Various Catechol Derivatives Obtained from Sclerotized Cuticles
During the process of sclerotization most of the catecho-lic material will be firmly linked to the cuticular matrix and can only be solubilized by degrading the cuticle, for instance by hydrolysis. A large variety of catecholic derivatives are released by acid hydrolysis of sclerotized cuticle; some are simple catechols and some catecholic adducts containing amino acids. The structures of the derivatives can indicate whether or not they are likely to be degradation products of materials derived from the sclerotizing precursors, and may also indicate which reactions are responsible for their formation.
Figure 5 Structure of adducts formed between W-acetylhistidine and NADA.
Figure 6 Structure of products formed from a-W-acetyllysine and oxidized NADA.
Figure 7 Products formed by incubation of NADA together with cuticle.
Among the compounds extracted from sclerotized cuticle we find unused sclerotization precursors, intermediates of the sclerotiza-tion process, by-products from the process, and degradation products of protein-bound cross-links and polymers. It is likely that some of the extracted compounds have functions unrelated to sclerotization; catechols can be precursors for pigments, such as papiliochromes (Umebachi, 1993), or they can function as antioxidants protecting the epicuticular lipids from autoxidation (Atkinson et al., 1973b). 3,4-Dihydroxyphenyl acetic acid, which is present in the solid cuticle of beetle species (Andersen, 1975), may serve the latter purpose, as it apparently does not take part in the sclerotization process (Barrett, 1984a, 1990). It can be problematic to decide whether catechols obtained from cuticles are related to the sclerotization process or not.
The mixture of compounds obtained by mild extraction of sclerotized cuticles (such as extraction in boiling water or neutral salt solutions) consists of compounds that are less modified than those obtained by acidic extraction at elevated temperatures, but a critical and careful interpretation of their structures will be necessary in all cases. Extraction with dilute acids tends to give higher yields of catechols than extraction with water, but the compounds identified in the extracts are often the same (Atkinson et al., 1973a). The higher yields obtained by acidic solvents may be due partly to swelling of the cuticular material at low pH values, resulting in easier liberation of trapped compounds, and partly to hydrolysis of acid-labile bonds. The sclerotization precursors NADA and NBAD, and their hydroxylated derivatives NANE and NBANE, are typical extraction products, and 3,4-dihydroxybenzoic acid and 3,4-dihydroxybenzaldehyde have been extracted from several types of sclerotized cuticle; they are probably formed during the sclerotization process by extensive oxidative degradation of the side chain of the sclerotizing precursors. The precise reaction pathway for their formation is not known, but their occurrence in sclerotized cuticles indicates that the intracuticular environment is highly oxidative during the sclerotization process. The presence in cuticular extracts of dopamine and norepinephrine may be due to deacetylation of the acylated forms, or they may have been transferred directly from epidermal cells to the cuticle, either by active transport across the apical cell membrane or by passive leakage through the cell membrane. The black cuticles of the Drosophila mutants black and ebony contain elevated levels of dopamine (Wright, 1987), which must have been transferred to the cuticle in the non-acylated state.
Quite large amounts of ketocatechols, such as arte-renone (29), DOPKET (30), N-acetylarterenone (33), 3,4-dihydroxyphenylglyoxal (31), 3,4-dihydroxyphenyl-glyoxylic acid (32), and 3,4-dihydroxyphenylketoethyl-acetate (34) (Figure 8), can be obtained from sclerotized cuticle by hydrolysis with dilute hydrochloric acid (Andersen, 1970, 1971; Andersen and Barrett, 1971; Andersen and Roepstorff, 1978). The yields of ketocatechols can amount to several percent of the cuticular dry weight (Andersen, 1975; Barrett, 1977), and the type of keto-catechols obtained depends upon the exact conditions of hydrolysis. It has been argued that they are degradation products of a common precursor in the cuticle (Andersen,1971).
More complex catechol derivatives can be obtained by using milder conditions to extract sclerotized cuticle, such as cold concentrated formic acid or boiling dilute acetic acid. From such extracts a number of dimeric compounds of the dihydroxyphenyl-dihydrobenzodioxine type were isolated and identified (Andersen and Roepstorff, 1981; Roepstorff and Andersen, 1981), and a related trimeric compound (35) (Figure 9) was obtained by formic acid extraction of sclerotized locust cuticle (Andersen et al., 1992a). Ketocatechols are readily formed when such dimers and oligomers are hydrolyzed with acid (Andersen and Roepstorff, 1981), but the amount of ketocatechols obtained by hydrolysis of the extracted benzodioxine derivatives is only a small fraction of the amount produced by acid hydrolysis of intact sclerotized cuticle, indicating that the major part of ketocatechols obtainable from cuticle is derived from catecholic material covalently linked to the cuticular proteins and chitin.
Figure 8 Ketocatechols obtained by acid hydrolysis of sclerotized cuticle. (29): arterenone;(30): 3,4-dihydroxyketoethanol (DOPKET);(31): 3,4-dihydroxyphenylglyoxal; (32): 3,4-dihydroxyphenylglyoxylic acid; (33): N-acetylarteremone; (34): O-acetyl-dihydroxyphenylketoethanol.
