Unique features of the structural model of ‘hard’ cuticle proteins: implications for chitin–protein interactions and cross-linking in cuticle
Introduction
Cuticle is a composite material made primarily of chitin filaments embedded in a proteinaceous matrix. It provides structural and mechanical support by serving functionally as both skin and skeleton to arthropods (Neville, 1975; Vincent and Wegst, 2004). The mechanical properties of the cuticle are conferred by the proportion of chitin, by the degree of sclerotization and by the sequences of its proteins.
The precise nature of the interaction of cuticular proteins with chitin fibers and the detailed structure of insect cuticle have not yet been resolved. Certain sequence motifs occur in cuticular proteins from even distantly related species and such conserved motifs have common and important roles for the proper function of cuticle (Andersen et al., 1995). The most prevalent motif is the “R&R Consensus sequence” first identified by Rebers and Riddiford (1988): G-x(8)-G-x(6)-Y-x-A-x-E-x-G-Y-x(7)-P-x(2)-P or a modification of it: G-x(7)-[DEN]-G-x(6)-[FY]-x-A-[DGN]-x(2,3)-G-[FY]-x-[AP]-x(6) (Willis, 1999) (where x represents any amino acid, the values in parentheses indicate the number of residues and brackets include alternative amino acids at the site). An extension of this motif is a stretch of approximately 68 amino acids, the “extended R&R Consensus” that was recognized by several groups (reviewed in Willis et al., 2005). Andersen recognized and named three distinct forms of the “extended consensus” RR-1, RR-2 (Andersen, 1998) and RR-3 (Andersen, 2000). RR-1 bearing proteins have been isolated from flexible cuticles, while RR-2 proteins have been associated with hard cuticle. The RR-3 form of the consensus has been based on but five sequences from postecdysial cuticle of insects plus sequences from other arthropod classes. The assignment of RR-1 proteins to “soft” cuticle and RR-2 to “hard” is based on limited data (discussed in Willis et al., 2005). There is no agreed upon definiton of this classification of cuticle. “Hard” cuticles are generally sclerotized and mechanically stiff. “Soft” cuticles include, but are not restricted to, those that can expand within an instar due to growth by intussusception. Further work is needed to learn if the assignment of protein class to cuticle type is universal.
The prevalence of the R&R Consensus led several authors to postulate that it served an important function, quite possibly chitin binding (Bouhin et al., 1992; Charles et al., 1992; Andersen et al., 1995).
The involvement of the extended consensus in chitin binding has been confirmed by direct experimentation (Rebers and Willis, 2001; Togawa et al. 2004). There were earlier experimental findings and proposals that -sheet should be involved in chitin–protein interactions (Fraenkel and Rudall, 1947; Atkins, 1985) and these have been amplified by secondary structure prediction and experimental data. This more recent work indicates that antiparallel -pleated sheet is most probably the underlying molecular conformation of a large part of this extended R&R Consensus, especially the part which contains the R&R Consensus itself. We also proposed that this conformation is most probably involved in -sheet-chitin chain interactions of the cuticular proteins with chitin filaments (Iconomidou et al., 1999, Iconomidou et al., 2001).
A more specific analysis of the nature of cuticular protein/chitin binding became possible when, unexpectedly, a distant (20%) sequence similarity was found between “soft” cuticle proteins and the crystallographically determined C-terminal (Zanotti et al., 1994), -barrel portion, of bovine plasma retinol binding protein (RBP). When, following alignment, both conservative substitutions and identities were combined, the similarity rises to 60% of the total HCCP12 sequence (Hamodrakas et al., 2002), a representative member of the “soft” cuticle (RR-1) proteins (Binger and Willis, 1994; Iconomidou et al., 1999). This similarity allowed the construction, by “homology” (comparative) modelling of a structural model of the “extended R&R Consensus” of cuticle proteins (Hamodrakas et al., 2002). This modelling was successful even though it seems that RBP and the R&R Consensus-bearing cuticular proteins are not strictly homologous.
Furthermore, modelling of HCCP66 (Entrez accession number 1169133) and AGCP2b (Entrez accession number 2961110), two “hard” cuticle proteins, showed that the “extended R&R Consensus” (Iconomidou et al., 1999) not only of “soft” but also of “hard” cuticle proteins might easily adopt the proposed conformation (Hamodrakas et al., 2002).
The RR-2 bearing proteins, associated with hard cuticles, are of particular interest because the extended R&R Consensus is virtually invariant in length and in the identity of one third of its amino acids and very limited variation in another third. Furthermore, it is RR-2 proteins, far more than RR-1, which may have numerous histidine residues that could participate in cross-linking (Willis et al., 2005). In this work, we present in detail several unique features of the proposed structural model for ‘hard’ cuticle proteins to serve as a chitin binding structural motif, thus providing the basis for elucidating cuticle's overall architecture and chitin–protein interactions in cuticle.
