The cuticular proteins of Tenebrio molitor: II. Patterns of synthesis during postembryonic development

The program of synthesis for the soluble cuticular proteins of Tenebrio molitor was determined by following the incorporation of labeled leucine after a 4-hr pulse in vivo. Soluble proteins were extracted from labeled cuticles and separated on SDS-polyacrylamide slab gels; individual gel slices were counted. The synthetic patterns of larvae and pupae were similar to one another but distinct from the adult pattern. At each stage, the preecdysial pattern was unlike that of postecdysial animals. Distinct periods of synthesis were detected for different proteins. One protein was synthesized and deposited throughout cuticle formation in all three metamorphic stages. One group was synthesized only after ecdysis, while synthesis and secretion of other proteins were restricted to the preecdysial period. Some cuticular proteins never acquired detectable label.     

Analysis of the cuticular proteins of Hyalophora cecropia with polyclonal antibodies

The cuticular proteins from different anatomical regions and metamorphic stages of Hyalophora cecropia were analyzed with polyclonal antibodies raised against cuticular protein extracts from each stage. Western blots of 2D gels coupled with detection of antibody-antigen binding with avidin-biotinylated-horseradish peroxidase complexes (ABC method) proved to be extremely sensitive. Reactions of polyclonal antisera with blots of extracts of different cuticular regions revealed the following: (1) glycosylated cuticular proteins were highly antigenic; (2) there was less cross-reaction between rigid and flexible cuticles from the same metamorphic stage than among cuticles with similar mechanical properties from different stages; (3) proteins with identical molecular weights and isoelectric points were antigenically indistinguishable.     

Spatial and temporal variations in cuticle proteins as revealed by monoclonal antibodies. Immunoblotting analysis and ultrastructural immunolocalization in a beetle, Tenebrio molitor

A library of monoclonal antibodies (Mabs) against adult cuticle of Tenebrio was used to visualize the secretion of cuticular antigens during metamorphosis. Immunoblots of water- and urea-soluble proteins, and high resolution immunogold labelling has shown that, except in one clone, the Mabs recognize antigens in the three developmental stages. However, the MW of larval and pupal antigens are different from the adult ones, though sharing common epitopes. Blots of cuticle proteins (CPs) bound to different lectins shown few water-soluble glycosylated proteins weakly or not recognized by the Mabs, suggesting that the majority of the Mabs do not recognize glycosylated epitopes. The immunolocalization of the different antigens suggests a molecular basis for both developmental and regional variations in cuticular architecture and to the modifications due to sclerotization, which differ between pre- and postecdysial cuticles of the three developmental stages.     

Partial amino acid sequences of cuticular proteins from Hyalophora cecropia

The amino-terminal amino acid sequences for seven cuticular proteins from Hyalophora cecropia are reported. Proteins were purified by blotting two dimensional acrylamide gels onto acid-etched glass fiber filters, and the proteins were sequenced without further elution. The sequences of the serine-rich proteins from rigid cuticles revealed a new family of cuticular proteins, with features reminiscent of the amino-termini of certain vertebrate neurofilament proteins, members of the intermediate filament protein family which includes keratins. The proteins from flexible cuticles showed sequence similarity to proteins previously sequenced for Drosophila, Manduca and Sarcophaga. Proteins with identical electrophoretic mobility from two different metamorphic stages or from two anatomical regions within a single stage had identical amino-terminal sequences.     

Enzymatic activities involved in incorporation of N-acetyldopamine into insect cuticle during sclerotization

During sclerotization of insect cuticle, N-acetyldopamine (NADA) is enzymatically oxidized before reaction with cuticular proteins. Not all oxidized NADA reacts with cuticular structural materials, a small fraction reacts with water or other available low molecular weight compounds to give soluble products. Various types of cuticle were incubated with excess NADA and the products studied by reversed phase high performance liquid chromatography (RP-HPLC) to obtain information on the enzymatic activities in the cuticle. The occurrence of at least two enzymes competing for NADA and present in different proportions in the various types of cuticle can explain the results. NADA may be incorporated into cuticle via α,β-dehydro-NADA (β-sclerotization) or via quinone methides and o-quinones, and the actual course of sclerotization will depend upon the relative activities of the enzymes involved. The various pathways may all be used simultaneously.     

