Lepidopteran wing scales contain abundant cross-linked film-forming histidine-rich cuticular proteins.

Scales are symbolic characteristic of Lepidoptera; however, nothing is known about the contribution of cuticular proteins (CPs) to the complex patterning of lepidopteran scales. This is because scales are resistant to solubilization, thus hindering molecular studies. Here we succeeded in dissolving developing wing scales from Bombyx mori, allowing analysis of their protein composition. We identified a distinctive class of histidine rich (His-rich) CPs (6%-45%) from developing lepidopteran scales by LC-MS/MS. Functional studies using RNAi revealed CPs with different histidine content play distinct and critical roles in constructing the microstructure of the scale surface. Moreover, we successfully synthesized films in vitro by crosslinking a 45% His-rich CP (BmorCPR152) with laccase2 using N-acetyl- dopamine or N-ß-alanyl-dopamine as the substrate. This molecular study of scales provides fundamental information about how such a fine microstructure is constructed and insights into the potential application of CPs as new biomaterials.

Proteomics reveals localization of cuticular proteins in Anopheles gambiae.

Anopheles gambiae devotes over 2% of its protein coding genes to its 298 structural cuticular proteins (CPs). This paper provides new LC-MS/MS data on two adult structures, proboscises and palps, as well as three larval samples - 4th instar larvae, just their terminal segment, and a preparation enriched in their tracheae. These data were combined with our previously published results of proteins from five other adult structures, whole adults, and two preparations chosen for their relatively clean cuticle, the larval head capsules left behind after ecdysis and the pupal cuticles left behind after adult eclosion. Peptides from 28 CPs were recovered in all adult structures; 24 CPs were identified for the first time, 6 of these were members of the TWDL family. Most newly identified proteins came from the larval sources. Based solely on peptide recovery, from our data and from other investigators, most available on VectorBase, there were only 4 CPs that were restricted to a single adult structure. More were restricted to a single metamorphic stage, 14 in larvae, 0 in pupae and 32 in adults. Expression data from our earlier RT-qPCR studies reduces these numbers. Charting restriction of CPs to stage or structure is a step forward in establishing their specific roles.

The Evolution and Metamorphosis of Arthropod Proteomics and Genomics.

This article presents an overview of the development of techniques for analyzing cuticular proteins (CPs), their transcripts, and their genes over the past 50 years based primarily on experience in the laboratory of J.H. Willis. It emphasizes changes in the kind of data that can be gathered and how such data provided insights into the molecular underpinnings of insect metamorphosis and cuticle structure. It describes the techniques that allowed visualization of the location of CPs at both the anatomical and intracuticular levels and measurement of the appearance and deployment of transcripts from CP genes as well as what was learned from genomic and transcriptomic data. Most of the early work was done with the cecropia silkmoth, Hyalophora cecropia, and later work was with Anopheles gambiae.

Properties of the cuticular proteins of Anopheles gambiae as revealed by serial extraction of adults.

How cuticular proteins (CPs) interact with chitin and with each other in the cuticle remains unresolved. We employed LC-MS/MS to identify CPs from 5-6 day-old adults of Anopheles gambiae released after serial extraction with PBS, EDTA, 2-8M urea, and SDS as well as those that remained unextracted. Results were compared to published data on time of transcript abundance, localization of proteins within structures and within the cuticle, as well as properties of individual proteins, length, pI, percent histidine, tyrosine, glutamine, and number of AAP[A/V/L] repeats. Thirteen proteins were solubilized completely, all were CPRs, most belonging to the RR-1 group. Eleven CPs were identified in both soluble fractions and the final pellet, including 5 from other CP families. Forty-three were only detected from the final pellet. These included CPRs and members of the CPAP1, CPF, CPFL, CPLCA, CPLCG, CPLCP, and TWDL families, as well as several low complexity CPs, not assigned to families and named CPLX. For a given protein, many histidines or tyrosines or glutamines appear to be potential participants in cross-linking since we could not identify any peptide bearing these residues that was consistently absent. We failed to recover peptides from the amino-terminus of any CP. Whether this implicates that location in sclerotization or some modification that prevents detection is not known. Soluble CPRs had lower isoelectric points than those that remained in the final pellet; most members of other CP families had isoelectric points of 8 or higher. Obviously, techniques beyond analysis of differential solubility will be needed to learn how CPs interact with each other and with chitin.

