanine-containing proteins.Analysis ofincorporation of [3H]leucine into protein inM. sextalarvae that had been injected with canavanine revealed that thisarginine analog stimulates protein synthesis. During the first 3hr after ijection of canavanine, canavanine-mediated netstimulation of protein formation was readily discerned. There.after, the stimulation of protein synthesis appeared to be offsetby the preferential degradation of anomalous proteins. Double-label protein-turnover experiments with larvae injected with[14C]canavanine- and [3Hlarginine-containing hemolymphproteins showed that canavanine-containing proteins weredegraded preferentially.

L-Canavanine [L-2-amino-4-(guanidinooxy)butyric acid] issynthesized by at least twelve hundred different legumes;often it is the principal nonprotein amino acid of the plant(1-3). This natural product is a structural analog of L-argininein which the terminal methylene group is replaced by oxygen.This arginine analog is activated and aminoacylated byarginyl-tRNA synthetase and then incorporated into nascentpolypeptide chains (4, 5). Canavanine-containing proteins("canavanyl proteins") exhibit altered physicochemicalproperties (6, 7); for example, the vitellogenin of Locustamigratoria migratorioides formed in the presence of cana-vanine possesses an electrophoretic mobility that differs fromthat of the normal macromolecule (8). Many essential func-tional properties of proteins synthesized in the presence ofcanavanine are affected adversely (9). Canavanine's toxiceffects are particularly pronounced in rapidly developinginsect larvae, and these invertebrates have become theorganisms of choice for probing the role of this analog in thechemical defense of higher plants.Toxins such as canavanine constitute the chemical barrier

to predation and disease that higher plants evolved forself-protection and improvement of their Darwinian fitness.Although much is known of the chemical nature of theseprotective allelochemicals, little is understood of the bio-chemical bases for their protective modes ofactions. Massivedevelopmental aberrations result when final (fifth) instars ofthe tobacco hornworm, Manduca sexta (L.) (Sphingidae),ingest a diet containing canavanine (9). Comparative studiesof canavanyl protein formation with the tobacco hornworm,a canavanine-sensitive insect, and the tobacco budworm,Heliothis virescens (Fabricius) (Noctuidae), a canavanine-resistant organism, have provided strong evidence that ab-

errant protein synthesis contributes significantly to canavan-ine's antimetabolic properties in canavanine-sensitive insects(10, 11). Canavanine can inhibit various reactions of macro-molecular synthesis that are part ofRNA or DNA formation(4). The severe developmental anomalies observed incanavanine-consuming M. sexta larvae may be related notonly to aberrant protein production but also to curtailedprotein synthesis. The studies reported in this communica-tion examine the relationship of canavanine and the ability ofthe developing larvae to synthesize proteins.

MATERIALS AND METHODSMaterials. L-[guanidinooxy-14C]Canavanine (58 ,uCi/

iAmol; 1 Ci = 37 GBq) was synthesized and purified by themethod of Rosenthal et al. (12). Other radiolabeled aminoacids were purchased from New England Nuclear. L-Canavanine was isolated from an acetone powder of jackbean (Canavalia ensiformis) seeds (13). Sigma supplied theremaining biochemicals.

Insects. Unless otherwise indicated, all experiments wereconducted with M. sexta larvae on the second day of theterminal, fifth instar. M. sexta larvae were obtained from acontinuous colony reared at the University of Kentucky;procedures for rearing the insects have been described fully(9). All insects were anesthetized with carbon dioxide beforeparenteral injection and hemolymph collection.

L-[3,4,5-3H]Leucine Incorporation Studies. Canavanine-treated larvae received 1 mg of L-canavanine/g of fresh bodyweight and various amounts of tritiated leucine. Since aninjected dose of arginine does not affect protein synthesis(unpublished data), insects injected with water served as thecontrols. At the indicated time, the hemolymph was obtainedby cutting a proleg and permitting the hemolymph to drainfrom the body cavity. Unless otherwise indicated, hemo-lymph proteins were obtained by precipitating 300 Al ofhemolymph with an equal volume of 25% (wt/vol) trichloro-acetic acid and stored at -600C until processed further.

