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 β-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 β-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 β-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.     

Annotation and expression analysis of cuticular proteins from the tobacco hornworm,Manduca sexta

The insect cuticle is a unique material that covers the exterior of the animal as well as lining the foregut, hindgut, and tracheae. It offers protection from predators and desiccation, defines body shape, and serves as an attachment site for internal organs and muscle. It has demonstrated remarkable variations in hardness, flexibility and elasticity, all the while being light weight, which allows for ease of movement and flight. It is composed primarily of chitin, proteins, catecholamines, and lipids. Proteomic analyses of cuticle from different life stages and species of insects has allowed for a more detailed examination of the protein content and how it relates to cuticle mechanical properties. It is now recognized that several groups of cuticular proteins exist and that they can be classified according to conserved amino acid sequence motifs. We have annotated the genome of the tobacco hornworm, Manduca sexta, for genes that encode putative cuticular proteins that belong to seven different groups: proteins with a Rebers and Riddiford motif (CPR), proteins analogous to peritrophins (CPAP), proteins with a tweedle motif (CPT), proteins with a 44 amino acid motif (CPF), proteins that are CPF-like (CPFL), proteins with an 18 amino acid motif (18 aa), and proteins with two to three copies of a C-X₅-C motif (CPCFC). In total we annotated 248 genes, of which 207 belong to the CPR family, the most for any insect genome annotated to date. Additionally, we discovered new members of the CPAP family and determined that orthologous genes are present in other insects. We established orthology between the M. sexta and Bombyx mori genes and identified duplication events that occurred after separation of the two species. Finally, we utilized 52 RNAseq libraries to ascertain gene expression profiles that revealed commonalities and differences between different tissues and developmental stages.     

Genome-wide identification and analysis of genes encoding cuticular proteins in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae)

The multifunctional insect cuticle serves as the exoskeleton, determines body shape, restricts water loss, provides attachment sites for muscles and internal organs and is a formidable barrier to invaders. It is morphologically divided into three layers, including envelope, epicuticle, and procuticle and is composed of chitin and cuticular proteins (CPs). Annotation of CPs and their cognate genes may help understand the structure and functions of insect cuticles. In this paper, we interrogated the genome of Pteromalus puparum, an endoparasitoid wasp that parasitizes Pieris rapae and Papilio xuthus pupae, and identified 82 genes encoding CPs belonging to six CP families, including 62 in the CPR family, 8 in CPAP3, 5 in CPF/CPFL, 2 low complexity proteins, 2 in TWDL, and 3 in Apidermin. We used six RNA-seq libraries to determine CP gene expression profiles through development and compared the cuticle hydrophobicity between the P. puparum and the ectoparasitoid Nasonia vitripennis based on GRAVY values of CPR sequences. In the Nasonia-Pteromalus comparison, we found in both N. vitripennis and P. puparum, the peak of their CPR hydrophobicity displayed at their pupal stage, whereas their adult stage showed the lowest level. Except at the adult stage, the CPR hydrophobicity in N. vitripennis is always higher than P. puparum. Finally, we identified three novel Apidermin genes, a family found solely in Hymenoptera and revealed a new sequence feature of this family. This new information contributes to a broader understanding of insect CPs generally.     

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

Background

Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.

Methodology/Principal Findings

Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.

Conclusions

This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.     

Shotgun proteomic analysis of wing discs from the domesticated silkworm (Bombyx mori) during metamorphosis

Proteomic profiles from the wing discs of silkworms at the larval, pupal, and adult moth stages were determined using shotgun proteomics and MS sequencing. We identified 241, 218, and 223 proteins from the larval, pupal, and adult moth stages, respectively, of which 139 were shared by all three stages. In addition, there were 55, 37, and 43 specific proteins identified at the larval, pupal, and adult moth stages, respectively. More metabolic enzymes were identified among the specific proteins expressed in the wing disc of larvae compared with pupae and moths. The identification of FKBP45 and the chitinase-like protein EN03 as two proteins solely expressed at the larval stage indicate these two proteins may be involved in the immunological functions of larvae. The myosin heavy chain was identified in the pupal wing disc, suggesting its involvement in the formation of wing muscle. Some proteins, such as proteasome alpha 3 subunits and ribosomal proteins, specifically identified from the moth stage may be involved in the degradation of old cuticle proteins and new cuticle protein synthesis. Gene ontology analysis of proteins specific to each of these three stages enabled their association with cellular component, molecular function, and biological process categories. The analysis of similarities and differences in these identified proteins will greatly further our understanding of wing disc development in silkworm and other insects.     

