N-acetyldopamine quinone methide/1,2-dehydro-N-acetyl dopamine tautomerase. A new enzyme involved in sclerotization of insect cuticle

The enzyme system causing the side chain desaturation of the sclerotizing precursor, N-acetyldopamine (NADA), was solubilized from the larval cuticle of Sarcophaga bullata and resolved into three components. The first enzyme, phenoloxidase, catalyzed conversion of NADA to NADA quinone and provided it for the second enzyme (NADA quinone isomerase), which makes the highly unstable NADA quinone methide. Quinone methide was hydrated rapidly and nonenzymatically to form N-acetylnorepinephrine. In addition, it also served as the substrate for the last enzyme, quinone methide tautomerase, which converted it to 1,2-dehydro-NADA. Reconstitution of NADA side chain desaturase activity was achieved by mixing the last enzyme fraction with NADA quinone isomerase, obtained from the hemolymph of the same organism, and mushroom tyrosinase. Therefore, NADA side chain desaturation observed in insects is caused by the combined action of three enzymes rather than the action of a single specific NADA desaturase, as previously thought.     

On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

3-O-Hydrosulphato-4-hydroxyphenethylamine (dopamine 3-O-sulphate), a metabolite involved in the sclerotization of insect cuticle

Dopamine 3-O-sulphate (3-O-hydrosulphato-4-hydroxyphenethylamine) was isolated from newly ecdysed cockroaches, Periplaneta americana (L.), and its structure established by chemical and physical techniques and by synthesis. Relatively high concentrations (about 1mumol/g wet. wt.) of dopamine 3-O-sulphate exist in the newly ecdysed insect, and these concentrations decrease sharply as sclerotization of the cuticle proceeds. At least 40% of the radioactivity of (14)C-labelled dopamine 3-O-sulphate injected into newly ecdysed nymphs was recovered in the sclerotized cuticle 7-12 days after the injection. However, less than 1% of the radioactivity of injected dopamine 3-O-[(35)S]sulphate was recovered, and this value was not appreciably different from that for the incorporation of Na(2) (35)SO(4). Apparently, little or none of the sulphate moiety of dopamine 3-O-sulphate is incorporated directly into the cuticle as the intact sulphate ester. These observations are discussed in relation to current concepts of cuticular sclerotization in insects.     

Protein cross-linking by peroxidase: Possible mechanism for sclerotization of insect cuticle

Incubation of test proteins with horseradish peroxidase in the presence of hydrogen peroxide and a catechol resulted in polymerization and precipitation of test proteins. SDS-PAGE readily revealed the generation of dimers, trimers, and higher oligomers in the reaction mixture. With the exception of 3,4-dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three catechols to accomplish polymerization is attributed to their high rate of intramolecular cyclization, which results in melanin formation. Radioactive studies with [³H]N-acetyldopamine clearly reveal both intermolecular and intramolecular cross-linking of test proteins by peroxidase. Based on these studies a possible mechanism for sclerotization and the biological significance of peroxidase in cuticle is discussed.     

An enzyme from locust cuticle involved in the formation of crosslinks from N-acetyldopamine

Locust cuticle is shown to contain an enzyme activating the β-position in the side chain of N-acetyldopamine. When isolated cuticle is incubated with N-acetyldopamine part of the substrate becomes incorporated into the cuticle and part of it forms soluble reaction products, one of which is identified as a dimer of N-acetyldopamine. In the structure suggested for the dimer both phenolic groups of one molecule of N-acetyldopamine are connected to the β-position of another.     

Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster.

The properties of cuticular enzymes involved in sclerotization of Drosophila melanogaster puparium were examined. The cuticle-bound phenoloxidase from the white puparium exhibited a pH optimum of 6.5 in phosphate buffer and oxidized a variety of catecholic substrates such as 4-methylcatechol, N-beta-alanyldopamine, dopa, dopamine, N-acetyldopamine, catechol, norepinephrine, 3,4-dihydroxyphenylglycol, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid. Phenoloxidase inhibitors such as potassium cyanide and sodium fluoride inhibited the enzyme activity drastically, but phenylthiourea showed marginal inhibition only. This result, coupled with the fact that syringaldazine served as the substrate for the insoluble enzyme, confirmed that cuticular phenoloxidase is of the "laccase" type. In addition, we also examined the mode of synthesis of the sclerotizing precursor, 1,2-dehydro-N-acetyldopamine. Our results indicate that this catecholamine derivative is biosynthesized from N-acetyldopamine through the intermediate formation of N-acetyldopamine quinone and N-acetyldopamine quinone methide as established for Sarcophaga bullata [Saul, S. and Sugumaran, M., F.E.B.S. Letters 251, 69-73 (1989)]. Accordingly, successful solubilization and fractionation of cuticular enzymes involved in the introduction of a double bond in the side chain of N-acetyldopamine indicated that they included o-diphenoloxidase, 4-alkyl-o-quinone:p-quinone methide isomerase, and N-acetyldopamine quinone methide:dehydro N-acetyldopamine isomerase and not any side chain desaturase.     

