Partial amino acid sequences of cuticular proteins from Hyalophora cecropia

The amino-terminal amino acid sequences for seven cuticular proteins from Hyalophora cecropia are reported. Proteins were purified by blotting two dimensional acrylamide gels onto acid-etched glass fiber filters, and the proteins were sequenced without further elution. The sequences of the serine-rich proteins from rigid cuticles revealed a new family of cuticular proteins, with features reminiscent of the amino-termini of certain vertebrate neurofilament proteins, members of the intermediate filament protein family which includes keratins. The proteins from flexible cuticles showed sequence similarity to proteins previously sequenced for Drosophila, Manduca and Sarcophaga. Proteins with identical electrophoretic mobility from two different metamorphic stages or from two anatomical regions within a single stage had identical amino-terminal sequences.     

Sequence determination of three cuticular proteins and isoforms from the migratory locust,Locusta migratoria,using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.     

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.     

Amino acid sequence determination of a protein purified from the shell of the shrimp,Pandalus borealis

One of the urea-extractable proteins in the shell of the shrimp Pandalus borealis has been purified, and the complete amino acid sequence has been determined by the combined use of mass spectrometry and Edman degradation of the intact protein as well as of enzymatically derived peptides.Characteristic features of the sequence are discussed and compared to sequences from insect cuticular proteins and other structural proteins.     

Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences

We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein.     

Involvement of tyrosine residues, N-terminal amino acids, and beta-alanine in insect cuticular sclerotization

During sclerotization of insect cuticle the acyldopamines, N-acetyldopamine (NADA) and N-beta-alanyldopamine (NBAD), are oxidatively incorporated into the cuticular matrix, thereby hardening and stabilizing the material by forming crosslinks between the proteins in the cuticular matrix and by forming polymers filling the intermolecular spaces in the cuticle. Sclerotized cuticle from the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor, was hydrolyzed in dilute hydrochloric acid, and from the hydrolysates some components presumably degradation products of cuticular crosslinks were isolated. In two of the components, the sidechain of 3,4-dihydroxyacetophenone was linked to the amino groups of glycine and beta-alanine, respectively, and in the third component to the phenolic group of tyrosine. These three compounds, glycino-dihydroxyacetophenone, beta-alanino-dihydroxyacetophenone, and O-tyrosino-dihydroxyacetophenone, as well as the previously reported compound, lysino-dihydroxyacetophenone [Andersen, S.O., Roepstorff, P., 2007. Aspects of cuticular sclerotization in the locust, Schistocerca gregaria, and the beetle, Tenebrio molitor. Insect Biochem. Mol. Biol. 37, 223-234], are suggested to be degradation products of cuticular crosslinks, in which amino acid residues formed linkages to both the alpha- and beta-positions of the sidechain of acyldopamines.     

The amino acid sequences of plastocyanin from Mercurialis perennis and Capsella bursa-pastoris

The amino acid sequences of the plastocyanins from Mercurialis perennis and Capsella bursa-pastoris have been determined. The amides at positions 64 and 68 in the Mercurialis sequence were positioned by ‘homology’ Both proteins are single polypeptide chains of 99 residues and are closely related to other higher plant plastocyanins.     

Characterization of cuticular proteins from the migratory locust,Locusta migratoria

Proteins were extracted from the still unhardened (teneral) cuticle of the migratory locust, Locusta migratoria. The proteins are soluble only at extreme pH-values and at low ionic strength, the solubility increases with decreasing temperature. The unhardened cuticle contains approx. 100 different proteins according to two-dimensional polyacrylamide gel electrophoresis. The majority of the proteins are very basic. The basicity and solubility properties of the proteins have necessitated development of modified electrophoretic procedures. The amino acid composition of the bulk protein shows that alanine, proline, glycine, valine and tyrosine constitute two thirds of the total amino acid content and that cysteine, methionine and tryptophan are absent.The proteins have been extracted from various parts of the cuticle and analysed by two-dimensional electrophoresis. Characteristic protein compositions were found for cuticle from the different body parts. Amino acid analyses of these extracts are strikingly similar. The only significant difference is in the glycine-alanine ratio. Cuticles that are destined to become hard are extremely rich in alanine, whereas the flexible parts of the cuticle are enriched in glycine. The results indicate that the proteins of locust cuticle constitute a group of structural proteins different from other known structural proteins.     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.     

