Comparative Proteomics Studies of Insect Cuticle by Tandem Mass Spectrometry: Application of a Novel Proteomics Approach to the Pea Aphid Cuticular Proteins

Since nearly all adult insects fly, the cuticle has to provide a very efficient and lightweight skeleton. Information is available about the mechanical properties of cuticle-Young's modulus of resilin is about 1 MPa, of soft cuticles about 1 kPa to 50 MPa, of sclerotised cuticles 1-20 GPa; Vicker's Hardness of sclerotised cuticle ranges between 25 and 80 kgf mm(-2); density is 1-1.3 kg m(-3)-and one of its components, chitin nanofibres, the Young's modulus of which is more than 150 GPa. Experiments based on fracture mechanics have not been performed although the layered structure probably provides some toughening. The structural performance of wings and legs has been measured, but our understanding of the importance of buckling is lacking: it can stiffen the structure (by elastic postbuckling in wings, for example) or be a failure mode. We know nothing of fatigue properties (yet, for instance, the insect wing must undergo millions of cycles, flexing or buckling on each cycle). The remarkable mechanical performance and efficiency of cuticle can be analysed and compared with those of other materials using material property charts and material indices. Presented in this paper are four: Young's modulus-density (stiffness per unit weight), specific Young's modulus-specific strength (elastic hinges, elastic energy storage per unit weight), toughness-Young's modulus (fracture resistance under various loading conditions), and hardness (wear resistance). In conjunction with a structural analysis of cuticle these charts help to understand the relevance of microstructure (fibre orientation effects in tendons, joints and sense organs, for example) and shape (including surface structure) of this fibrous composite for a given function. With modern techniques for analysis of structure and material, and emphasis on nanocomposites and self-assembly, insect cuticle should be the archetype for composites at all levels of scale.

For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu(2+) (12-31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms.

The availability of whole genome sequences of several arthropods has provided new insights into structural cuticular proteins (CPs), in particular the distribution of different families, the recognition that these proteins may comprise almost 2% of the protein coding genes of some species, and the identification of features that should aid in the annotation of new genomes and EST libraries as they become available. Twelve CP families are described: CPR (named after the Rebers and Riddiford Consensus); CPF (named because it has a highly conserved region consisting of about forty-four amino acids); CPFL (like the CPFs in a conserved C-terminal region); the TWDL family, named after a picturesque phenotype of one mutant member; four families in addition to TWDL with a preponderance of low complexity sequence that are not member of the families listed above. These were named after particular diagnostic features as CPLCA, CPLCG, CPLCW, CPLCP. There are also CPG, a lepidopteran family with an abundance of glycines, the apidermin family, named after three proteins in Apis mellifera, and CPAP1 and CPAP3, named because they have features analogous to peritrophins, namely one or three chitin-binding domains. Also described are common motifs and features. Four unusual CPs are discussed in detail. Data that facilitated the analysis of sequence variation of single CP genes in natural populations are analyzed. (c) 2010 Elsevier Ltd. All rights reserved.