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.     

Analysis of the cuticular proteins of Hyalophora cecropia with two dimensional electrophoresis

The soluble cuticular proteins of defined anatomical regions from different metamorphic stages of the giant silkmoth, Hyalophora cecropia, were characterized by two dimensional electrophoresis. As urea concentrations in 2D gels were increased, some of the cuticular proteins from the larval dorsal abdomen decreased in mobility relative to the molecular weight standards. This decrease was also influenced by the pH and ionic strength of the resolving gel. Clustering of proteins into groups, whose members showed similar behavior under different electrophoretic conditions, was indicative of membership in multigene families. By such criteria, common families were found in cuticles with similar mechanical properties from different metamorphic stages, yet there was evidence that different members of a single family were independently regulated.     

In vitro translation of epidermal RNA from different anatomical regions and metamorphic stages of Hyalophora cecropia

Epidermal RNA isolated from different anatomical regions and metamorphic stages of Hyalophora cecropia was translated in vitro with commercial wheat germ and rabbit reticulocyte systems. The translation products were analyzed by 2D gel electrophoresis. The two systems yielded identical products if canine microsomal membranes were added to remove signal peptides from the reticulocyte products. The endogenous processing by the wheat germ extract occurred even in the presence of protease inhibitors. Some of the processed translation products co-migrated with unlabeled cuticular protein standards. All of the major cuticular proteins could be identified, but only when translations were carried out with RNA from epidermis underlying cuticle containing these proteins. Hence, cuticular protein distribution is due to differential synthesis and not to differential extractability. For larval abdominal RNA, most of the major translation products did not co-migrate with known larval cuticular proteins or with proteins synthesized and retained by the epidermis. These unknown products were lower in apparent molecular weight than the cuticular proteins. Their identity remains unknown; they may be premature translation products, but altering translation conditions did not reduce their abundance.     

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.     

DOPA and tyrosine decarboxylase activity in tissues of Periplanet a americana in relation to cuticle formation and ecdysis

DOPA decarboxylase activity in haemolymph and integument was low in last instar and early pharate adult Periplaneta americana, but began to increase shortly before ecdysis. Decarboxylation rates of l-DOPA, about 10 times the larval level by the start of ecdysis, reached a peak about 6 hr afterward, coinciding with the main period of cuticular sclerotization. Activity decreased rapidly during the next 18 hr, then decreased gradually for several days. Haemolymph DOPA decarboxylase activity was about four times greater than the integument, based on tissue dry weights. The fat body and gut tissues had low DOPA decarboxylase activity in all ages tested, and this did not increase at ecdysis. Tyrosine decarboxylase activity was significant only in the haemolymph and at consistently low levels.DOPA decarboxylase, therefore, apparently plays a major rôle in production of catecholamine derivatives for cuticular sclerotization in P. americana, while tyrosine decarboxylation is minor. Both haemolymph and integument appear to be important sites of dopamine biosynthesis.     

Larval cuticle proteins of Lucilia cuprina: Electrophoretic separation,quantification and developmental changes

Proteins from isolated cuticles of third instar larvae of the sheep blowfly, Lucilia cuprina, have been solubilized with water or 7 M urea or 2% SDS. While 7 M urea or 2% SDS extract significantly more protein than water, the same major proteins, in the same relative proportions, are extracted by all three solutions. More than 80% of the cuticular protein is extracted by 7 M urea or 2% SDS. Extracted proteins resolve into nine major bands when analysed by gradient polyacrylamide gel electrophoresis. These proteins are anionic, relatively low in molecular weight (13–28 kd) and are essentially free of carbohydrate. Only minor differences exist between the proteins of two morphologically distinct cuticular regions. Cuticle proteins, extracted from larvae at different developmental stages (first, second and third instars) display quantitatively and qualitatively unique electrophoretic profiles. A number of proteins are common to all stages however. The electrophoretic profiles of proteins extracted from larval cuticles at various times within an instar also differ although the differences are largely quantitative. This is particularly evident during the transition from the feeding to the wandering stages of the third instar; the weight of the cuticle relative to that of the larva increases and this is accompanied by marked changes in the electrophoretic profile of the cuticle proteins.     

