Theodore L. Hopkins

Oxidative conjugation of catechols with proteins in insect skeletal systems

Abstract Cuticle sclerotization or tanning is a vital process that occurs during each stage of insect development to harden and stabilize the newly secreted exoskeleton. The structural polymers protein and chitin make up the bulk of the cuticle, and chemical interactions between these biopolymers with quinonoid tanning agents are largely responsible for the physical properties of the mature exoskeleton. The oxidative conjugation of catechols with cuticular proteins plays an important role in this metabolism. The main hypothesis for cuticle sclerotization involves the formation of adducts and cross-links between nucleophilic imidazole nitrogens of histidyl residues in the proteins and electrophilic ring or side-chain carbons of ortho-quinones and para-quinone methides derived from the catechols, N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenylethanol. C–N and C–O linkages between these quinone tanning agents and proteins in cuticles from a variety of insects from several orders have been elucidated. cDNAs for both the tyrosinase and laccase types of phenoloxidases that catalyze the cross-linking reactions have been isolated and sequenced. The sequences of laccase cDNAs from two insect species were more similar to fungal laccases than to those from plants. These results provide insights into how insects use structural proteins, catechols, and oxidative enzymes to form catechol–amino acid adducts during sclerotization.

N-β-Alanyldopamine: Major Role in Insect Cuticle Tanning

N-β-Alanyldopamine is the major tyrosine metabolite in the hemolymph and cuticle during pupal tanning in the tobacco hornworm, Manduca sexta L. Its concentration in hemolymph increases over 800-fold above larval levels by the start of tanning and decreases as the pupal cuticle darkens and hardens. It is a major catechol in species representing several insect orders and is the preferred substrate for pupal cuticular o-diphenol oxidase. In insects, N-β-alanyldopamine appears to be the main precursor for tanning chemicals at certain developmental stages.

Catecholamines in haemolymph and cuticle during larval, pupal and adult development of Manduca sexta (L.)

Catecholamines in haemolymph and cuticle of the tobacco hornworm, Manduca sexta (L.), were analyzed by liquid chromatography with electrochemical detection. During pupal tanning, N-β-alanyldopamine (NBAD) was the major dopamine derivative in haemolymph. It increased to maximal titres by the time of larval-pupal ecdysis, and subsequently decreased as pupal cuticle tanned. NBAD progressively increased in cuticle for several days after ecdysis. N-acetyldopamine (NADA), although present in only minor amounts in pupae, was the primary catecholamine in haemolymph of both larvae and adults shortly before and after ecdysis. NADA predominated in the hard colourless cuticle of the larval head capsule, and in the wings and abdominal tergites of the adult. NBAD, however, was predominant in the dark cuticle of larval mandibles, and in the thin soft larval abdominal cuticle after stabilization. Dopamine, the precursor of NBAD and NADA, was usually the second or third most abundant catecholamine in both haemolymph and cuticle. The kinds of sclerotins produced may depend in part upon the relative abundance of the different catecholamines which are selectively incorporated into the various regions of the exoskeleton. The catecholamines in haemolymph occurred mainly as ring hydroxyl conjugates that were probably β-glucosides. The relative percentage of free or unconjugated compounds increased after ecdysis and accumulated in cuticle. Conjugation, therefore, appears to be important for sequestration of tanning precursors in haemolymph, followed by hydrolysis and uptake of free catecholamines for stabilization and pigmentation of new cuticle.

Characterization of haemolymph protyrosinase and a cuticular activator from Manduca sexta (L.)

Abstract A protyrosinase has been isolated from fifth instar larval haemolymph of the tobacco hornworm, Manduca sexta (L.) by ammonium sulphate fractionation, hydroxylapatite chromatography and gel filtration. It exhibited a single band after polyacrylamide gel electrophoresis at pH 8.5 and two bands in the presence of sodium dodecyl sulphate with apparent molecular weights of 7.7 × 10 4 and 7.1 × 10 4 . The proenzyme is a metalloprotein containing 0.18% copper. The activating enzyme was partially purified from pharate pupal cuticle by ammonium sulphate precipitation and hydroxylapatite chromatography. The activation was inhibited by di iso propylphosphorofluoridate and had a pH optimum of 8.8. Chymotrypsin also activated the proenzyme. The cuticular activator is probably a serine protease. Activated protyrosinase exhibited physical, chemical and kinetic properties very similar to those of tyrosinase extracted from pharate pupal cuticle. Haemolymph protyrosinase may serve as a precursor for both haemolymph and cuticular tyrosinases that synthesize catecholamines and quinones used for wound healing, parasite encapsulation as well as for cuticular stabilization and pigmentation.