Localization of RR-1 and RR-2 cuticular proteins within the cuticle of Anopheles gambiae.

The largest arthropod cuticular protein family, CPR, has the Rebers and Riddiford (R&R) Consensus that in an extended form confers chitin-binding properties. Two forms of the Consensus, RR-1 and RR-2, have been recognized and initial data suggested that the RR-1 and RR-2 proteins were present in different regions within the cuticle itself. Thus, RR-2 proteins would contribute to exocuticle that becomes sclerotized, while RR-1s would be found in endocuticle that remains soft. An alternative, and more common, suggestion is that RR-1 proteins are used for soft, flexible cuticles such as intersegmental membranes, while RR-2s are associated with hard cuticle such as sclerites and head capsules. We used TEM immunogold detection to localize the position of several RR-1 and RR-2 proteins in the cuticle of Anopheles gambiae. RR-1s were localized in the procuticle of the soft intersegmental membrane except for one protein found in the endocuticle of hard cuticle. RR-2s were consistently found in hard cuticle and not in flexible cuticle. All RR-2 antibodies localized to the exocuticle and four out of six were also found in the endocuticle. Hence the location of RR-1s and RR-2s depends more on properties of individual proteins than on either hypothesis.

Immunolocalization of cuticular proteins in Johnston's organ and the corneal lens of Anopheles gambiae.

Previous work with EM immunolocalization examined the intracuticular placement of several antibodies directed against cuticular proteins (CPs) in various structures of Anopheles gambiae. Those structures had long stretches of fairly uniform cuticle. We have now used 19 antibodies directed against members of five CP families on two adult structures with considerable complexity, Johnston's organ and the corneal lens of the compound eye. We also localized chitin with colloidal-gold labeled wheat germ agglutinin. Twelve of these antibodies recognized structures in Johnston's organ. Only 6 were detected in the outer pedicel wall, but the internal structures were more complex with distinct distributions of members of the five CP families in six different structures. The corneal lens had four distinct regions of laminar cuticle. Thirteen of the 15 members of the CPR family were detected, none from the other CP families. Specific antibodies were localized to different regions and in different laminae within a region. The specificity of deployment of cuticular proteins revealed in this study is helping to explain why An. gambiae allocates about 2% of its protein coding genes to structural CPs.

Distribution of cuticular proteins in different structures of adult Anopheles gambiae.

Anopheles gambiae devotes over 2% (295) of its protein coding genes to structural cuticular proteins (CPs) that have been classified into 13 different families plus ten low complexity proteins not assigned to families. Small groups of genes code for identical proteins reducing the total number of unique cuticular proteins to 282. Is the large number because different structures utilize different CPs, or are all of the genes widely expressed? We used LC-MS/MS to learn how many products of these genes were found in five adult structures: Johnston's organs, the remainder of the male antennae, eye lenses, legs, and wings. Data were analyzed against both the entire proteome and a smaller database of just CPs. We recovered unique peptides for 97 CPs and shared peptides for another 35. Members of 11 of the 13 families were recovered as well as some unclassified. Only 11 CPs were present exclusively in only one structure while 43 CPs were recovered from all five structures. A quantitative analysis, using normalized spectral counts, revealed that only a few CPs were abundant in each structure. When the MS/MS data were run against the entire proteome, the majority of the top hits were to CPs, but peptides were recovered from an additional 467 proteins. CP peptides were frequently recovered from chitin-binding domains, confirming that protein-chitin interactions are not mediated by covalent bonds. Comparison with three other MS/MS analyses of cuticles or cuticle-rich structures augmented the current analysis. Our findings provide new insights into the composition of different mosquito structures and reveal the complexity of selection and utilization of genes coding for structural cuticular proteins.