Processing the L-PH]Leucine-Containing Proteins. The pre-cipitated hemolymph proteins were collected by centrifuga-tion with a Beckman model B Microfuge. After carefulremoval of the supernatant solution, the pellet was extractedtwice with 10% (wt/vol) C13CCOOH, twice with 5% (wt/vol)C13CCOOH, once with anhydrous ether/absolute ethanol(1:1, vol/vol), and once with anhydrous ether. The pellet wasair-dried and treated with 0.3 ml of TS-1 tissue solubilizer(Research Products International) at 500C overnight. Thedissolved pellet was transferred to a liquid scintillation vial,using three 1-ml portions of Bray's scintillation medium (14),for measurement of radioactivity.

L-[guanidinooxy-'4C]Canavanine Content of Proteins.Trichloracetic acid-precipitated proteins from [14C]canavan-ine-treated (58 ;kCi/;kmol) larvae were hydrolyzed with 6 MHC1. Hydrolysates were freed of arginine by ion-exchangechromatography (15, 16) and then incubated with arginase

14

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Proc. Natl. Acad. Sci. USA 83 (1986) 15

and urease (15, 16). Arginase (EC 3.5.3.1) converts L-[guanidinooxy-'4C]canavanine to L-canaline and ['4C]urea;urease (EC 3.5.1.5) hydrolyzes the latter to ammonia and'4Co2. The labeled carbon dioxide is then trapped inHyamine hydroxide and quantitated by liquid scintillationspectroscopy.

Degradation of Radiolabeled Hemolymph Proteins. Therelative degradation of [14C]canavanine- and [3Hlarginine-containing hemolymph proteins was determined by injecting2-day-old flfth-instar larvae (=4 g fresh body weight each)with either 10 gCi of L-[2,3-3H]arginine or 12 ,uCi of L-[guanidinooxy-14C]canavanine. After 7 hr, =7 ml of hemo-lymph was collected from each group of larvae and treatedwith phenylthiourea to prevent hemolymph melanization.The hemolymph was centrifuged for 2 min with a Microfuge,and the clarified hemolymph was dialyzed against 500 ml ofsaline solution (Cherbas'; ref. 24) overnight; the saline waschanged after the initial 4 hr of dialysis. The dialyzedhemolymph was centrifuged for 2 min as above and thehemolymph radioactivity from the arginine- and thecanavanine-treated insects was determined by liquid scintil-lation spectroscopy. The hemolymph samples then werepooled so that each isotope contributed about equally to thetotal radioactivity. The combined hemolymph samples werefiltered through a sterile disposable 0.2-,um cellulose acetatemicrofilter (Schleicher & Schuell) before injection into 1-day-old fifth-instar larvae. At various times, hemolymph wascollected from groups of three larvae, pooled, and processedas described above.

['4C]Canavanine-containing protein degradation by cana-vanine-treated and control larvae was assessed by the aboveprocedures with several exceptions. [14C]Canavanine-con-taining hemolymph proteins were collected after 24 hr,dialyzed as above, and injected into 2-day-old fifth-instarlarvae that had been injected 4.5 hr earlier with canavanine(1 mg/g of body weight) or water. For the experiments ofTable 4, duplicate 0.5-ml hemolymph samples were collectedfrom five insects at the indicated times and processed asdescribed above but were dissolved with 0.6 ml ofTS-1 tissuesolubilizer and placed at 60'C for 2-3 days prior to assay by

liquid scintillation spectroscopy. For the experiments ofTable 5, only 0.25 ml of hemolymph was collected, dissolvedin 0.3 ml of TS-1, and placed at 60°C for 1 day.