昆虫表皮蛋白及其基因表达调控机理的研究进展

昆虫表皮蛋白(insectcuticular proteins,ICP)是结构蛋白,它和几丁质一起组成昆虫抵御外界环境的屏障——角质层。根据保守性基序的不同,ICP可分为CPR、CPF、CPFL、CPG和CPT5家族。它们都有独特的结构与性质。环境、激素、转录因子和内含子等共同影响昆虫表皮蛋白基因(insect cuticular protein genes,ICPG)的表达,进而使ICPG具有时期和组织特异性。ICP和ICPG被认为是研究昆虫蜕皮与变态的调控机理和理解昆虫发育期角质层在生物化学、物理化学以及结构上的修饰的重要模型,受到越来越多的重视。本文综述了ICP的分类以及环境、激素、转录因子和内含子等对ICPG表达的调控。     

Pupal and larval cuticle proteins of Drosophila melanogaster

Proteins, soluble in 7 M urea, were extracted from third-instar larval and pupal cuticles of Drosophila melanogaster. Both extracts contain a limited number of polypeptides resolved by one- or two-dimensional electrophoresis. The five major larval proteins have low molecular weights (less than 20000) and are not glycosylated. The major pupal cuticle proteins fall into two size classes: two with apparent molecular weights of 56K and 82K and four with molecular weights between 15K and 25K. The proteins with high apparent molecular weights are glycosylated. In nondenaturing gels, no components of the larval and pupal cuticle extracts comigrate. One-dimensional "fingerprints" indicate that cuticle proteins from these two stages have unique primary structures. Immunological results indicate that the major low molecular weight larval and pupal cuticle proteins are comprised of two families of proteins that share antigenic determinants. The high molecular weight pupal cuticle proteins are immunologically unrelated to the low molecular weight components. We conclude that the pupal and larval proteins are encoded in part by multigene families that have arisen by gene duplication and evolutionary divergence.     

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.     

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.     

Two gene families clustered in a small region of the Drosophila genome

Three Drosophila genes that are clustered within 8 X 10(3) bases of DNA at the chromosomal region 44D have been identified and mapped, and the gene cluster entirely sequenced. The three genes are 55 to 60% homologous in DNA sequence. One gene contains an intron in its 5'-proximal protein coding sequence while the other two have none at this position; similarly, another gene has an intron in its 3'-proximal protein coding sequence which is not found in the other genes. All three genes are abundantly expressed together in Drosophila first, second, and early third instar larval stages and in adults, but they are not abundantly expressed in either embryonic, late third instar larval, or pupal stages. This gene family lies 11 X 10(3) bases away from another cluster containing four Drosophila larval cuticle protein genes plus a pseudogene. The cuticle genes are all abundantly expressed throughout third instar larval development. Thus, at least seven protein-coding genes and one pseudogene lie within 27 X 10(3) bases of DNA. Moreover, two small gene families can lie adjacent on a chromosome and exhibit different patterns of developmental regulation, even though individual genes within each clustered family are co-ordinately expressed.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Identity and expression pattern of chemosensory proteins in Heliothis virescens (Lepidoptera, Noctuidae)

Analyzing the chemosensory organs of the moth Heliothis virescens, three proteins belonging to the family of insect chemosensory proteins (CSPs) have been cloned; they are called HvirCSP1, HvirCSP2 and HvirCSP3. The HvirCSPs show about 50% identity between each other and 30–76% identity to CSPs from other species. Overall, they are rather hydrophilic proteins but include a conserved hydrophobic motif. Tissue distribution and temporal expression pattern during the last pupal stages were assessed by Northern blots. HvirCSP mRNAs were detected in various parts of the adult body with a particular high expression level in legs. The expression of HvirCSP1 in legs started early during adult development, in parallel with the appearance of the cuticle. HvirCSP1 mRNA was detectable five days before eclosion (day E-5), increased dramatically on day E-3 and remained at high level into adult life. The tissue distribution and the time course of appearance of HvirCSPs are in agreement with a possible role in contact chemosensation.     