The CPCFC cuticular protein family: Anatomical and cuticular locations in Anopheles gambiae and distribution throughout Pancrustacea.

Arthropod cuticles have, in addition to chitin, many structural proteins belonging to diverse families. Information is sparse about how these different cuticular proteins contribute to the cuticle. Most cuticular proteins lack cysteine with the exception of two families (CPAP1 and CPAP3), recently described, and the one other that we now report on that has a motif of 16 amino acids first identified in a protein, Bc-NCP1, from the cuticle of nymphs of the cockroach, Blaberus craniifer (Jensen et al., 1997). This motif turns out to be present as two or three copies in one or two proteins in species from many orders of Hexapoda. We have named the family of cuticular proteins with this motif CPCFC, based on its unique feature of having two cysteines interrupted by five amino acids (C-X(5)-C). Analysis of the single member of the family in Anopheles gambiae (AgamCPCFC1) revealed that its mRNA is most abundant immediately following ecdysis in larvae, pupae and adults. The mRNA is localized primarily in epidermis that secretes hard cuticle, sclerites, setae, head capsules, appendages and spermatheca. EM immunolocalization revealed the presence of the protein, generally in endocuticle of legs and antennae. A phylogenetic analysis found proteins bearing this motif in 14 orders of Hexapoda, but not in some species for which there are complete genomic data. Proteins were much longer in Coleoptera and Diptera than in other orders. In contrast to the 1 and occasionally 2 copies in other species, a dragonfly, Ladona fulva, has at least 14 genes coding for family members. CPCFC proteins were present in four classes of Crustacea with 5 repeats in one species, and motifs that ended C-X(7)-C in Malacostraca. They were not detected, except as obvious contaminants, in any other arthropod subphyla or in any other phylum. The conservation of CPCFC proteins throughout the Pancrustacea and the small number of copies in individual species indicate that, when present, these proteins are serving important functions worthy of further study.

CutProtFam-Pred: detection and classification of putative structural cuticular proteins from sequence alone, based on profile hidden Markov models.

The arthropod cuticle is a composite, bipartite system, made of chitin filaments embedded in a proteinaceous matrix. The physical properties of cuticle are determined by the structure and the interactions of its two major components, cuticular proteins (CPs) and chitin. The proteinaceous matrix consists mainly of structural cuticular proteins. The majority of the structural proteins that have been described to date belong to the CPR family, and they are identified by the conserved R&R region (Rebers and Riddiford Consensus). Two major subfamilies of the CPR family RR-1 and RR-2, have also been identified from conservation at sequence level and some correlation with the cuticle type. Recently, several novel families, also containing characteristic conserved regions, have been described. The package HMMER v3.0 (http://hmmer.janelia.org/) was used to build characteristic profile Hidden Markov Models based on the characteristic regions for 8 of these families, (CPF, CPAP3, CPAP1, CPCFC, CPLCA, CPLCG, CPLCW, Tweedle). In brief, these families can be described as having: CPF (a conserved region with 44 amino acids); CPAP1 and CPAP-3 (analogous to peritrophins, with 1 and 3 chitin-binding domains, respectively); CPCFC (2 or 3 C-x(5)-C repeats); and four of five low complexity (LC) families, each with characteristic domains. Using these models, as well as the models previously created for the two major subfamilies of the CPR family, RR-1 and RR-2 (Karouzou et al., 2007), we developed CutProtFam-Pred, an on-line tool (http://bioinformatics.biol.uoa.gr/CutProtFam-Pred) that allows one to query sequences from proteomes or translated transcriptomes, for the accurate detection and classification of putative structural cuticular proteins. The tool has been applied successfully to diverse arthropod proteomes including a crustacean (Daphnia pulex) and a chelicerate (Tetranychus urticae), but at this taxonomic distance only CPRs and CPAPs were recovered.