Applications of Solids NMR to the Analysis of Insect Sclerotized Structures

Abstract This article reviews the solids NMR research conducted on insect sclerotized structures in the last 10 years and previews some of the experiments that will be conducted in the future. Solids NMR has been used as a noninvasive approach to investigate the chemical compositions of, and some covalent interactions that occur in, several types of sclerotized structures that are otherwise highly intractable to conventional chemical analyses. Sclerotization is a complex process used by insects to confer stability and mechanical versatility to their cuticular exoskeletons and certain other proteinaceous structures. Samples analyzed include cuticular exoskeletons, egg cases, egg shells, cocoons and peritrophic membranes. Cross polarization, dipolar decoupling, magic angle spinning, magnetization dephasing, and isotropic enrichment were used to obtain high resolution spectra that provide information about the types and relative concentrations of carbon atoms as well as internuclear distances and covalent bonds between carbon and nitrogen atoms. Relative amounts of protein, chitin, catechols, lipids, pigment, and oxalate were estimated. Covalent interactions between protein nitrogens and catechol carbons were detected in the stiff brown pupal cuticle of the tobacco hornworm, Manduca sexta . The results of these solids NMR studies support the hypothesis that sclerotization of insect structures occurs primarily when quinones derived from N -acylcatecholamines form cross-links and adducts with functional groups of proteins deposited in the structures. Future applications of solids NMR will utilize advanced techniques for further probing the covalent interactions of 13 C, 15 N and 17 O-labeled catechols, chitin and protein in sclerotized structures.

Roles of dopa decarboxylase and phenoloxidase in the melanization of the tobacco hornworm and their control by 20-hydroxyecdysone

SummaryWhenManduca sexta larvae are allatectomized 5 h before head capsule slippage (HCS) in the final larval molt, the new larval cuticle contains granules that melanize 3 h before ecdysis when the ecdysteroid titer falls (Curtis et al. 1984). In both the epidermis and hemolymph of these allatectomized larvae dopamine was higher than dopa prior to and at the time of melanization. Dopamine also increased in the new cuticle as melanization began. Dopa decarboxylase (DDC) activity increased in the epidermis, cuticle, and fat body beginning 16 h after HCS, with a two-fold greater increase in the epidermis of allatectomized larvae. Both α-MDH and α-fluoromethyl-dopa inhibited epidermal DDC activity and inhibited melanization in vitro when dopa was used as a precursor. Addition of dopamine to the medium allowed melanization in the presence of the inhibitors. All these results indicate that dopamine is likely the primary precursor of cuticular melanin. The diphenoloxidase in the premelanin granules was activated in vivo between 19 and 21 h after HCS and was found to prefer dopamine to dopa and not to convert tyrosine to melanin. The activation of the prophenoloxidase was inhibited by 20-hydroxyecdysone (20-HE), both in vivo and in vitro, if hormone was given by 16 h after HCS. Infusion of 1.2 μg/ml 20-HE into allatectomized larvae for 24 h from HCS prevented both the increase in DDC activity and the activation of the premelanin granules. Although the larvae ecdysed after a 15 h delay, melanization never occurred.

A trypsin-solubilized laccase from pharate pupal integument of the tobacco hornworm, Manduca sexta☆

A laccase-like enzyme was solubilized from Manduca sexta pharate pupal integument by trypsin treatment and partially purified by ammonium sulfate precipitation, gel filtration, ultracentrifugation and anion exchange high performance liquid chromatography (HPLC). The molecular weight of the laccase as determined by gel filtration HPLC was 250 ± 30 kDa. The most purified preparation contained a single oxidase with typical laccase activity as determined by substrate and inhibitor specificities, enzyme kinetics, and a single band of enzyme activity on nondenaturing polyacrylamide gels with syringaldazine (SYR) and 3,4-dihydroxyphenylalanine (DOPA) substrates. The specific activity of the most purified enzyme preparation was 18.0 μmol of quinone and 1.1 μmol of ϱ-quinoneimine formed min−1 mg−1 from 2-methylhydroquinone (MHQ) and DOPA, respectively, at pH 6. The pH optimum of the laccase was 6 for MHQ and about 7 for N-acetyldopamine. Incubation of the M. sexta laccase with selective laccase and tyrosinase inhibitors demonstrated that the enzyme is generally inhibited like fungal laccase with p-diphenolic substrates and like mushroom tyrosinase with o-diphenolic substrates. Manduca laccase affinity for MHQ was 400-fold greater than that for DOPA and the catalytic efficiency (VmaxKm) for oxidation of the p-diphenol was 2000-fold higher than that for the o-diphenol. Topical application of SYR to pharate pupal forewing cuticle revealed laccase activity in situ. The natural tanning precursor, N-β-alanyldopamine (NBAD), was the best o-diphenolic substrate tested. N-β-Alanylnorepinephrine was also formed at a high rate when NBAD was incubated with M. sexta laccase, but at a low rate with fungal laccase, suggesting specificity for side chain oxidation through a p-quinone methide intermediate. The results indicate that M. sexta laccase is important for oxidizing cuticular o-diphenols to o-quinone and p-quinone methide tanning agents for sclerotization of the M. sexta pupal exoskeleton.