Changes in translatable mRNAs during the larval-pupal transformation of the epidermis of the tobacco hornworm

At the initiation of metamorphosis when exposed to ecdysteroid in the absence of juvenile hormone (JH), the lepidopteran epidermis changes its commitment from one for larval differentiation to one for pupal differentiation. Changes in mRNA populations during this change both in vivo and in vitro were followed by a one-dimensional SDS-gel electrophoretic analysis of translation products made in a mRNA-dependent rabbit reticulocyte lysate system. The larval epidermal cell was found to lose its translatable mRNAs for larval cuticular proteins and the larval-specific pigment insecticyanin during the change in commitment; these never reappeared. For Class I cuticular proteins and for insecticyanin, this loss occurred during the exposure to ecdysteroid, each with a differing time course. By contrast, Class II cuticular mRNAs first increased during this time, then also disappeared by the time the cells were pupally committed. In vitro these mRNAs appeared in only trace amounts in response to 20-hydroxyecdysone (20-HE). The pupally committed cell (late in the wandering stage) contained mRNAs for three low-molecular-weight proteins which were precipitable with the pupal cuticular antiserum. The remainder of the pupal cuticular mRNAs were not translatable until the third day after wandering, a time when pupal cuticle is being deposited in response to a molting surge of ecdysteroid. The pupally committed cell also had at least one new noncuticular mRNA which coded for a 34K protein and which was absent from both larval and pupal epidermal cells making cuticle. Since its appearance in response to 20-HE in vitro is repressed by JH, it is called a pupal commitment-specific protein. Thus, during the change of commitment 20-HE inactivates larval-specific genes irreversibly in a sequential cascade of events. The activation of most pupal-specific genes then requires a subsequent exposure to more ecdysteroid.     

Action in vivo d'ecdysones sur la morphogénèse imaginale d'Aeshna cyanea (Odonata)

Single amounts of α or β ecdysone were injected during the last larval instar of Aeshna cyanea at various times after ecdysis. In these experimental conditions, α and β ecdysone had similar effects. Very large amounts of brown or black cuticle appeared on the tarsal claws soon after hormone injection, so that the cuticular synthesis of the larvae which were injected at the beginning of the last stage appears about two or three times more quickly than in controls. Nearly all the larval characters were exhibited by animals injected on the day of or the day after the last larval ecdysis. If the hormonal injection was further delayed, only adultoid forms were obtained. No perfect adults appeared. The effects evoked by α or β ecdysone may be different from one organ to another.On the other hand, some results were different according to the type of ecdysone. Darkening of the tarsal claws (and perhaps sclerotization) appears sooner when β ecdysone is supplied. The morphology of the external organs which degenerate during metamorphosis is not always the same after injection of equal amounts of α or β ecdysone at the same time. The regression of the larval organs seems to be more explicit and appears sooner when β ecdysone was administrated. The morphogenesis of the organs which grow during metamorphosis was either weaker or non-existent with β ecdysone.These results are discussed with regard to previous work.     

Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine epsilon-amino groups during the first 24h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in beta-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the epsilon-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free beta-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no beta-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the beta-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its epsilon-amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101-108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229-237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392], but the lysine-dihydroxyacetophenone compound and the histidine-DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.     

Cuticular strength and pigmentation of rust-red and black strains of Tribolium castaneum: Correlation with catecholamine and β-alanine content

Rust-red wild and black mutant strains of the red flour beetle, Tribolium castaneum, were used to investigate temporal patterns of catecholamine and β-alanine content during sclerotization and pigmentation of adult cuticle and to relate these patterns to corresponding changes in cuticle resistance to puncture. Rust-red elytral cuticle sclerotized more rapidly than black cuticle until 6 days after adult eclosion when both became equal in puncture resistance. The cuticular concentrations of $\text{N-β-}\text{alanyldopamine}$ (NBAD), β-alanine and 3,4-dihydroxyphenylacetic acid (DOPAC) increased more rapidly in the rust-red strain than in the black strain during the first 7 days following adult eclosion. Conversely, cuticular dopamine increased more rapidly in black than in the red strain. Thus the rust-red pigmentation and rapid sclerotization appear to be related to the availability of β-alanine, $\text{N-β-}\text{alanyldopamine}$ and DOPAC. Melanization was prevented and rust-red pigmentation induced by injections of β-alanine or NBAD into newly ecdysed black mutant beetles. Crosses of the two strains generally had intermediate levels of cuticular dopamine and β-alanine, but the NBAD levels were similar to those of the rust-red strain. Dopamine, NBAD and DOPAC levels became similar in both black and rust-red strains about 6 days after adult ecdysis as did resistance to puncture. Therefore, dopamine appears to be directed initially into the melanin pathway in black adults due to a temporary lack of $\text{N-}\text{acylation}$ with β-alanine. After the melanization phase, dopamine is metabolized to sclerotization precursors eventually resulting in normal physical properties of the exoskeleton.     