RESULTS

Canavanine provided by parenteral injection established theLD50 by this administrative route to be 1.0 mg/g offresh bodyweight. Mortality characteristically occurs at larval-pupalecdysis; such canavanine-treated larvae therefore survive atleast 48 hr after injection. Injection of an LD50 dose ofcanavanine containing L-[guanidinooxy-'4C]canavanine re-sults in incorporation ofcanavanine into hemolymph proteins(Fig. 1).

In an initial experiment intended to assess whethercanavanine inhibits protein synthesis, an LD50 dose ofL-canavanine was injected along with [3H]leucine. Analysisof hemolymph proteins after 12 or 24 hr disclosed nosignificant effect of canavanine on the level of [3H]leucine-containing proteins (Table 1). The amount of 3H in thesesamples represents a balance between the reactions ofprotein synthesis and those fostering the degradation of thelabeled proteins at the time the insects are sacrificed. Thus,when the larvae are treated simultaneously with canavanineand radioactive leucine, so that sufficient (nonradioactive)canavanine is present to ensure production of canavanylproteins, the amount of leucine-labeled proteins is the samefor both the 12- and the 24-hr samples as when canavanine isomitted.A comparable evaluation has been made with the proteins

constituting the larval musculature. The musculature con-sists oftissues remaining after removal ofthe hemolymph andinternal body organs such as the fat body, digestive tract, andMalpighian tubules. The 3H content of the musculatureproteins was consistently the same for canavanine-treatedand control larvae.The finding of similar [3H]leucine incorporation into the

proteins of the hemolymph and musculature from bothcanavanine-treated and control larvae was unexpected.There is compelling evidence that living systems degrade

1.8k

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1.5 F

1.2F

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FIG. 1. Incorporation of L-[guanidinooxy-14C]canavanine into hemolymph proteins. Each point represents the mean (±SEM) of threesamples. Each sample (0.6 ml) contained 200 ,l of hemolymph from each of three larvae. The labeled hemolymph proteins were processed asdescribed in the text except for the use of 2 M NaOH to dissolve the precipitated proteins. The average larval fresh weight at the initiation ofthe experiment was 2.26 g; initial larval fresh weight did not vary by more than 2% between insects. On the average, each larva received 694nCi of label as well as 1 mg of carrier L-canavanine/g of fresh weight.

Biochemistry: Rosenthal and Dahlman

0

16 Biochemistry: Rosenthal and Dahlman

Table 1. Incorporation of L-[PH]leucine into larvalhemolymph proteins

3H, nCi/300 ALI of hemolymphTime, hr Control Canavanine-treated

12 0.84 ± 0.16 0.86 ± 0.0524 1.08 ± 0.16 1.13 ± 0.05

Each larva received 1.0 ,UCi of L-[3H]leucine/g of fresh weight. Atthe same time, canavanine-treated larvae each received 1 mg of thecompound/g of fresh weight. Each value is the mean ± SEM for ninelarvae.

preferentially their aberrant macromolecules (17-19). Thishas been established in studies involving reticulocytes,prokaryotes, and numerous cultured mammalian cell types.Thus, it was expected that the [3H]leucine-labeled larvalproteins, synthesized under conditions in which canavanineis also incorporated (Fig. 1), would be degraded preferentiallyrelative to their canavanine-free counterparts.We therefore tested whether M. sexta does in fact degrade

canavanine-containing proteins faster than normal proteins.Larvae were injected with either L-[guanidinooxy-14C]-canavanine or L-[3H]arginine. After 7 hr, hemolymph pro-teins were prepared from each group and were processed toremove any radioactive free amino acids, filter-sterilized,combined, and introduced into the hemolymph of recipientlarvae. The hemolymph proteins of the recipient larvae wereisolated at various times and evaluated for their 14C/3H ratio(Table 2). It is evident that tobacco hornworm larvae degradepreferentially their aberrant, canavanine-containing proteins.As a result, we would expect significantly less net incorpo-ration of [3H]leucine in those insects that received canavan-ine than in control organisms.The finding (Table 1) that this is not the case can be