Section snippets
Materials and methods
A sensitive alignment of a representative set of 44 ‘hard’ cuticle protein sequences (Table 1) was produced with CLUSTAL W (Thompson et al., 1994). The BLOSUM 62 similarity matrix was used and all other parameters were the default parameters of CLUSTAL W (Thompson et al., 1994).
A structural model for ‘hard’ cuticle proteins was then derived by homology modelling, utilizing the program WHAT IF (Vriend, 1990), using as template the structural model proposed for ‘soft’ cuticle proteins (Hamodrakas
Results
The extended RR-2 consensus region of 44 proteins with 36 different sequences were aligned (Fig. 1). What is extraordinary about the RR-2 consensus is its conservation across 14 species from six orders of insects. Only two single amino acid gaps are required to accommodate all 44 RR-2 sequences. Twenty-two of the 70 residues (31%) in the extended consensus are virtually invariant and an additional 23 are represented by a single amino acid in over half of the proteins (displayed by red and green
Discussion
There are now 139 sequences available for what are known or postulated to be cuticle structural proteins (Willis et al., 2005). These numbers do not include almost 200 more that have been identified by protein prediction programs used to annotate the D. melanogaster and A. gambiae genomes. These have been omitted because their annotation is still in a state of flux.
The R&R Consensus is a common feature of cuticle structural proteins, from all six orders of insects examined to date (Fig. 1) and
Acknowledgements
This work was supported in part by grant AI055624 from the US National Institutes of Health. We thank the University of Athens for financial support. We also thank Drs. G. Vriend, I. Vakser and A. Nicholls for providing us with their computer programs, and the anonymous referees for their constructive criticism.
References (38)
Amino acid sequence studies on endocuticular proteins from the desert locust, Schistocerca gregaria
Insect Biochem. Molec. Biol.
(1998)Studies on proteins in post-ecdysial nymphal cuticle of locust, Locusta migratoria, and cockroach, Blaberus cranifer
Insect Biochem. Molec. Biol.
(2000)Cuticular sclerotization and tanning
- et al.
Insect cuticular proteins
Insect Biochem. Mol. Biol.
(1995) - et al.
Identification of the cDNA, gene and promoter for a major protein from flexible cuticles of the giant silkmoth Hyalophora cecropia
Insect Biochem. Mol. Biol.
(1994) - et al.
Structure of chitin–protein complexes: ovipositor of the ichneumon fly Megarhyssa
J. Mol. Biol.
(1980) - et al.
Some conformational studies of larval cuticular proteins from Calliphora vicina
Insect Biochem.
(1979) Twisted β-pleated sheet: the molecular conformation which possibly dictates the formation of the helicoidal architecture of several proteinaceous eggshells
Int. J. Biol. Macromol.
(1984)- et al.
The crystal structure of the complex of concanavalin A with 4-methylumbelliferyl-α-d-glucopyranoside
J. Struct. Biol.
(1997) - et al.
A structural model of the chitin-binding domain of cuticle proteins
Insect Biochem. Mol. Biol.
(2002)
Homology modeling of the insect chitinase catalytic domain-oligosaccharide complex and the role of a putative active site tryptophan in catalysis
Insect Biochem. Mol. Biol.
Is ß-pleated sheet the molecular conformation which dictates the formation of the helicoidal cuticle?
Insect Biochem. Mol. Biol.
“Soft”-cuticle protein secondary structure as revealed by FT-Raman, ATR FT-IR and CD spectroscopy
Insect Biochem. Mol. Biol.
Mass spectrometric analysis of catechol-histidine adducts from insect cuticle
Anal. Biochem.
Oxidative conjugation of catechols with proteins in insect skeletal systems
Tetrahedron
Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes
J. Mol. Biol.
A conserved domain in arthropod cuticular proteins binds chitin
Insect Biochem. Mol. Biol.
Chitin-binding proteins in invertebrates and plants comprise a common chitin-binding structural motif
J. Biol. Chem.
Analysis for the chitin recognition mechanism of cuticle proteins from the soft cuticle of the silkworm, Bombyx mori
Insect Biochem. Mol. Biol.
Cited by (87)
Spatio-temporal distribution and genetic background of elastic proteins inside the chitin/chitosan matrix of insects including their functional significance for locomotion
2024, Insect Biochemistry and Molecular BiologyThe hypothetical cuticular protein, CPH19, is involved in cuticle formation during molt of silkworm Bombyx mori
2023, Journal of Asia-Pacific EntomologyGenome-wide annotation of cuticular protein genes in non-biting midge Propsilocerus akamusi and transcriptome analysis of their response to heavy metal pollution
2022, International Journal of Biological MacromoleculesChitin and cuticle proteins form the cuticular layer in the spinning duct of silkworm
2022, Acta BiomaterialiaTranscriptome analysis reveals the role of Zelda in the regulation of embryonic and wing development of Tribolium castaneum
2023, Bulletin of Entomological Research