In a canavanine-sensitive insect such as the tobacco hornworm, Manduca sexta [Sphingidae], this arginine antagonist markedly reduces larval growth, delays the onset and otherwise disrupts larval-pupal ecdysis, adversely affects pupal development and metamorphosis to the adult, and reduces female fecundity and fertility (Dahlman and Rosenthal, 1975; Dahlman and Rosenthal, 1976 ).

Replacement of the terminal methylene group of L-arginine with oxygen is such a subtle structural alteration that most arginyl-tRNA synthetases readily accept canavanine as a substrate (Allende and Allende, 1964; Mitra and Mehler, 1967). Indeed, all insects studied to date that are adapted to or are otherwise resistant to this toxicant, do not assimilate canavanine into newly synthesized proteins (Bleiler et al., l988; Rosenthal et al., 1987).

On the other hand, M. sexta larvae, which are sensitive to canavanine, synthesize appreciable canavanine-containing proteins. Provided at 1 mg/g fresh body weight, the highest canavanine concentration that M. sexta larvae can tolerate, the ratio of canavanine to arginine for the various insectan proteins is:

This substitution for arginine by canavanine results in the formation of structurally aberrant, canavanine-containing proteins throughout the larval body (Rosenthal et al., 1987).

STUDIES OF LOCUSTA: ALTERED THREE DIMENSIONAL CONFORMATION IN CANAVANINE-CONTAINING PROTEINS

To determine the biochemical consequence of canavanyl protein formation, a number of larval proteins were examined in detail. In the first study, canavanine was provided to gravid females of the locust, Locusta migratoria migratorioides [Orthoptera] whose ovarian mass had been removed surgically on the day of adult emergence.

This surgical procedure results in a pronounced accumulation of vitellogenin in the hemolymph. Analysis of canavanyl vitellogenin, purified from the hemolymph of these canavanine-treated locusts, shows that on average 18 of the 200 arginyl residues of vitellogenin or about one in 225 amino acids are replaced by canavanine (Rosenthal et al., 1989a). Even this level of replacement is sufficient to elicit a dramatic alteration in protein structure that can be seen by electrophoretic analysis of these proteins. The altered fragmentation pattern undoubtedly reflects a profound change in vitellogenin structure due to canavanine incorporation.

These disparate forms of vitellogenin were also treated with chemicals able to react with surface-exposed amino acid residues to form new amino acids. For example, treatment with cyanate carbamylates reactive lysyl residues and converts them to homocitrullyl. About one-quarter of the lysyl residues of native vitellogenin do not react with cyanate; presumably, these are inaccessible and buried within the interior of the macromolecule. These residues are readily carbamylated in canavanyl vitellogenin.

(Fig. 1)

A similar chemical approach establishes that nearly twice as many surface-exposed tyrosine residues are acetylated as compared to the native protein.

(Fig. 2)

Figure 3 depicts the emission fluorescence spectrum of canavanyl and native vitellogenin. The greater fluorescent intensity of canavanyl vitellogenin relative to the native macromolecule undoubtedly reflects the larger number of exposed aromatic residues in canavanyl vitellogenin.

(Fig. 3)

These experiments establish that canavanine incorporation into vitellogenin alters the three dimensional conformation of the protein, a property essential for normal function (Rosenthal et al., 1989b).

THIS STUDY PROVIDED THE FIRST DIRECT EXPERIMENTAL EVIDENCE THAT CANAVANINE INCORPORATION CAN ALTER PROTEIN CONFORMATION.

Microbial infection or mechanical injury to larvae of the fly, Phormia terranovae [Diptera] induces a family of protective, antibacterial proteins known trivially as the diptericins (Keppi et al., 1986).

If canavanine is provided at the time of mechanically injury, it is incorporated into all of these protective proteins (Rosenthal et al., 1989b). This assimilation causes a total loss of detectable antibacterial activity for nearly all the diptericins; only a single diptericin-diptericin A retains demonstrable biological activity.