Developmental changes in cuticular proteins of Ascaris suum

1. Cuticles were isolated from developmental stages of the swine nematode Ascaris suum by a combination of mechanical disruption and detergent treatment of larvae or by surgical removal of cuticle from adults. Proteins from the isolated cuticles were solubilized with 2-mercaptoethanol (2ME) and analyzed by SDS-PAGE. 2. 2ME soluble, cuticular proteins from adults consisted of 5 to 6 bands with 80% of proteins in 2 bands with mol. wts of 106,000 and 93,000. Cuticular proteins from the third and fourth larval stages (L3 and L4) were comparable to adult, but differences in the number of bands were observed. The soluble proteins from the adult, L3 and L4 were readily degraded by a bacterial collagenase suggesting that these proteins are collagen-like structural elements of the cuticle. 3. The soluble proteins from the second stage (L2) differed from the adult and other larval stages in both the number and mol. wt of protein bands and their lack of degradation by bacterial collagenase. Amino acid composition of soluble cuticular proteins were similar for adult and L4, but glycine and proline were present in lower amounts in the L2. 4. These results support a hypothesis that there are stage specific differences in cuticular proteins from A. suum and that the greatest differences appear to exist between L2 and other stages.     

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.     

Involvement of tyrosine residues, N-terminal amino acids, and beta-alanine in insect cuticular sclerotization

During sclerotization of insect cuticle the acyldopamines, N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD), are oxidatively incorporated into the cuticular matrix, thereby hardening and stabilizing the material by forming crosslinks between the proteins in the cuticular matrix and by forming polymers filling the intermolecular spaces in the cuticle. Sclerotized cuticle from the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor, was hydrolyzed in dilute hydrochloric acid, and from the hydrolysates some components presumably degradation products of cuticular crosslinks were isolated. In two of the components, the sidechain of 3,4-dihydroxyacetophenone was linked to the amino groups of glycine and beta-alanine, respectively, and in the third component to the phenolic group of tyrosine. These three compounds, glycino-dihydroxyacetophenone, beta-alanino-dihydroxyacetophenone, and O-tyrosino-dihydroxyacetophenone, as well as the previously reported compound, lysino-dihydroxyacetophenone [Andersen, S.O., Roepstorff, P., 2007. Aspects of cuticular sclerotization in the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor. Insect Biochem. Mol. Biol. 37, 223-234], are suggested to be degradation products of cuticular crosslinks, in which amino acid residues formed linkages to both the alpha- and beta-positions of the sidechain of acyldopamines.     

Tyrosinase catalyzed protein polymerization as an in vitro model for quinone tanning of insect cuticle

The validity of Pryor's widely accepted quinone tanning hypothesis for the sclerotization of insect cuticle was examined using an in vitro model system. Quinones generated in situ by the oxidation of catechols with mushroom tyrosinase and molecular oxygen readily reacted with test proteins such as lysozyme, ribonuclease and cytochrome-c, producing dimers, trimers, and higher oligomers. With the exception of 3,4-dihydroxyphenylalanine, dopamine, and norepinephrine, most other catechols tested participated in protein polymerization. The inability of these three compounds to support oligomerization of test protein was attributed to their high rate of intramolecular cyclization reaction. Radioactive incorporation studies reveal the formation of catechol-protomer adducts, as well as aryl-protein crosslinks in the reaction mixture. The above results strongly support the quinone tanning hypothesis. Based on these findings, the mechanism of cuticular sclerotization is discussed.