Temporal and spatial expression of cuticular proteins of Anopheles gambiae implicated in insecticide resistance or differentiation of M/S incipient species.

BACKGROUND: Published data revealed that two of the 243 structural cuticular proteins of Anopheles gambiae, CPLCG3 and CPLCG4, are implicated in insecticide resistance and a third, CPF3, has far higher transcript levels in M than in S incipient species. We studied the distribution of transcripts for these three genes in the tissues of An. gambiae and the location of the proteins in the cuticle itself to gain information about how these cuticular proteins contribute to their important roles. Our data are consistent with CPLCG3/4 contributing to a thicker cuticle thus slowing penetration of insecticides and CPF3 possibly having a role in the greater desiccation tolerance of the M form. METHODS: Using RT-qPCR, we established the temporal expression of the genes and by in situ hybridization we revealed the main tissues where their mRNAs are found. Electron microscopy immunolocalization, using secondary antibodies labeled with colloidal gold, allowed us to localize these proteins within different regions of the cuticle. RESULTS: The temporal expression of these genes overlaps, albeit with higher levels of transcripts from CPF3 in pharate adults and both CPLCG3 and CPLCG4 are higher in animals immediately after adult eclosion. The main location of mRNAs for all three genes is in appendages and genitalia. In contrast, the location of their proteins within the cuticle is completely different. CPF3 is found exclusively in exocuticle and CPLCG3/4 is restricted to the endocuticle. The other CPF gene expressed at the same times, CPF4, in addition to appendages, has message in pharate adult sclerites. CONCLUSIONS: The temporal and spatial differences in transcript abundance and protein localization help to account for An. gambiae devoting about 2% of its protein coding genes to structural cuticular proteins. The location of CPLCG3/4 in the endocuticle may contribute to the thickness of the cuticle, one of the recently appreciated components of insecticide resistance, while the location of CPF3 might be related to the greater desiccation resistance of the M form.

Changes in transcript abundance for cuticular proteins and other genes three hours after a blood meal in Anopheles gambiae.

Numerous studies have examined changes in transcript levels after Anopheles gambiae takes a blood meal. Marinotti et al. (2006) used microarrays and reported massive changes in transcript levels 3 h after feeding (BF3h) compared to non-blood fed (NBF). We were intrigued by the number of transcripts for structural cuticular proteins (CPs) that showed such major differences in levels and employed paired-end (50 bp) RNA-seq technology to compare whole body transcriptomes from 5-day-old females NBF and BF3h. We detected transcripts for the majority of CPs (164/243) but levels of only 12 were significantly altered by the blood meal. While relative transcript levels of NBF females were somewhat similar to the microarray data, there were major differences in BF3h animals, resulting in levels of many transcripts, both for CPs and other genes changing in the opposite direction. We compared our data also to other studies done with both microarrays and RNA-seq. Findings were consistent that a small number of CP genes have transcripts that persist even in 5-day-old adults. Some of these transcripts showed diurnal rhythms (Rund et al., 2013; Rinker et al., 2013). In situ hybridization revealed that transcripts for several of these CP genes were found exclusively or predominantly in the eye. Transcripts other than for CPs that changed in response to blood-feeding were predominantly expressed in midgut and Malpighian tubules. Even in these tissues, genes responsible for proteins with similar functions, such as immunity or digestion, responded differently, with transcript levels for some rising and others falling. These data demonstrate that genes coding for some CPs are dynamic in expression even in adults and that the response to a blood meal is rapid and precisely orchestrated.

A possible structural model of members of the CPF family of cuticular proteins implicating binding to components other than chitin.