Sequence determination of three cuticular proteins and isoforms from the migratory locust,Locusta migratoria,using a combination of Edman degradation and mass spectrometric techniques

The cuticle (exoskeleton) is a characteristic structure of insects and other arthropods. It is an extracellular layer which surrounds and protects the insect, and it is composed of proteins, lipids, water molecules, phenolic materials and chitin. Four proteins isolated from the thorax and femur cuticle of pharate adult migratory locust, Locusta migratoria, have been purified by ion-exchange chromatography and reversed-phase high performance liquid chromatography (RP-HPLC). Their amino acid sequences were determined by combined use of mass spectrometry and automated Edman degradation. The cuticular extract was also separated by two-dimensional gel electrophoresis. In order to localize and identify the position of the proteins in the gel, a number of gel spots were excised and the proteins electroeluted. The molecular mass of some of the electroeluted proteins was determined by means of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) as well as by electrospray mass spectrometry (ESI-MS). Two of the sequenced proteins exist as pairs of closely related isoforms; one of the pairs contains the conserved 68-residue RR-2 motif, common for proteins from solid cuticles, and the other proteins contain the short motif Ala-Ala-Pro-Ala/Val repeatedly throughout the sequence.     

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.     

Intriguing similarities between two novel odorant-binding proteins of locusts

Two novel odorant-binding proteins (OBPs) of locust, LmigOBP2 and LmigOBP3 are very different from each other and from the previously reported LmigOBP1 in their amino acid sequences. Moreover, OBP3 contains three additional cysteines, a fact not previously recorded in standard length OBPs. However, these two proteins exhibit remarkably similar binding affinities to a set of organic compounds. Such behaviour is supported by three-dimensional models, showing very similar folding for LmigOBP2 and LmigOBP3, but clearly different for LmigOBP1. Also several amino acid residues lining the binding pockets of the three proteins appear conserved in LmigOBP2 and LmigOBP3, but not in LmigOBP1. Western blot experiments revealed the presence of LmigOBP2 in antennae, mouth parts and cerci, but could not detected LmigOBP3 in any of these tissues. In immunocytochemistry, antibodies against LmigOBP2 strongly stained the outer lymph of sensilla chaetica of the antennae, in contrast with LmigOBP1, previously reported in sensilla basiconica.     

Structure of rabbit skeletal myosin: Analysis of the amino acid sequences of two fragments from the rod region

The amino acid sequences of fragments from light meromyosin and heavy meromyosin subfragment-2 have been analysed and structural features noted. As with other α-fibrous protein sequences, there is a regular disposition of apolar residues in positions a and d of the heptapeptide-type repeat characteristic of the coiled-coil conformation. The common occurrence of acidic and basic residues in the e and g positions, respectively, give rise to a maximum number of interchain ionic interactions when the two parallel chains of myosin are in axial register. Although the quasi-repeating heptapeptides in the sequences both have two points of discontinuity (unlike that in most other α-fibrous proteins), secondary structure prediction methods indicate that the fragments will be 90 to 100% α-helical. Fast Fourier transform techniques have revealed a significant periodicity of about 27.4 ± 0.3 residues (~41 Å) in the linear disposition of the acidic residues and the basic residues in both of the fragments. This period is compatible with similarly directed myosin molecules in the thick filament being axially staggered with respect to one another by an odd multiple of 143 Å. Preliminary evidence is also presented to show that the sequence of the rod region of myosin may have a 28 residue gene duplication repeat.     