The isolation of ketocatechols from insect cuticle and their possible rôle in sclerotization

Several catechol-derivatives have been isolated from acid hydrolysates of insect hard cuticle, and the two major compounds were identified as a hydroxy-ketocatechol, 2-hydroxy-3′,4′-dihydroxyacetophenone, and the corresponding aldehyde, 3,4-dihydroxyphenylglyoxal. It is shown that the glyoxal is formed from the former compound during hydrolysis.     

Enzymatic processes involved in the incorporation of hydroxycinnamates into grass cell walls

Many plant species have one or more types of acylation of cell wall polymers. Grasses (Poaceae family) are unique with abundant acylation of specific cell wall polymers by hydroxycinnamates. The most common hydroxycinnamates found in a wide range of grasses are ferulates (trans-4-hydroxy-3-methoxycinnamate) and p-coumarates (trans-4-hydroxycinnamate). These two hydroxycinnamates are synthesized by the phenylpropanoid pathway. Though structurally related, they seem to have different functional roles within the cell wall. Ferulates have been shown to have a critical role in cross-linking cell wall components; forming links between structural polysaccharides and links between structural polysaccharides and lignin. They are incorporated into the cell wall by distinctly different mechanisms. Ferulic acid is incorporated into cell walls as ester linked substituents on arabinoxylans. The exact role p-coumarates play in plant cell walls is unknown, but it has been shown that p-coumaric acid is ester-linked to monolignols and shuttled out to the wall to become incorporated into newly forming lignin polymers. Both processes require the activity of specific hydroxycinnamoyl transferases utilizing CoA derivatives to drive the transferase reactions.     

Pupal cuticle proteins of Manduca sexta: characterization and profiles during sclerotization

Proteins in pupal abdominal cuticle of the tobacco hornworm, Manduca sexta, were characterized during the pre-ecdysial and post-ecdysial periods of sclerotization and endocuticle formation. Protein extractability decreased dramatically as the cuticle became sclerotized through 6 h post-ecdysis, but increased rapidly from 9 to 48 h as endocuticular layers were secreted. Nearly 100 proteins that were extracted from pre-ecdysial cuticle became largely insoluble during sclerotization. Three major proteins in this group destined to become exocuticle had apparent molecular masses (Mapp) of 20, 27 and 36 kDa, and were designated MS-PCP20, MS-PCP27, and MS-PCP36. Amino acid analysis revealed glycine to predominate in all three proteins, and alanine, aspartate, glutamate, proline and serine were also relatively abundant. Histidine residues, which provide sites for adduct and cross-link formation with quinone metabolites of N-beta-alanyldopamine during sclerotization of pupal cuticle, ranged from 2 to 3 mol %. N-Terminal amino acid analysis of MSPC-20 and MSPC-36 also revealed some sequence similarities indicating they may be related. An almost entirely new group of proteins appeared by 9 h as endocuticule secretion began, and these increased in abundance through 48 h post-ecdysis. Two of these were major proteins with Mapps of 33 and 34 kDa, and they also had close similarities in their N-terminal amino acid sequences. This study showed that the large number of proteins secreted into the presumptive exocuticle of the pupa before ecdysis are involved in sclerotization reactions and as a consequence become largely insoluble. The epidermis then switches to the secretion of an entirely new group of proteins that are involved in formation of the endocuticle.     

The incorporation of precursors into Drosophila larval cuticle

Incorporation of tritiated leucine, tyrosine and glucosamine into the integument of larval Drosophila melanogaster was followed by electron-microscope autoradiography. Tritiated leucine, tyrosine, and glucosamine were incorporated into the endocuticle by apposition, giving rise to a distinct band of label in the endocuticle at a level which depended on the time between labelling and fixation. The labelled amino acids, but not glucosamine, were also detected in the epicuticle and both above and below the distinct labelled band in the endocuticle. The results indicate that the epicuticle grows within the third instar by intussusception of new materials which are transported from the epidermal cells through the endocuticle to the epicuticle. Breakdown of cuticle which was radioactively labelled by feeding larvae tritiated precursors was also followed by autoradiography. The results indicate that the breakdown products from the old cuticle may be reutilized in the synthesis of new cuticle.     

Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon, but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon, we identified the Drosophila melanogaster gene CG13419 as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-beta (TGF-beta) and glycoprotein hormones. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased bursicon bioactivity. In situ hybridization and immunocytochemistry revealed that bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons also lacked bursicon bioactivity. Our results indicate that CG13419 encodes bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to have a defined function in invertebrates. Mutants show that the spectrum of bursicon actions is broader than formerly demonstrated.     

黄粉虫幼虫体壁硬化过程中酚氧化酶活性的变化

为研究酚氧化酶(PO)在昆虫蜕皮过程中的功能和作用, 采用微量测定法研究了黄粉虫Tenebrio molitor体壁硬化过程中血淋巴和表皮中的PO活性变化。结果表明：初蜕皮幼虫血淋巴中PO活性较高, 但随着体壁的不断黑化与硬化, 其活性呈现下降趋势, 在3～4 h内达到最低点, 而后PO活性逐渐上升, 7 h左右活性上升至最高, 并接近于正常幼虫的水平;在刚蜕完皮后的1 h内, 体壁中 PO活性基本无变化, 但随后即开始下降, 3 h左右降到最低点, 然后开始回升, 6~7 h左右恢复到正常水平, 并趋于稳定;以L-DOPA为底物, 通过双倒数曲线作图法求得黄粉虫血淋巴PO的Km＝1.176 mmol/L, 体壁PO的Km＝0.881 mmol/L, 表明体壁PO与底物L-DOPA的亲和力要高于血淋巴PO。研究表明两种来源的酚氧化酶均参与了黄粉虫幼虫的体壁硬化过程, 但在作用方式及与底物的亲和力方面存在差异。     

Autolysis of cell walls in pea epicotyls during growth. Enzymatic activities involved

Pisum sativum L. (cv. Lincoln) epicotyl cell walls show autohydrolysis and release into the incubation medium up to 120 μg of sugar per mg of cell wall dry weight in 30 h. Cell walls from younger epicotyls with high growth capacity showed higher auto-lytic capacity than older epicotyls. This suggests that both processes, growth and au-tolysis, are related. The proteins responsible for autolysis were extracted from the wall fraction with high saline solution (3 M LiCl) and enzymatic activities associated with the proteins were studied. The highest activity corresponded to α-galactosidase; lower activities were found for β-galactosidase, a-arabinosidase and exoglucanase. Changes in enzymatic activities and changes in the proportion of sugars released in autolysis by cell walls during the growth of epicotyls support the notion that α-galac-tosidase is one of the enzymes involved in the process of autolysis, and that the liberation of arabinose and galactose in this process occurs as arabinogalactan.     

Model reactions for insect cuticle sclerotization: cross-linking of recombinant cuticular proteins upon their laccase-catalyzed oxidative conjugation with catechols

The quinone-tanning hypothesis for insect cuticle sclerotization proposes that N-acylcatecholamines are oxidized by a phenoloxidase to quinones and quinone methides, which serve as electrophilic cross-linking agents to form covalent cross-links between cuticular proteins. We investigated model reactions for protein cross-linking that occurs during insect cuticle sclerotization using recombinant pupal cuticular proteins from the tobacco hornworm, Manduca sexta, fungal or recombinant hornworm laccase-type phenoloxidase, and the cross-linking agent precursor N-acylcatecholamines, N-beta-alanydopamine (NBAD) or N-acetyldopamine (NADA). Recombinant M. sexta pupal cuticular proteins MsCP36, MsCP20, and MsCP27 were expressed and purified to near homogeneity. Polyclonal antisera to these recombinant proteins recognized the native proteins in crude pharate brown-colored pupal cuticle homogenates. Furthermore, antisera to MsCP36, which contains a type-1 Rebers and Riddiford (RR-1) consensus sequence, also recognized an immunoreactive protein in homogenates of larval head capsule exuviae, indicating the presence of an RR-1 cuticular protein in a very hard, sclerotized and nonpigmented cuticle. All three of the proteins formed small and large oligomers stable to boiling SDS treatment under reducing conditions after reaction with laccase and the N-acylcatecholamines. The optimal reaction conditions for MsCP36 polymerization were 0.3mM MsCP36, 7.4mM NBAD and 1.0U/mul fungal laccase. Approximately 5-10% of the monomer reacted to yield insoluble oligomers and polymers during the reaction, and the monomer also became increasingly insoluble in SDS solution after reaction with the oxidized NBAD. When NADA was used instead of NBAD, less oligomer formation occurred, and most of the protein remained soluble. Radiolabeled NADA became covalently bound to the MsCP36 monomer and oligomers during cross-linking. Recombinant Manduca laccase (MsLac2) also catalyzed the polymerization of MsCP36. These results support the hypothesis that during sclerotization, insect cuticular proteins are oxidatively conjugated with catechols, a posttranslational process termed catecholation, and then become cross-linked, forming oligomers and subsequently polymers.     