The physical properties of cuticle are determined by the structure of its two major components, cuticular proteins (CPs) and chitin, and, also, by their interactions. A common consensus region (extended R&R Consensus) found in the majority of cuticular proteins, the CPRs, binds to chitin. Previous work established that beta-pleated sheet predominates in the Consensus region and we proposed that it is responsible for the formation of helicoidal cuticle. Remote sequence similarity between CPRs and a lipocalin, bovine plasma retinol binding protein (RBP), led us to suggest an antiparallel beta-sheet half-barrel structure as the basic folding motif of the R&R Consensus. There are several other families of cuticular proteins. One of the best defined is CPF. Its four members in Anopheles gambiae are expressed during the early stages of either pharate pupal or pharate adult development, suggesting that the proteins contribute to the outer regions of the cuticle, the epi- and/or exo-cuticle. These proteins did not bind to chitin in the same assay used successfully for CPRs. Although CPFs are distinct in sequence from CPRs, the same lipocalin could also be used to derive homology models for one A. gambiae and one Drosophila melanogaster CPF. For the CPFs, the basic folding motif predicted is an eight-stranded, antiparallel beta-sheet, full-barrel structure. Possible implications of this structure are discussed and docking experiments were carried out with one possible Drosophila ligand, 7(Z),11(Z)-heptacosadiene.

Structural cuticular proteins from arthropods: annotation, nomenclature, and sequence characteristics in the genomics era.

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.

Extensive gene amplification and concerted evolution within the CPR family of cuticular proteins in mosquitoes.

Annotation of the Anopheles gambiae genome has revealed a large increase in the number of genes encoding cuticular proteins with the Rebers and Riddiford Consensus (the CPR gene family) relative to Drosophila melanogaster. This increase reflects an expansion of the RR-2 group of CPR genes, particularly the amplification of sets of highly similar paralogs. Patterns of nucleotide variation indicate that extensive concerted evolution is occurring within these clusters. The pattern of concerted evolution is complex, however, as sequence similarity within clusters is uncorrelated with gene order and orientation, and no comparable clusters occur within similarly compact arrays of the RR-1 group in mosquitoes or in either group in D. melanogaster. The dearth of pseudogenes suggests that sequence clusters are maintained by selection for high gene-copy number, perhaps due to selection for high expression rates. This hypothesis is consistent with the apparently parallel evolution of compact gene architectures within sequence clusters relative to single-copy genes. We show that RR-2 proteins from sequence-cluster genes have complex repeats and extreme amino-acid compositions relative to single-copy CPR proteins in An. gambiae, and that the amino-acid composition of the N-terminal and C-terminal sequence flanking the chitin-binding consensus region evolves in a correlated fashion.

Developmental expression patterns of cuticular protein genes with the R&R Consensus from Anopheles gambiae.

CPR proteins are the largest cuticular protein family in arthropods. The whole genome sequence of Anopheles gambiae revealed 156 genes that code for proteins with the R&R Consensus and named CPRs. This protein family can be divided into RR-1 and RR-2 subgroups, postulated to contribute to different regions of the cuticle. We determined the temporal expression patterns of these genes throughout post-embryonic development by means of real-time qRT-PCR. Based on expression profiles, these genes were grouped into 21 clusters. Most of the genes were expressed with sharp peaks at single or multiple periods associated with molting. Genes coding for RR-1 and RR-2 proteins were found together in several co-expression clusters. Twenty-five genes were expressed exclusively at one metamorphic stage. Five out of six X-linked genes showed equal expression in males and females, supporting the presence of a gene dosage compensation system in A. gambiae. Many RR-2 genes are organized into sequence clusters whose members are extremely similar to each other and generally closely associated on a chromosome. Most genes in each sequence cluster are expressed with the same temporal expression pattern and at the same level, suggesting a shared mechanism to regulate their expression.

Annotation and analysis of a large cuticular protein family with the R&R Consensus in Anopheles gambiae.