Temporal programming of epidermal cell protein synthesis during the larval-pupal transformation ofManduca sexta

Summary During the final larval instar the epidermis of the tobacco hornworm,Manduca sexta, synthesizes the larval cuticular proteins and the pigment insecticyanin. Then at the onset of metamorphosis the cells first become pupally-committed, then later produce the pupal cuticle. The changes in the pattern of epidermal protein synthesis during this period were followed by incubating the integument in vitro with either³H-leucine or³⁵S-methionine, then analyzing the proteins by 2-dimensional gel electrophoresis. Precipitation by larval and pupal cuticular antisera and by insecticyanin antibody identified these proteins. Three distinct changes in epidermal protein synthesis were noted: 1) Stage-specific proteins, some of which are larval cuticular proteins, appear just before and during the change of commitment on day 3. (2) By late the following day (wandering stage), synthesis of these and many other proteins including all the identified larval cuticular proteins and insecticyanin was undetectable. Several noncuticular proteins were transiently synthesized by this pupally committed cell during wandering and sometimes the following day. (3) During the production of pupal cuticle a new set of pupal-specific cuticular proteins as well as some common cuticular proteins (precipitated by both antisera) were synthesized. Some of the latter were also synthesized during the period between pupal commitment and pupal cuticle deposition.In spite of an apparent absence of methionine in both larval and pupal cuticle, many cuticular proteins incorporated³⁵S-methionine. Thus they may be synthesized as proproteins.Insecticyanin was shown to have two forms differing in isoelectric point, the cellular form being more acidic than the hemolymph form. Synthesis of the cellular form ceased before that of the hemolymph form.     

Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry

Identification of authenticated cuticular proteins has been based on isolation and sequencing of individual proteins extracted from cleaned cuticles. These data facilitated classification of sequences from conceptual translation of cDNA or genomic sequences. The question arises whether such putative cuticular proteins actually are incorporated into the cuticle. This paper describes the profiling of cuticular proteins from Anopheles gambiae starting with cuticle cleaned by the insect itself in the course of molting. Proteins extracted from cast larval head capsules and cast pupal cuticles were fractionated by 1D SDS gel electrophoresis. Large gel slices were reduced, carbamidomethylated and digested with trypsin. The pellet remaining after SDS extraction was also treated with trypsin. The resulting peptides were separated on a C18 column and then analyzed by tandem mass spectrometry. Two-hundred-ninety-five peptides from putative cuticular proteins were identified; these corresponded to a minimum of 69 and a maximum of 119 different proteins. Each is reported as an authentic Anopheles cuticular protein for the first time. In addition to members of two known cuticular protein families, members of additional families likely to be structural components of the cuticle were identified. Furthermore, other peptides were identified that can be attributed to molting fluid, muscle and sclerotizing agents.     

Aspects of cuticular sclerotization in the locust, Scistocerca gregaria, and the beetle, Tenebrio molitor