Enzymatic incorporation into oligonucleotides of modified nucleosides

Behaviour of modified nucleosides, tRNA components, and their analogues has been studied in the internucleotide bond formation catalysed by ribonucleases of various substrate specificity, polynucleotide phosphorylases, and T4 RNA ligase and the results are summarised in this paper. Pseudouridine, dihydrouridine, ribothymidine, 5-methylcytidine, inosine, and 6-methyladenosine can participate in the reaction of internucleotide bond formation the presence of most ribonucleases used, viz. Pb2, Pcl2, Pb1, Pch1, C2, T1, pancreatic RNase. 3-Methylcytidine and 4-acetylcytidine form internucleotide bond (as phosphate acceptors) usually by means of guanyl-specific ribonucleases, whereas 1-methylandenosine is incorporated with ribonuclease Pel2. 7-Methylguanosine and 1-methylguynosine 2',3'-cyclophosphates can be used as phosphate donors in the presence of ribonuclease Pb2; in the similar enzymatic reaction 6-isopentenyladenosine is an uneffective acceptor.     

Enzymic activities involved in the oothecal sclerotization of the praying mantid,Tenodera aridifolia sinensis saussure

The enzyme(s) responsible for the sclerotization of mantid ootheca is secreted by the left colleterial gland. From an extract of the glands of Tenodera aridifolia sinensis, two soluble enzyme fractions of different activities were obtained. One fraction acted on N-acetyldopamine (NADA), a precursor of a representative sclerotizing agent, and produced NADA-quinone. The other did not act on NADA itself but converted the quinone to a highly reactive intermediate, such as quinone methide, which was able to react nonenzymically with nucleophilic compounds. Other insoluble enzyme preparations obtained from the silk and pupal cuticle of the Japanese giant silk moth, Dictyoploca japonica, also had these two activities.     

Catechols involved in sclerotization of cuticle and egg pods of the grasshopper,Melanoplus sanguinipes,and their interactions with cuticular proteins

N‐Acetyldopamine (NADA) is the major catechol in the hemolymph of nymphal and adult grasshoppers, Melanoplus sanguinipes (F.), and mainly occurs as an acid‐labile conjugate indicated to be a sulfate ester. Its concentration increases in last instar nymphs and peaks during adult cuticle sclerotization. Dopamine (DA), the precursor of NADA and melanic pigments, is about 10 times lower in concentration than NADA, but shows a similar pattern of accumulation. NADA also predominates in cuticle, but its concentration is lowest during the active period of sclerotization, reflecting its role as a precursor for quinonoid tanning agents. Two other catechols, 3,4‐dihydroxybenzoic acid (DOBA) and 3,4‐dihydroxyphenylethanol (DOPET), also occur in hemolymph and cuticle, and their profiles suggest a role in cuticle stabilization. Solid‐state NMR analysis of sclerotized grasshopper cuticle (fifth instar exuviae) estimated the relative abundances of organic components to be 59% protein, 33% chitin, 6% catechols, and 2% lipid. About 99% of the catechols are covalently bound in the cuticle, and therefore are involved in sclerotization of the protein‐chitin matrix. To determine the types of catechol covalent interactions in the exocuticle, samples of powdered exuviae were heated in Hcl under different hydrolytic conditions to release adducts and cross‐linked products. 3,4‐Dihydroxyphenylketoethanol (DOPKET) and 3,4‐dihydroxyphenylketoethylamine (arterenone) are the major hydrolysis products in weak and strong acid, respectively, and primarily represent NADA oligomers that apparently serve as cross‐links and filler material in sclerotized cuticle. Intermediate amounts of norepinephrine (NE) are released, which represent N‐acetylnorepinephrine (NANE), a hydrolysis product of NADA bonded by the b‐carbon to cuticular proteins and possibly chitin. Small quantities of histidyl‐DA and histidyl‐DOPET ring and side‐chain C‐N adducts are released by strong acid hydrolysis. Therefore, grasshopper cuticle appears to be sclerotized by both o‐quinones and p‐quinone methides of NADA and dehydro‐NADA, which results in a variety of C‐O and C‐N covalent bonds linked primarily through the side‐chain carbons of the catechol moiety to amino acid residues in cuticular proteins. The primary catechol extracted from both the female accessory glands/calyx and the proteinaceous frothy material of the egg pod is DOBA, which also commonly occurs in cockroach accessory glands and oothecae, presumably as a tanning agent precursor. 3,4‐Dihydroxyphenylalanine (DOPA) was also detected in extracts of the accessory glands/calyx of grasshoppers, and may serve as a precursor for DOBA synthesis. Arch. Insect Biochem. Physiol. 40:119–128, 1999. © 1999 Wiley‐Liss, Inc.