STUDIES OF MANDUCA SEXTA LYSOZYME: LOSS OF CATALYTIC ACTIVITY IN CANAVANINE-CONTAINING PROTEINS.

Canavanine-mediated impairment in function was also demonstrated with a catalytic protein, lysozyme (EC 3.2.1.17) that is induced by injection of fragments of the cell wall of the bacterium, Micrococcus lutea into M. sexta larvae (Keppi et al., 1986). If these larvae are provided canavanine when challenged, it is readily incorporated into de novo-synthesized lysozyme. The ratio of canavanine to arginine in this aberrant lysozyme is 1: 3.8; assay of canavanyl lysozyme discloses a 48% loss in catalytic activity. This study provided the first demonstration of the ability of canavanine, through aberrant protein formation, to adversely affect the catalytic activity of an enzyme.

THE RELATIONSHIP OF CANAVANYL PROTEIN FORMATION TO CANAVANINE-SENSITIVITY

The importance of aberrant protein formation in the expression of canavanine’s antimetabolic effect is also supported convincingly by a radically different experimental approach that determines how frequently canavanine replaces arginine in the proteins of various insects, i.e. the substitution error frequency (SEF). Manduca sexta larvae incorporate about 3.5% of the administered radiolabeled canavanine into hemolymphic proteins after 24 h (Rosenthal et al., 1987). This results in a SEF that varies according to the insectan tissue but is about 1 in 2 for proteins of the larval body wall and musculature (Rosenthal et al., 1987).

The bruchid beetle, Caryedes brasiliensis, an inhabitant of the neotropic forests of Costa Rica, develops from larva to adult in the canavanine-laden seeds of Dioclea megacarpa.

The weevil, Sternechus tuberculatus, oviposits on the pericarp of Canavalia brasiliensis; larvae forage within the fruit whose seeds also store canavanine.

These two seed predators are examples of insects that are adapted biochemically to canavanine (Bleiler et al., l988), and can therefore tolerate, even flourish with this normally poisonous natural product (Rosenthal, 1983; Rosenthal et al., 1982).

The SEF for C. brasiliensis is 1 in 365 whilst that of S. tuberculatus is 1 in 500-1,000. The tobacco budworm, Heliothis virescens, does not consume canavanine-containing plants; however, it is naturally resistant to this potentially toxic allelochemical (Berge et al., 1986). This aggressive generalist herbivore exhibits a SEF of 1 in 65.

THUS, CANAVANINE-ADAPTED OR -RESISTANT INSECTS MINIMIZE OR AVOID CANAVANYL PROTEIN FORMATION WHILST CANAVANINE-SENSITIVE INSECTS READILY INCORPORATED THIS ARGININE ANTAGONIST.

L-HOMOARGININE STUDIES

Another interesting finding of studies of nonprotein amino acid interaction with M. sexta arises from analysis of the effect of L-homoarginine, the higher homolog of L-arginine, on larval growth and maturation (Rosenthal and Harper, 1996). L-[Guanidino-¹⁴C]homoarginine is incorporated readily into the newly synthesized hemolymph as well as body wall and musculature proteins of M. sexta larvae without effecting adversely larval growth and development. This radioactive, potential arginine antagonist is incorporated into M. sexta lysozyme and P. terranovae diptericins without adversely affecting biological activity.

The innocuous nature of homoarginine as compared to canavanine appears to reflect homoarginine’s basicity-its elevated pK_a that arguably exceeds that of arginine (Rosenthal and Harper, 1996). As a result, homoarginine does not disrupt essential residue interactions. In contrast, canavanine, with a pK_a (link to be created here) for the guanidinooxy group near 7 (Boyar and Marsh, 1982), is much less basic than arginine (pK_a = 12.48); this decreased basicity disrupts R group interactions forming the requisite interactions essential for protein conformation (Rosenthal and Harper, 1996).