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.     

The sialidase gene from Clostridium septicum: cloning, sequencing, expression in Escherichia coli and identification of conserved sequences in sialidases and other proteins

Summary An oligonucleotide mixture corresponding to the codons for conserved and repeated amino acid sequences of bacterial sialidases (Roggentin et al. 1989) was used to clone a 4.3 kb PstI restriction fragment of Clostridium septicum DNA in Escherichia coli. The complete nucleotide sequence of the sialidase gene was determined from this fragment. The derived amino acid sequence corresponds to a protein of 110000 Da. The ribosomal binding site and promoter-like consensus sequences were identified upstream from the putative ATG initiation codon. The molecular and immunological properties of the sialidase expressed by E. coli are similar to those of the sialidase as isolated from C. septicum. The newly synthesized protein is assumed to include a leader peptide of 26 amino acids. On sequence alignment, the sialidases from C. septicum, C. sordellii and C. perfringens show significant homologies. As in other bacterial sialidases, conserved amino acid sequences occur at four positions in the protein. Aside from the consensus sequences, only poor homology to other bacterial and viral sialidases was found. The consensus sequence could be identified even in other, non-sialidase proteins, indicating a common function or the evolutionary relatedness of these proteins.     

The sequence,organization, and evolution of the Locusta migratoria mitochondrial genome

The sequencing of the cloned Locusta migratoria mitochondrial genome has been completed. The sequence is 15,722 by in length and contains 75.3% A+T, the lowest value in any of the five insect mitochondrial sequences so far determined. The protein coding genes have a similar A+T content (74.1%) but are distinguished by a high cytosine content at the third codon position. The gene content and organization are the same as in Drosophila yakuba except for a rearrangement of the two tRNA genes tRNA^lys and tRNA^asp. The A+T-rich region has a lower A+T nucleotide content than in other insects, and this is largely due to the presence of two G+C-rich 155-bp repetitive sequences at the 5 end of this section and the beginning of the adjacent small rRNA gene. The sizes of the large and small rRNA genes are 1,314 and 827 bp, respectively, and both sequences can be folded to form secondary structures similar to those previously predicted for Drosophila. The tRNA genes have also been modeled and these show a strong resemblance to the dipteran tRNAs, all anticodons apparently being conserved between the two species. A comparison of the protein coding nucleotide sequences of the locust DNA with the homologous sequences of five other arthropods (Drosophila yakuba, Anopheles quadrimaculatus, Anopheles gambiae, Apis mellifera, and Artemia franciscana) was performed. The amino acid composition of the encoded proteins in Locusta is similar to that of Drosophila, with a Dayhoff distance twice that of the distance between the fruit fly and the mosquitoes. A phylogenetic analysis revealed the locust genes to be more similar to those of the Dipterans than to those of the honeybee at both the nucleotide and amino acid levels. A comparative analysis of tRNA orders, using crustacean mtDNAs as outgroups, supported this. This high level of divergence in the Apis genome has been noted elsewhere and is possibly an effect of directional mutation pressure having resulted in an accelerated pattern of sequence evolution. If the general assumption that the Holometabola are monophyletic holds, then these results emphasize the difficulties of reconstructing phylogenies that include lineages with variable substitution rates and base composition biases. The need to exercise caution in using information about tRNA gene orders in phylogenetic analysis is also illustrated. However, if the honeybee sequence is excluded, the correspondence between the other five arthropod sequences supports the findings of previous studies which have endorsed the use of mtDNA sequences for studies of phylogeny at deep levels of taxonomy when mutation rates are equivalent. Correspondence to: P.K. Flook     

Molecular and Functional Analysis of UDP-N-Acetylglucosamine Pyrophosphorylases from the Migratory Locust,Locusta migratoria

UDP-N-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA’s derived from two different genes (LmUAP1 and LmUAP2) in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with LmUAP1 double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species.