Svend Olav Andersen

Insect cuticular proteins

Insect cuticles are composite structural materials with mechanical properties optimal for their biological functions. The bulk properties of cuticles are to a large extent determined by the interactions between the various components, mainly the chitin filament system and the proteins. The various cuticular types show pronounced differences in mechanical properties, and it is suggested that these differences can be related to the properties of the individual proteins and to the degree of secondary stabilization (sclerotization). The amino acid sequences, which have been obtained for insect cuticular proteins either by direct sequencing of purified proteins or by deduction from corresponding DNA-sequences, are listed according to insect order and species. Extensive sequence similarity is observed among several cuticular proteins obtained from different insect orders. Other cuticular proteins are characterized by repeated occurrence of a few small motifs consisting mainly of hydrophobic residues. The latter group of proteins has so far only been reported from stiff cuticles. The possible relevance of the various motifs and repeats for protein interaction and the mechanical properties of cuticles is discussed.

Insect cuticular sclerotization: A review

Different regions of an insect cuticle have different mechanical properties, partly due to different degrees of stabilization and hardening occurring during the process of sclerotization, whereby phenolic material is incorporated into the cuticular proteins. Our understanding of the chemistry of cuticular sclerotization has increased considerably since Mark Pryor in 1940 suggested that enzymatically generated ortho-quinones react with free amino groups, thereby crosslinking the cuticular proteins. The results obtained since then have confirmed the essential features of Pryors suggestion, and the many observations and experiments, which have been obtained, have led to a detailed and rather complex picture of the sclerotization process, as described in this review. However, many important questions still remain unanswered, especially regarding the precise regional and temporal regulation of the various steps in the process.

Resilin. A Rubberlike Protein in Arthropod Cuticle

Publisher Summary Resilin is a structural protein, which was discovered recently as a major constituent of certain elastic hinges and tendons in the cuticle of locusts and dragonflies. It is also present as an insoluble gel-like component in certain patches of the cuticle of insects and crayfish. In many respects, it resembles elastin from vertebrates and may well be called “arthropod elastin,” but it differs from elastin in many important ways and lends itself more easily to studies of the rubberlike state in proteins, the number and nature of cross-links, and the formation of three-dimensional networks in biological structures. Pure unstrained resilin appears as a mass of optically and mechanically isotropic protein, both in situ and when dissected free. In the dry state, it is hard and brittle and is insoluble in all solvents that do not break peptide bonds. In aqueous media and also in many anhydrous hydrophilic liquids, it swells isotropically and reversibly; it then becomes rubbery, and then exhibits typically long-range deformability and complete elastic recovery. Both in situ and when free, resilin is easily digested by all proteinases.After complete acid or basic hydrolysis, it yields only amino acids of which fifteen are ordinary amino acids (no sulfur-containing ones and no tryptophan or hydroxyproline) and two are unusual and specific. When resilin is mixed with other substances, the bulk properties of the cuticle question may of course differ appreciably from those of the pure material, but in most cases, its presence can be established by means of simple tests—such as mechanical behavior, strain birefringence, color reactions, swelling, and fluorescence.

Characterization of a new type of cross-linkage in resilin, a rubber-like protein

Abstract Native resilin as found in elastic ligaments in insects shows a bright blue fluorescence due to two compounds which are firmly connected to the protein, but which can be liberated by prolonged hydrolysis with acid or alkali. The two compounds have characteristic ultraviolet-absorption spectra with maximum absorption in acid solution at 286 and 283 mμ, respectively, and at 322 and 377 mμ in alkaline solution. This shift in the absorption maxima occurs for both compounds near neutrality and it is accompanied by an increased fluorescence. It is assumed that the group responsible for this shift is of phenolic nature. Spot tests indicate that both compounds are aromatic α-amino acids containing a phenolic group; tests for diphenols and for a middle nucleus are negative. One of the compounds is shown to be a diaminodicarboxylic acid and the other a triaminotricarboxylic acid. In the native protein the amino groups of the compounds do not react with dinitrofluorobenzene, indicating that they are built into the peptide structure, in this way linking the chains in a 3-dimensional network. The degree of cross-linking calculated from the amounts of these compounds-present in resilin agrees well with the estimates from physical measurements.

Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus craniifer

Proteins were extracted from the cuticle of mid-instar nymphs of locusts, Locusta migratoria, and cockroaches, Blaberus craniifer. Seven proteins were purified from the locust extract and five from the cockroach extract, and their amino acid sequences were determined. Polyacrylamide gel electrophoresis indicates that the proteins are present only in the post-ecdysially deposited layer of the nymphal cuticles. One of the locust and one of the cockroach nymphal proteins contain a 68-residue motif, the RR-2 sequence, which has been reported for several proteins from the solid cuticles of other insect species. Two of the cockroach proteins contain a 75-residue motif, which is also present in a protein from the larval/pupal cuticle of a beetle, Tenebrio molitor, and in proteins from the exoskeletons of a lobster, Homarus americanus, and a spider, Araneus diadematus. The motif contains a variant of the Rebers-Riddiford consensus sequence, and is called the RR-3 motif. One of the locust and three of the cockroach post-ecdysial proteins contain one or more copies of an 18-residue motif, previously reported in a protein from Bombyx mori pupal cuticle. The nymphal post-ecdysial proteins from both species have features in common with pre-ecdysial proteins (pharate proteins) in cuticles destined to be sclerotised; they show little similarity to the post-ecdysial cuticular proteins from adult locusts or to proteins from soft, pliable cuticles. Possible roles for post-ecdysial cuticular proteins are discussed in relation to the reported structures.