rationalized if canavanine enhances de novo protein produc-tion. This point can be evaluated experimentally if the[3H]leucine-incorporation period is kept very short, allowingthe reactions of protein production to be manifested overthose of protein degradation. In a preliminary experiment(data not shown), canavanine-mediated enhancement ofprotein synthesis was detected within 15 min of canavanineinjection into the hemolymph. Within the first 30 min,canavanine-enhanced protein synthesis reached a value 54%more than that of the control larvae, and after 60 min, 22%more than that of the controls.To determine how long canavanine-mediated stimulation

of protein synthesis continues, we tested the effect ofcanavanine injection prior to injection of [3H]leucine. Inthese experiments, treated larvae were administered L-canavanine (1 mg/g of body weight). At various timesthereafter, the larvae were injected with [3H]leucine; 30 minlater, hemolymph protein was isolated and the amount ofradioactive amino acid incorporated was determined (Table3). Canavanine-mediated stimulation of hemolymph proteinsynthesis was observed only when [3H]leucine was admin-

Table 2. Relative stability of [14C]canavanine- and[3H]arginine-containing hemolymph proteinsinjected into larvae

Time, hr 14C/3H ratio

0 1.2112 1.0524 0.9336 0.8148 0.55

Each value is the mean for three samples; each sample wasprepared from three larvae.

istered <3 hr after canavanine. A 30-min pretreatment withcanavanine increased the incorporation of radioactiveleucine by 67%, and a 90-min pretreatment increased incor-poration by 62%. However, pretreatment >3 hr prior toinjection of [3H]leucine failed to produce enhanced leucineincorporation (Table 3). These results indicate that canava-

nine-linked net stimulation of protein synthesis lasts <3 hr.The short duration of stimulated protein synthesis might

result from depletion of the injected canavanine. To evaluatethis possibility, we injected L-[guanidinooxy-14Clcanavanineinto larvae and the hemolymph was analyzed for radioactivecanavanine (Fig. 2). Analysis of the hemolymph 1.5 hr aftercanavanine injection showed that 68% ofthe original materialremained; this value compares to 57% after 3 hr. It is unlikelythat this decrease in insect canavanine content, as judged byhemolymph analysis, could account for the loss in discerniblecanavanine-mediated stimulation of protein synthesis be-tween 1.5 and 3 hr after the administration of canavanine.

It is possible that canavanine increases hemolymph proteinsynthesis in the fat body by elevating the level ofhemolymphamino acids available to the fat body. No significant reduc-tion in levels of hemolymph or fat body amino acids wasdetected after canavanine treatment (amino acid analysis ofacid-soluble material; data not shown). It may also be thatcanavanine affects fat body uptake of [3H]leucine from thehemolymph and enriches the specific activity of the leucineavailable to the fat body for the production of hemolymphproteins. To evaluate this possibility, we determined theuptake oflabeled leucine into the fat body and the [3H]leucinecontent of the tRNA pool of the fat body (see ref. 21 formethods). To be certain that the monitored RNA radioactiv-ity represented the [3H]leucyl-tRNALeu pool, the radioactiveRNA sample was uncharged by treatment at pH 10 for 30 minat 37°C. The results indicate that canavanine does not affectthe charged leucine pool of the fat body (data not shown).Finally, during the fifth larval instar, dietary canavanine doesnot alter the hemolymph concentration ofcations, such as K+or Mg2+, that can affect protein synthesis by the fat body (22).During the course of this investigation, we learned of the

experimental efforts of Lindquist and her associates with theso-called "heat shock" proteins ofDrosophila melanogaster(23). In response to a stress-inducing stimulus such as heat,this insect rapidly produces a group of stress-induced pro-teins; canavanine was reported to induce these proteins inDrosophila (23). As a result, we evaluated the capacity ofcanavanine to elicit synthesis ofthese stress-induced proteinsin M. sexta. Although these heat shock proteins are inducedin M. sexta when the larvae are injected with a nonsterilesolution, parenterally administered canavanine itself in a

sterile solution does not induce heat shock proteins. Forma-tion of these proteins cannot explain canavanine-dependentstimulation of hemolymph protein synthesis.