Figure 9 Structure of NADA-trimer.
Since the various benzo-dioxine dimers can be formed in vitro by reacting oxidized dehydro-NADA with catechols, it is likely that oxidized dehydro-NADA will react with protein-linked catechols to form protein-linked benzodioxine dimers and higher oligomers.
Acid hydrolysis of M. sexta pupal cuticle has yielded adducts containing histidine linked to either the C-6 ring position (36) or the P-position (37) of dopamine, or to the corresponding positions in DOPET (38 and 39) (Figure 10), demonstrating that adduct formation to histidine residues occurs during in vivo sclerotization (Xu et al., 1997; Kerwin et al, 1999), and indicating that DOPET may have a role as a sclerotization precursor. Direct evidence that covalent bonds are formed between acyldopamines and histidine residues during sclerotization had previously been obtained by solid state NMR studies, utilizing incorporation of isotopically labeled dopamine, histidine, and P-alanine into sclero-tizing pupal cuticle of M. sexta (Schaefer et al., 1987; Christensen et al., 1991). The NMR spectra demonstrated the presence of bonds between nitrogen atoms in the imidazole ring of histidine and ring-positions or the P-position of the dopamine side chain. Formation of covalent bonds involving the amino group of P-alanine and the e-amino group of lysine was also indicated. Furthermore, catecholamine-containing proteins from sclerotizing M. sexta pupal cuticle have been purified and partially characterized, and NBANE was released from these proteins by mild acid hydrolysis, indicating the presence of a bond between the P-position of the side chain of NBAD and some amino acid residue in the proteins (Okot-Kotber et al, 1996).
Several amino acid-containing adducts, some of which have a ketocatecholic structure, were obtained by acid hydrolysis of locust and Tenebrio sclerotized cuticle. Some of the adducts contained histidine residues linked via their imidazole ring to the P-position of various catechols, such as dopamine (37), DOPET (39), and 3,4-dihy-droxyphenyl-acetaldehyde (DOPALD, 40) (Andersen and Roepstorff, 2007; Figure 10). Other adducts were derivatives of 3,4-dihydroxyacetophenone with an amino acid residue linked to the a-carbon atom, and the amino acid could be glycine and P-alanine (41 and 42) linked via their amino groups, lysine linked via its e-amino group (43), or tyrosine linked via its phenolic group (44) (Andersen, 2007; Andersen and Roepstorff, 2007; Figure 11). Several more amino acid-containing adducts were released during the hydrolysis, but they have not been identified. No adducts with an amino acid residue linked to the aromatic ring have yet been obtained from hydrolysates of S. gregaria and T. molitor sclerotized cuticles, and neither have adducts containing histidine linked to the a-carbon atom of 3,4-dihydroxyacetophenone been observed, whereas histidine is the only amino acid that so far has been found linked to the P-position of the side chain.
The adducts containing an amino acid residue linked to 3,4-dihydroxyacetophenone are a sort of ketocatechol, and presumably the keto-group is formed during hydrolysis of the cuticles, as keto-groups are not normally present in sclerotized cuticle. The adducts have been suggested to be hydrolytic degradation products of cross-linking materials, consisting of NADA residues linked to cuticular proteins via both a- and P-positions (Andersen, 2007; Andersen and Roepstorff, 2007). Such cross-links will be formed if oxidized dehydro-NADA residues react with two nucleophilic amino acids in the cuticular proteins, and a scheme was proposed for how such cross-links are degraded to ketocatecholic adducts during acid hydrolysis.
The histidine-dopamine P-adduct is presumably a hydrolytic degradation product of NADA and NBAD adducts formed when the corresponding p-quinone methides reacted with histidine residues in the cuticular proteins. Such protein-linked catechols may possibly be reoxidized to quinones and react with another nucleo-philic group, thereby forming a cross-link, but no evidence has yet been presented for the existence of such cross-links.
Figure 10 Structure of histidine-containing catecholic adducts obtained by hydrolysis of sclerotized cuticles.
Figure 11 Structure of amino acid-containing derivatives of 3, 4-dihydroxyacetophenone obtained by hydrolysis of sclerotized cuticles.
Quantitative determination of the various amino acid-containing adducts obtained by hydrolysis of 16 different types of sclerotized cuticle showed that cuticles from adult locusts and cockroaches yielded large amounts of keto-catecholic adducts and only little P-histidine-dopamine, indicating that utilization of dehydro-NADA is the main pathway for their sclerotization. Cuticles from M. sexta and H. cecropia pupae, T. molitor larvae and pupae, and C. vicina puparia gave mainly P-histidine-dopamine and only small amounts of the ketocatecholic adducts, indicating that sclerotization of these cuticles mainly involves p-quinone methides. Adult beetles (T. molitor and Pachyn-oda sinuata) gave significant amounts of both types of adducts, indicating that both p-quinone methides and dehydro-NADA are to a significant extent involved in the sclerotization (Andersen, 2008).