Effect of wheat germ agglutinin on formation and structure of the peritrophic membrane in european corn borer (Ostrinia nubilalis) larvae

European corn borer (ECB; Ostrinia nubilalis (Hubner)) larvae (third instar) fed 0.05% w/w wheat germ agglutinin (WGA) in their diet for 72 h showed very little increase in body weight, whereas weight of control larvae increased nearly fourfold. Light and transmission electron microscopy studies showed that the morphology of the peritrophic membrane (PM) changed within 24 h after ECB larvae fed on the WGA diet. Whereas the PM in the anterior region of the midgut was a thin membranous structure in control larvae, the WGA-fed larvae secreted a multiple-layered and unorganized PM that contained embedded food particles, bacteria, and pieces of disintegrated microvilli. Gold-labeled WGA was localized specifically in the PM and microvilli. The PM of WGA-fed larvae was inundated with dark-staining amorphous structures that, when incubated with anti-WGA, showed heavy WGA localization. The antibody label indicated that most of the ingested WGA was found in the PM, with lesser amounts on the microvillar surface and the least amount within the epithelium. After 72 h, the middle portion of the mesenteron revealed a thin, compact PM in the control larvae, whereas the PM of the WGA-fed larvae was multilayered and discontinuous, which allowed plant cell-wall fragments to penetrate into the microvilli of the epithelium. Scanning electron microscopy of PMs from fifth instar ECB larvae fed the WGA diet revealed a breakdown in the chitinous meshwork by 48 h after initiation of feeding. The endo-PM surface from control larvae was smooth and intact, whereas the PM of WGA-fed larvae showed disintegration of the meshwork and a reduced proteinaceous matrix. This allowed bacteria and food particles to penetrate through the PM into the ectoperitrophic space and directly contact the microvilli. Therefore, WGA, a protein inhibitor of larval growth, interferes with the formation and integrity of the PM, which exposes the brush border to ingested material. This, in turn, appears to stimulate secretion of additional PM layers, the concomitant disintegration of the microvilli, and cessation of feeding.

N-β-alanylnorepinephrine: Biosynthesis in insect cuticle and possible role in sclerotization

N-β-alanylnorepinephrine (NBANE), a metabolite of N-β-alanyldopamine (NBAD) in pupal cuticle of the tobacco hornworm, Manduca sexta, was identified by analysis of hydrolytic products, by comparison of HPLC retention times with a synthetic standard and by fast atom bombardment mass spectrometry. NBANE and NBAD were abundant in the brown cuticle of M. sexta pupae, Periplaneta americana adults, Tribolium castaneum adults and Musca domestica puparia. Side chain modification of NBAD was rapid during the first few hours of sclerotization as evidenced by a dramatic increase in NBANE extracted from M. sexta pupal cuticle with cold 1.2 M HCl. In later stages of sclerotization less NBANE was recovered indicating further metabolism and possible involvement in the formation of crosslinks to cuticular components. Although similar amounts of the two catecholamines were extracted by 80% methanol from pupal cuticle which was just starting to tan, over 15-fold more NBANE than NBAD was recovered with cold dilute HCl. Low pH may be necessary to remove physically entrapped NBANE from the insoluble polymeric matrix of the cuticle, or NBANE released by the acid treatment may be a hydrolytic product from NBAD covalently linked by its β-carbon to cuticular components. NBANE was synthesized from NBAD by an insoluble enzyme in tanning pupal cuticle that also catalyzed to a lesser extent the β-hydroxylation of N-acetyldopamine (NADA). However, NADA was a better precursor than NBAD for compounds that yield 3,4-dihydroxphenylketoethanol after heating in 1 M HCl. Therefore, two pathways were observed in tanning cuticle for side chain metabolism of the N-acylated dopamines, and the type of acyl group influenced the relative amounts of different sclerotizing agents produced.

Properties of tyrosinase and dopa quinone imine conversion factor from pharate pupal cuticle of Manduca sexta L.

Tyrosinase was partially purified from tobacco hornworm [Manduca sexta (L.)] pharate pupal cuticle by ammonium sulphate precipitation and anion-exchange chromatography. The enzyme hydroxylated tyrosine and tyramine to dihydroxyphenylalanine (DOPA) and dopamine respectively, and also oxidized DOPA, dopamine and other catecholamines including Nacetyldopamine (NADA) and N-β-alanyldopamine (NBAD) to o-quinone and p-quinone imine products. 5,6-Dihydroxyindole (DHI) was also dehydrogenated by tyrosinase to indole quinone. The order of substrate preference was NBAD > NADA > dopamine > DHI > DOPA. Maximum activity occurred at pH6. Catalytic amounts of DOPA stimulated the hydroxylation of tyrosine and tyramine. Tyrosinase inhibitors were diethyl-dithiocarbamate, phenylthiourea, cyanide and benzoic acid. DOPA quinone imine conversion factor (QICF), a heat-labile and protease-susceptible substance that accelerates the decarboxylation of DOPA quinone imine to DHI, was partially purified from extracts of pharate pupal cuticle. Tyrosinase and QICF may play critical roles in generating catecholamine metabolites required for the sclerotization and melanization of insect cuticle.