Table 3. Canavanine-mediated stimulation of incorporation ofL-[3H]leucine into hemolymph proteins

3H, nCi/300 ,ul of hemolymphTime, hr Control Canavanine-treated

0.5 1.05 ± 0.04 1.74 ± 0.071.5 1.17 ± 0.03 1.89 ± 0.053.0 1.19 ± 0.09 1.11 ± 0.046.0 1.09 ± 0.06 0.99 ± 0.05

12.0 1.21 ± 0.03 1.21 ± 0.0324.0 1.88 ± 0.13 1.91 ± 0.05

At the specified times after injection of canavanine or water(control), each larva received 2.0 ,uCi of L-[3H]leucine/g of freshbody weight. Hemolymph was collected 30 min later. Each value isthe mean ± SEM for nine larvae.

Proc. Natl. Acad. Sci. USA 83 (1986)

Proc. Natl. Acad. Sci. USA 83 (1986) 17

c._

l 60

U 40

20

Time, hr

FIG. 2. Loss of free L-[guanidinooxy-14C]canavanine from the hemolymph of larvae. Each value represents the mean ± SEM of three 150-iIsamples; each sample was obtained by pooling the hemolymph of three larvae and precipitating the proteins with trichloroacetic acid. Afterremoval of the precipitated material by centrifugation, the trichloroacetic acid was extracted from the solution with anhydrous ether. TheL-[guanidinooxy-'4C]canavanine content of the solution was determined by enzymatic hydrolysis and assay of the evolved 14CO2 trapped withHyamine hydroxide by liquid scintillation spectroscopy (14). Each larva in the experiment received the same amount (0.79 ,Ci) of labeled freecanavanine. The 15-min sample was considered to be a measure of the total injected radioactivity. This was necessary because hemolymphvolume changed during the course of the experiment as the result of larval growth. However, the same hemolymph volume was taken at eachsampling time, making it necessary to correct for the change in volume before the percent free [14C]canavanine remaining in the hemolymphcould be calculated. Larval hemolymph volume was calculated from the relationship of hemolymph volume to larval fresh weight (20).

A second important observation emanates from these inves-tigations. Over our 48-hr experimental period, the amount of[3H]leucine in hemolymph proteins is the same in canavanine-treated and control insects (Table 1). Yet, during this experi-mental period, preferential degradation of canavanine-contain-ing proteins in larvae that had not been pretreated withcanavanine occurs (Table 2). These experimental findings canbe reconciled by one oftwo alternatives. First, canavanine mayinhibit preferential degradation of aberrant proteins. This inhi-bition would negate preferential degradation of canavanine-containing proteins and account for the experimental findingsshown in Table 1. Second, if canavanine does not affectpreferential degradation, canavanine-mediated stimulation ofprotein synthesis must continue for longer than 3 hr (see Table3) but do so at a rate that just balances the reactions ofpreferential protein degradation.To distinguish between these possibilities, we injected