L-HOMOCANAVANINE: X-RAY CRYSTALLOGRAPHY

X-Ray crystallographic analysis of canavanine disclosed that the distance between the terminal methylene carbon and the guanidinooxy carbon was less than the distance between the penultimate methylene carbon and the carbon of the guanidino group of arginine (Boyar and Marsh, 1982). This property of canavanine instigated development of synthetic methods for the production of the higher homolog of canavanine, homocanavanine, L-2-amino-5-(guanidinooxy)pentanoate (Rosenthal et al., 1995). Since homocanavanine is longer than canavanine, it may accomodate a better "steric fit" into the active site of arginyl-tRNA synthetase, we reasoned that this compound may prove a more potent arginine antagonist than canavanine.

At a dietary concentration of 2.5 mM, L-homocanavanine does not disrupt larval growth. At the same time, homocanavanine produces greater growth deformities than that observed with canavanine-treated insects; this factor causes the death of a larger number of pupae. Overall, while chain elongation to produce the higher homolog of canavanine does not significantly alter canavanine’s intrinsic toxicity, it does yield a derivative with equal toxicity toward M. sexta larvae (Rosenthal et al., 1995).

Methods for preparing D-canavanine were also pursued to determine if it exhibits biological activity, and how the activity of this stereoisomeric form compares to that of its naturally occurring antipode. Common wisdom suggests that D-canavanine would not be activated and aminoacylated by arginyl-tRNA synthetase; thus, any adverse biological effects from exposure to the D-enantiomer would not result from the incorporation of canavanine into an insectan protein. Thus, the D-enantiomer provides a means of determining canavanine’s toxicity divorced from aberrant protein formation.

D-Canavanine had little ability to elicit the larval growth inhibition and pupal deformity that are hallmarks of canavanine toxicosis. This finding is important because these adverse effects of canavanine consumption have been attributed to anomalous, canavanyl protein formation (Rosenthal, 1992a; Rosenthal, 1992b; Rosenthal and Dahlman, 1991). On the other hand, D-canavanine is biologically active as it readily induces larval edema. L- canavanine-mediated edema is caused by biochemical processes not requiring protein synthesis (Racioppi et al., 1981).

THUS, WHILE D-CANAVANINE IS ACTIVE BIOLOGICALLY, IT DOES NOT ELICIT THE ANTIMETABOLIC EFFECTS ASSOCIATED WITH ABERRANT PROTEIN FORMATION.

Finally, the 1-methyl and 1-ethyl esters of canavanine were synthesized and tested for their insecticidal potential. These esters possess greater hydrophobicity than the parent compound and may elicit enhanced toxicity as a result of greater penetration of the cellular membrane. Insects are able to deesterify xenobiotics (Yu, 1984; Yu, 1990). Thus, these esters may facilitate canavanine transport and cellular uptake before hydrolysis by intracellular esterases to the parent toxicant. Conversion of L-canavanine to its 1-methyl or 1-ethyl ester produces derivatives with demonstrable toxicity but less than that observed with the parent compound. The ethyl ester is significantly more toxic than the methyl ester as judged by larval growth dynamics (Rosenthal et al., 1995). The enhanced toxicity of the ethyl ester relative to its methyl counterpart suggests that the intrinsic toxicity of these derivatives may be enhanced by increased hydrophobicity. However, the greater steric bulk of the ethyl ester compared to the methyl ester should have produced a compound exhibiting diminished activity with canavanine-utilizing enzymes and consequently should be less toxic. The observed toxicity of the ethyl ester may reflect enhanced uptake into body cells where esterase activity may have produced higher levels of cellular canavanine.

SUMMARY

LITERATURE CITED

Allende, C. C., and Allende, J. E. (1964) Purification and substrate specificity of arginyl-ribonucleic acid synthetase from rat liver. J. Biol. Chem. 239: 1102-1106.

Berge, M. A., Rosenthal, G. A., and Dahlman, D. L. (1986) Tobacco budworm, Heliothis virescens [Noctuidae] resistance to L-canavanine, a protective allelochemical. Pestic. Biochem. Physiol. 25: 319-326.

Bleiler, J., Rosenthal, G. A., and Janzen, D. H. (l988) Biochemical ecology of canavanine-eating seed predators. Ecology 69: 427-433.