BACKGROUND: The most abundant family of insect cuticular proteins, the CPR family, is recognized by the R&R Consensus, a domain of about 64 amino acids that binds to chitin and is present throughout arthropods. Several species have now been shown to have more than 100 CPR genes, inviting speculation as to the functional importance of this large number and diversity. RESULTS: We have identified 156 genes in Anopheles gambiae that code for putative cuticular proteins in this CPR family, over 1% of the total number of predicted genes in this species. Annotation was verified using several criteria including identification of TATA boxes, INRs, and DPEs plus support from proteomic and gene expression analyses. Two previously recognized CPR classes, RR-1 and RR-2, form separate, well-supported clades with the exception of a small set of genes with long branches whose relationships are poorly resolved. Several of these outliers have clear orthologs in other species. Although both clades are under purifying selection, the RR-1 variant of the R&R Consensus is evolving at twice the rate of the RR-2 variant and is structurally more labile. In contrast, the regions flanking the R&R Consensus have diversified in amino-acid composition to a much greater extent in RR-2 genes compared with RR-1 genes. Many genes are found in compact tandem arrays that may include similar or dissimilar genes but always include just one of the two classes. Tandem arrays of RR-2 genes frequently contain subsets of genes coding for highly similar proteins (sequence clusters). Properties of the proteins indicated that each cluster may serve a distinct function in the cuticle. CONCLUSION: The complete annotation of this large gene family provides insight on the mechanisms of gene family evolution and clues about the need for so many CPR genes. These data also should assist annotation of other Anopheles genes.

Drosophila cuticular proteins with the R&R Consensus: annotation and classification with a new tool for discriminating RR-1 and RR-2 sequences.

The majority of cuticular protein sequences identified to date from a diversity of arthropods have a conserved region known as the Rebers and Riddiford Consensus (R&R Consensus). This consensus region has been used to query the whole genome sequence of Drosophila melanogaster. One hundred one putative cuticular proteins have been annotated. Of these, 29 had been annotated previously, and for several their authenticity as cuticular proteins had been verified by protein sequence data from isolated cuticles or by localization of their transcripts in epidermis when cuticle synthesis was occurring. The original names have been retained, and the 72 newly annotated proteins have been given names beginning with Cpr followed by the chromosomal band in which the gene is located. Proteins with the R&R Consensus can be split into three groups RR-1, RR-2 and RR-3, with some correlation to the type or region of the cuticle in which they occur. Previous classification was manual and subjective. We now have developed a tool using profile hidden Markov models that allows more objective classification. We describe the development and verification of the validity of this tool that is available at the cuticleDB website http://bioinformatics2.biol.uoa.gr/cuticleDB/index.jsp.

CPF and CPFL, two related gene families encoding cuticular proteins of Anopheles gambiae and other insects.

Cuticular proteins (CPs) are structural proteins of insects as well as other arthropods. Several CP families have been described, among them a small family defined by a 51 amino acid motif [Andersen, S.O., Rafn, K., Roepstorff, P., 1997. Sequence studies of proteins from larval and pupal cuticle of the yellow meal worm, Tenebrio molitor. Insect Biochem. Mol. Biol. 27, 121-131]. We identified four proteins of this family in Anopheles gambiae that we have named CPF. We have also identified CPFs from other insects by searching databases. Alignment of these CPF proteins showed that the conserved region is only 44 aa long and revealed another conserved motif at the C-terminus. A dendrogram divided the CPF proteins into four groups, one basal and three specialized. We also identified several proteins of another CP family, CPFL, which has similarities to CPFs. CPFs and CPFLs share some protein motifs. Expression studies with real-time qRT-PCR of the A. gambiae CPFs and CPFLs showed that the four CPFs and one CPFL gene are expressed just before pupal or adult ecdysis, suggesting that they are components of the outer layer of pupal and adult cuticles. The other CPFLs appear to contribute to larval cuticle. Recombinant CPF proteins did not bind to chitin in the assay we used.