The number of reactive amino groups in cuticular proteins decreases during the early period of insect cuticular sclerotization, presumably due to reaction with oxidation products of N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD). We have quantitated the decrease in cuticular N-terminal amino groups and lysine epsilon-amino groups during the first 24h of sclerotization in adult locusts, Schistocerca gregaria, and in larval and adult beetles, Tenebrio molitor, as well as the increase in beta-alanine amino groups in Tenebrio cuticle. The results indicate that nearly all glycine N-terminal groups and a significant part of the epsilon-amino groups from lysine residues are involved in the sclerotization process in both locusts and Tenebrio. A pronounced increase in the amount of free beta-alanine amino groups was observed in cuticle from adult Tenebrio and to a lesser extent also in Tenebrio larval cuticle, but from locust cuticle no beta-alanine was obtained. Hydrolysis of sclerotized cuticles from locusts and Tenebrio by dilute hydrochloric acid released a large number of compounds containing amino acids linked to catecholic moieties. Products have been identified which contain histidine residues linked via their imidazole group to the beta-position of various catechols, such as dopamine, 3,4-dihydroxyphenyl-ethanol (DOPET), and 3,4-dihydroxyphenyl-acetaldehyde (DOPALD), and a ketocatecholic compound has also been identified composed of lysine linked via its epsilon-amino group to the alpha-carbon atom of 3,4-dihydroxyacetophenone. Some of the hydrolysis products have previously been obtained from sclerotized pupal cuticle of Manduca sexta [Xu, R., Huang, X., Hopkins, T.L., Kramer, K.J., 1997. Catecholamine and histidyl protein cross-linked structures in sclerotized insect cuticle. Insect Biochemistry and Molecular Biology 27, 101-108; Kerwin, J.L., Turecek, F., Xu, R., Kramer, K.J., Hopkins, T.L., Gatlin, C.L., Yates, J.R., 1999. Mass spectrometric analysis of catechol-histidine adducts from insect cuticle. Analytical Biochemistry 268, 229-237; Kramer, K.J., Kanost, M.R., Hopkins, T.L., Jiang, H., Zhu, Y.C., Xu, R., Kerwin, J.L., Turecek, F., 2001. Oxidative conjugation of catechols with proteins in insect skeletal systems. Tetrahedron 57, 385-392], but the lysine-dihydroxyacetophenone compound and the histidine-DOPALD adduct have not been reported before. It is suggested that the compounds are derived from NADA and NBAD residues which were incorporated into the cuticle during sclerotization, and that the lysine-dihydroxyacetophenone as well as the DOPET and DOPALD containing adducts are degradation products derived from cross-links between the cuticular proteins, whereas the dopamine-containing adducts are derived from a non-crosslinking reaction product.     

FURTHER STUDY RE-EXAMINING WHETHER LARVAL EPIDERMIS CAN OMIT THE SECRETION OF A PUPAL CUTICLE, USING THE SILKWORM, BOMBYX MORI

When larval tissue is exposed to a hormonal milieu lacking juvenile hormone, adult characters appear directly, omitting the pupal stage, in some insects but not in others, including Bombyx mori. An attempt was made to induce omission of pupal characters in this species by varying the stage of the larval epidermis to be tested. Pieces of larval integument taken from fourth- and fifth-instar larvae of various stages were transplanted to developing adults. Although the number of cuticle layers and the types of cuticle produced differed depending on the age of the donors, none of the pieces omitted secreting the pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle, and that a transition of tissue competence may play an important part in the sequential appearance of larval, pupal, and adult characters.     

Protein crosslinking by transglutaminase controls cuticle morphogenesis in Drosophila

Transglutaminase (TG) plays important and diverse roles in mammals, such as blood coagulation and formation of the skin barrier, by catalyzing protein crosslinking. In invertebrates, TG is known to be involved in immobilization of invading pathogens at sites of injury. Here we demonstrate that Drosophila TG is an important enzyme for cuticle morphogenesis. Although TG activity was undetectable before the second instar larval stage, it dramatically increased in the third instar larval stage. RNA interference (RNAi) of the TG gene caused a pupal semi-lethal phenotype and abnormal morphology. Furthermore, TG-RNAi flies showed a significantly shorter life span than their counterparts, and approximately 90% of flies died within 30 days after eclosion. Stage-specific TG-RNAi before the third instar larval stage resulted in cuticle abnormality, but the TG-RNAi after the late pupal stage did not, indicating that TG plays a key role at or before the early pupal stage. Immediately following eclosion, acid-extractable protein from wild-type wings was nearly all converted to non-extractable protein due to wing maturation, whereas several proteins remained acid-extractable in the mature wings of TG-RNAi flies. We identified four proteins--two cuticular chitin-binding proteins, larval serum protein 2, and a putative C-type lectin-as TG substrates. RNAi of their corresponding genes caused a lethal phenotype or cuticle abnormality. Our results indicate that TG-dependent protein crosslinking in Drosophila plays a key role in cuticle morphogenesis and sclerotization.