Phenoloxidases in larval cuticle of the blowfly, Calliphora vicina

Three different phenoloxidases have been purified from the cuticle of mature larvae of the blowfly, Calliphora vicina. Enzyme A is a typical tyrosinase (EC 1.10.3.1., o-diphenol: O2 oxidoreductase). It oxidizes both monophenols and o-diphenols, is inactive towards p-diphenols, and is readily inhibited by thiourea and phenylthiourea. Enzymes B and C are laccases (EC 1.10.3.2., p-diphenol: O2 oxidoreductase), that will oxidize both o- and p-diphenols but are inactive towards monophenols. The molecular weight of enzyme B was estimated to be 90,000. The pH-optima for enzymes A and C are close to neutrality, whereas enzyme B has its pH-optimum at 4.5. It is suggested that enzyme A is involved in wound healing, and that enzyme B plays a role in sclerotization of the puparium.

Comparison between the sclerotization of adult and larval cuticle in Schistocerca gregaria

Abstract Femur cuticle from fifth instar larvae of the desert locust, Schistocerca gregaria, has been characterized with respect to composition, rate of deposition, and rate of sclerotization. The results are compared with those from adult cuticle of the same species. The protein compositions of the two types of cuticle are very similar, but the rates of deposition of both protein and chitin are different. The main difference is, however, that sclerotization is restricted to the first day after ecdysis in larval cuticle, whereas in adult cuticle sclerotization continues for at least a couple of weeks. The result is that the endocuticle remains untanned in the larvae, whereas in the adults the whole cuticle becomes tanned.

Characterization of a trypsin-solubilized phenoloxidase from locust cuticle

A diphenoloxidase has been dissolved from pre-sclerotized locust cuticle by means of trypsin digestion. The solubilized enzyme has been extensively purified and characterized with respect to substrate and inhibitor specificity, pH-optimum and pH-stability and thermostability. The enzyme will oxidize a number of both ortho- and para-diphenols, and in contrast to untreated locust cuticle it is more active in releasing tritium located on the aromatic ring of N-acetyldopamine than in releasing tritium from the aliphatic side-chain of the same compound. The possible relationship between the trypsin-solubilized activity and the insoluble sclerotization enzyme in intact cuticle is discussed.

Characterization of cuticular proteins from the migratory locust, Locusta migratoria

Proteins were extracted from the still unhardened (teneral) cuticle of the migratory locust, Locusta migratoria. The proteins are soluble only at extreme pH-values and at low ionic strength, the solubility increases with decreasing temperature. The unhardened cuticle contains approx. 100 different proteins according to two-dimensional polyacrylamide gel electrophoresis. The majority of the proteins are very basic. The basicity and solubility properties of the proteins have necessitated development of modified electrophoretic procedures. The amino acid composition of the bulk protein shows that alanine, proline, glycine, valine and tyrosine constitute two thirds of the total amino acid content and that cysteine, methionine and tryptophan are absent. The proteins have been extracted from various parts of the cuticle and analysed by two-dimensional electrophoresis. Characteristic protein compositions were found for cuticle from the different body parts. Amino acid analyses of these extracts are strikingly similar. The only significant difference is in the glycine-alanine ratio. Cuticles that are destined to become hard are extremely rich in alanine, whereas the flexible parts of the cuticle are enriched in glycine. The results indicate that the proteins of locust cuticle constitute a group of structural proteins different from other known structural proteins.

Cuticular sclerotization in the beetles Pachynoda epphipiata and Tenebrio molitor

Abstract The sclerotization of cuticle in two species of beetles, Pachynoda epphipiata and Tenebrio molitor, has been investigated and compared with the sclerotization in the locust, Schistocerca gregaria. Two types of sclerotization, β-sclerotization and quinone tanning, occur in all three species. The main type is β-sclerotization, i.e. cross-linking of proteins by means of N-acetyldopamine which is connected to the proteins through the β-position of its side chain. β-Sclerotization is completed in P. epphipiata when it leaves its cocoon, whereas in adult locusts and in adult Tenebrio β-sclerotization continues for several weeks. The cuticle of all three species contains an insoluble enzyme which activates the β-position of N-acetyldopamine and is presumably responsible for the formation of the cross-links. Locust cuticle contains also small amounts of another enzyme which activates the aromatic ring of N-acetyldopamine, resulting in the formation of an o-quinone, which may be involved in quinone tanning of the cuticle. At emergence adult Tenebrio cuticle is rich in both enzymes, but the quinone-forming enzyme is inactivated after a few days, whereas the β-enzyme first decreases and later increases in activity, so that the β-enzyme is the dominating activity in the cuticle of mature adult Tenebrio. The quinone-forming enzyme is presumably responsible for the formation of the brown colour of Tenebrio exocuticle. The exocuticle of adult beetles contains 3,4-dihydroxyphenylacetic acid, which, although it is not easily extracted from the cuticle, is not covalently bound to cuticular components. In Tenebrio it appears in the cuticle a few days after the final ecdysis. The amino acid compositions of both larval, pupal, and adult cuticle from P. epphipiata have been determined, and they are compared with the composition of the cuticle of the corresponding stages of Tenebrio.