Boyar, A., and Marsh, R. E. (1982) L-Canavanine, a paradigm for the structures of substituted guanidines. J. Am. Chem. Soc. 104: 1995-1998.

Dahlman, D. L., and Rosenthal, G. A. (1975) Non-protein amino acid-insect interactions. I. Growth effects and symptomology of L-canavanine consumption by the tobacco hornworm, Manduca sexta (L.). Comp. Biochem. Physiol. 51A : 33-36.

Dahlman, D. L., and Rosenthal, G. A. (1976 ) Further studies on the effect of L-canavanine on the tobacco hornworm, Manduca sexta (L.) (Sphing- idae). . J. Insect Physiol. 22 : 265-271.

Keppi, E., Zachary, D., Robertson, M., Hoffmann, D., and Hoffmann, J. (1986) Induced antibacterial proteins in the hemolymph of Phormia terranovae [Diptera]. Purification and possible origin of one protein. Insect Biochem. 16: 395-402.

Mitra, S. K., and Mehler, A. H. (1967) The arginyl transfer ribonucleic acid synthetase of Escherichia coli. J. Biol. Chem. 242: 5490-5494.

Racioppi, J. V., Dahlman, D. L., and Neukranz, R. K. (1981) The effect of L-canavanine consumption on arginine metabolism in Manduca sexta (Sphingidae; Lepidoptera). Comp. Biochem. Physiol. 70B: 639-642.

Rosenthal, G. A. (1977) The biological effects and mode of action of L-canavanine, a structural analogue of L-arginine. Q. Rev. Biol. 52: 155-178.

Rosenthal, G. A. (1983) The adaptation of a beetle to a poisonous plant. Sci. Amer. 249: 164-171.

Rosenthal, G. A. (1992a). The biochemical basis for the insecticidal properties of L-canavanine, a higher plant protective allelochemical. Insecticides: Mechanisms of Action and Resistance, D. Otto and B. Weber, eds., Intercept Ltd., Andover, England.

Rosenthal, G. A. (1992b). L-Canavanine and chemical defense in higher plants. Frontiers and New Horizons in Amino Acid Research, K. Takai, ed., Elsevier, New York.

Rosenthal, G. A., Berge, M. A., Bleiler, J. A., and Rudd, T. (1987) Avoidance of aberrant protein production and an organism's ability to utilize or tolerate L-canavanine. Experientia 43: 558-561.

Rosenthal, G. A., and Dahlman, D. L. (1991) Studies of L-canavanine incorporation into insectan lysozyme. J. Biol. Chem. 266: 15684-15687.

Rosenthal, G. A., and Harper, L. (1996) L-Homoarginine studies provide insight into the antimetabolic properties of L-canavanine. Insect Biochem. Molec. Biol. in press.

Rosenthal, G. A., Hughes, C. G., and Janzen, D. H. (1982) L-Canavanine, a dietary nitrogen source for the seed predator, Caryedes brasiliensis [Bruchidae]. Science 217: 353-355.

Rosenthal, G. A., Reichhart, J.-M., and Hoffmann, J. A. (1989a) L-Canavanine incorporation into vitellogenin and macromolecular conformation. J. Biol. Chem. 264: 13693-13696.

Rosenthal, G. A., Lambert, J., and Hoffmann, D. (1989b) L-Canavanine incorporation into protein can impair macromolecular function. J. Biol. Chem. 264: 9768-9771.

Rosenthal, G. A., Dahlman, D. L., Crooks, P. A., Na Phuket, S., and Trifonov, L. S. (1995) Insecticidal properties of some derivatives of L-canavanine. J. Food Agr. Chem. 43: 2728-2734.

Yu, S. J. (1984) Detoxication capacity in the honey bee, Apis mellifera L. Pest. Biochem. Physiol. 22: 360-368.

Yu, S. J. (1990) Liquid chromatographic determination of permethrin esterase activity in six phytophagous and entomophagous insects. Pest. Biochem. Physiol. 36: 237-241.