The number of insect cuticles that have been analyzed is too small to allow any firm conclusions, but the results obtained so far indicate that the p-quinone methide of dehydro-NADA is the main sclerotizing agent of cuticles where NADA is the only or the dominating sclerotization precursor, and that the o-quinones and p-quinone methides formed from NADA in these cuticles mainly play roles as precursors for dehydro-NADA. The o-quinones and p-quinone methides of NBAD appear to be the dominating sclerotization agents in those cuticles where NBAD is the important precursor for sclerotiza-tion. In cuticles where both NADA and NBAD are involved to a significant extent in sclerotization, NBAD o-quinones and p-quinone methides, as well as dehydro-NADA p-quinone methide, will contribute more or less equally to the process.
Control of Sclerotization
The mechanical properties of the various cuticular regions tend to differ significantly, varying from the very hard and resistant mandibles to the soft and flexible arthrodial membranes, and the cuticular properties are likely to be optimized with respect to the functions of the regions. The mechanical properties of cuticles are to a major extent determined by the degree of sclerotization, indicating that initiation as well as duration and degree of sclerotization are precisely controlled. In some cases initiation of sclero-tization is controlled by hormones: ecdysone in the case of puparium sclerotization; and bursicon in the case of post-ecdysial sclerotization. The duration of sclerotization may also be hormonally controlled, perhaps by a decrease in hormone titer. The amounts of sclerotizing material incorporated per milligram of cuticle can be used as a measure of the degree of sclerotization, and is probably determined by local factors and not by hormones. Factors such as the amounts of sclerotizing precursors available and the capacity of the epidermis to transport sclerotizing precursors into the cuticle are likely to be important. The degree of sclerotization is apparently not determined by the amount cross-linking enzymes present in the cuticle, since the enzyme activity in various cuticular regions of adult locust does not correlate with the amounts of keto-catechols obtainable (Andersen, 1974b).
Post-Ecdysial Sclerotization
Many cuticular regions are expanded to a new and larger size when the insect has emerged from the old cuticle (Cottrell, 1964), and most insects start cuticular expansion as soon as they have escaped from the exuvium; however, in some insects the period from end of ecdysis to fully expanded cuticle can be prolonged – for instance in flies, where the newly emerged adult has to dig its way through the substratum in which it pupariated before it can expand and harden its wings. The signal for initiating sclerotization of the expanded cuticle after ecdysis is release of the neurohormone bursicon from the central nervous system. Bursicon has a pronounced influence on the activities of the epidermal cells; it has been reported that absence of bursicon results in the failure of endocu-ticle deposition, as well as lack of melanin production and of sclerotization of the cuticle (Fraenkel and Hsiao, 1965; Fogal and Fraenkel, 1969), and it was suggested that bur-sicon is involved in the control of tyrosine hydroxylation to DOPA (Seligman et al., 1969). The molecular structure of bursicon was later established to be a heterodimer of two cystine knot protein subunits (Luo et al., 2005; Mendive et al., 2005), and it was shown that the hormone is responsible for initiating a number of processes occurring in insects immediately or soon after ecdysis (Dai et al., 2008; Honegger et al., 2008), one of them being activation of the epidermal tyrosine hydroxylase by phos-phorylation of a serine residue (Davis et al., 2007).
In some insects sclerotization stops when the pre-ecdys-ially deposited cuticle has been sclerotized to form exocu-ticle, although deposition of endocuticle may continue for several days, resulting in a sclerotized exocuticle and a non-sclerotized endocuticle. In other insects cuticular sclerotization continues during endocuticle deposition, with the result that both exo- and endocuticle become sclerotized, although not to the same extent. Sclerotiza-tion of femur cuticle in adult locusts (S. gregaria) continues for at least 10-12 days after ecdysis, and both exo- and endocuticle are sclerotized (Andersen and Barrett, 1971), in contrast to sclerotization of femur cuticle of fifth instar nymphs of the same species, which lasts for only a single day, and deposition of unsclerotized endocuticle continues for about 4-5 days (Andersen, 1973). Accordingly, the endocuticular proteins are readily extracted from femurs of mature nymphs, whereas little protein can be extracted from femurs of mature adults. This difference is probably related to the different fate of these two types of cuticle. The nymphal cuticle will to a large extent be degraded in preparation for the next ecdysis, and sclero-tized cuticle is more resistant to enzymatic degradation than non-sclerotized cuticle. The adult cuticle has to last for the remaining life of the animal, and there is no apparent advantage in having an easily degradable endocuticle. The leg cuticle of adult locusts is also exposed to stronger mechanical forces than the cuticle of nymphal legs, and so it may be an advantage for adult locusts to have both layers of the leg cuticle sclerotized, although not to the same extent. A similar difference in sclerotization in adults and younger instars is probably present in other insect species. It has not yet been established how the duration of the post-ecdysial sclerotization period is controlled, but it could well be by regulation of the bursicon titer.