[14C]canavanine-containing hemolymph proteins into controllarvae and into larvae that had received 1 mg of L-canavanine/g of body weight 4.5 hr earlier. Analysis of thedegradation of these 14C-labeled, aberrant proteins revealedthat their elimination was the same in canavanine-treated andnontreated larvae (Table 4). Clearly, canavanine treatmentdoes not impair larval ability to degrade canavanine-contain-ing proteins. In fact, these data indicate that the canavanine-treated larvae degrade more aberrant protein than the con-trols. For each [14C]canavanine-labeled protein moleculeinjected into the larva, there existed at least five unlabeledcanavanine-containing protein molecules. This estimation ispredicated upon the amount ofcanavanyl protein synthesizedin 4.5 hr (Fig. 1) relative to the known amount of labeledprotein injected. The specific radioactivity of the aberrantproteins was lower in the canavanine-treated larvae than in

the control larva, yet treated and control larvae exhibitedsimilar rates of loss of protein radioactivity. Thus, thecanavanine-treated insects degraded more aberrant proteinmolecules than did control larvae.To test this important point further, the unlabeled canavanyl

protein content was increased by extending the time betweencanavanine administration and injection ofthe [14C]canavanine-labeled proteins from 4.5 to 12 hr. This should provide =1.5times as much aberrant unlabeled protein (Fig. 1). The amountof [14C]canavanine-labeled protein was similar in canavanine-treated and in control insects 24 and 36 hr after administrationof the labeled proteins (Table 5).

DISCUSSIONEvidence has been presented to establish that parenterallyinjected canavanine stimulates protein synthesis by M. sexta

Table 4. Degradation of [14C]canavanine-labeled proteins inlarvae pretreated with unlabeled canavanine

14C, nCi/500 1.l of hemolymphTime, hr Control Canavanine-treated

0 0.65 ± 0.0412 0.59 ± 0.03 0.57 ± 0.0224 0.53 ± 0.01 0.50 ± 0.0336 0.36 ± 0.04 0.39 ± 0.0248 0.25 ± 0.01 0.23 ± 0.03

[14C]Canavanine-labeled hemolymph proteins were injected intolarvae 4.5 hr after injection of unlabeled canavanine (1 mg/g of bodyweight) or water (control). At the times indicated, duplicatehemolymph samples were taken from each of five larvae; thus, eachvalue is the mean ± SEM for 10 samples.

Biochemistry: Rosenthal and Dahlman

18 Biochemistry: Rosenthal and Dahlman

Table 5. Degradation of [14C]canavanine-labeled proteins after12-hr pretreatment with nonradioactive canavanine

14C, nCi/250 ,ul of hemolymphTime, hr Control Canavanine-treated

0 0.38 ± 0.0224 0.29 ± 0.01 0.30 ± 0.0336 0.23 ± 0.01 0.20 ± 0.01

[14C]Canavanine-labeled hemolymph proteins were injected 12 hrafter administration of unlabeled canavanine. For other details, seethe legend to Table 4.

larvae. Canavanine is incorporated into hemolymph proteinsbut these aberrant proteins are degraded preferentially.Three hours after canavanine injection, canavanine-mediatedstimulation of protein synthesis is matched by preferentialdegradation. It may be that canavanine-mediated net stimu-lation of protein synthesis can be discerned only for 3 hrbecause it takes this long for the reactions of proteindegradation to build to a level where they offset enhancedprotein formation. Moreover, anomalous protein degradationby canavanine-treated larvae may be linked to the amount ofaberrant protein produced.The ability of an insect such as M. sexta to degrade so

efficiently its aberrant, canavanine-containing proteins canbe important in higher-plant chemical defense and insectadaptation to protective allelochemicals. This ability maycontribute to insect capacity to tolerate canavanine. Signif-icantly diminished capacity to degrade canavanine-contain-ing proteins would make canavanine a more potentiallydeleterious natural product.We thank Andrew D. Hanson for his suggesting the evaluation of

fat body charging of [3H]leucine and Peter E. Dunn for disclosing thatadministration of canavanine in sterile solution does not induce heatshock proteins in M. sexta. We gratefully acknowledge the supportof National'Institutes of Health Grant AM17322, National ScienceFoundation Grant PCM 82-19721, and Department of AgricultureAgreement 59-2213-1-1-763-0. This communication is paper number84-7-38 of the Kentucky Agricultural Experimental Station atLexington.

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Proc. Natl. Acad. Sci. USA 83 (1986)