Characterization of proteins from arthrodial membranes of the lobster, Homarus americanus

A total of six proteins from the abdominal arthrodial membrane (intersegmental membrane) of the lobster, Homarus americanus, were purified and their amino acid sequences were determined by a combination of mass spectrometry and Edman degradation. The proteins are acidic with pI-values close to 4 and they all have molecular masses approximately 12 kDa. The sequences of five of the proteins differ in only a few residues, while the sixth protein differs from the others in more than half of the positions. Only little similarity is observed between the sequences of the arthrodial membrane proteins and those of proteins purified from the calcified parts of the exoskeleton of H. americanus. The arthrodial membrane proteins contain the Rebers-Riddiford consensus sequence common in proteins from insect cuticles. Comparison of the complete sequences to the sequences available in databases shows that the lobster membrane proteins are more closely related to proteins from insect pliant cuticles than to proteins derived from cuticles destined for sclerotization. Characteristic features in the protein sequences are discussed, and it is suggested that the various sequence regions have specific roles in determining the mechanical properties of arthrodial membranes.     

Exoskeletal proteins from the crab, Cancer pagurus.

Twelve proteins from calcified regions and five from flexible regions (arthrodial membranes) of the exoskeleton of Cancer pagurus have been purified and sequenced. One of the proteins from calcified exoskeleton is identical to one of the arthrodial membrane proteins. Several of the proteins from the calcified regions resemble proteins from corresponding regions of the exoskeleton of the lobster, Homarus americanus, in containing either two or four copies of an 18-residue sequence motif, which so far has been found only in crustacean calcified exoskeletons. The proteins obtained from the flexible arthrodial membranes resemble the proteins from lobster arthrodial membranes, and the similarities are shared with a number of proteins from flexible cuticles in insects, indicating that the common features in these proteins may be important for the mechanical properties of the materials in which they occur.     

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.     

Ribosomal Proteins S27E, P2, and L37A from Marine Invertebrates

Single complementary DNAs encoding sequences for 40S ribosomal proteins related to S27E from the American lobster Homarus americanus and mussel Mytilus galloprovincialis were characterized. Single genes for ribosomal proteins L37A and P2 from the gumboot chiton Cryptochiton stellerii are similarly described. The lobster S27E protein contains the highly conserved cysteine residues, suggesting its likely designation in the C₄ protein family containing zinc finger motifs. The lobster S27E protein also appears to have an intermediate gene copy number between lower and higher euckaryotes. Expression of the S27E protein in lobster hepatopancreas was slightly elevated during several postmolt and premolt stages. Chlorinated pesticide treatment significantly reduced S27E expression in hepatopancreas, indicating that this gene is responsive to endogenous and exogenous cues. Received March 6, 1998; accepted October 2, 1998.     

Cuticular Proteins in Insects and Crustaceans

Comparisons between crustacean and insect cuticles are hamperedby the paucity of cuticular protein sequences for the former.Sufficient complete sequences are available for insect cuticularproteins to allow recognition of conserved motifs and relationshipsamong proteins that reflect the type of cuticle from which theyhave been extracted. All five sequences from an arachnid andtwo of 14 from crustaceans have a motif found in the largestgroup of insect cuticular proteins. Numerous insights have beengained from studying insect cuticular proteins and their genes.These insights have been summarized in hopes of encouraginginterest in building on the foundations laid by Dorothy Skinnerwith the exoskeleton of Gecarcinus.     

Molecular cloning of a lobster Gβ subunit and Gβ expression in olfactory receptor neuron dendrites and brain neuropil

We have isolated from the olfactory organ of the American lobster (Homarus americanus) two cDNA clones with homology to β subunits of G proteins. LobG_β1 contained a complete open reading frame that predicted an amino acid sequence with >80% identity to G_β sequences from other species. LobG_β2 was a fragment of an open reading frame whose predicted amino acid sequence had 65–69% identity to other G_β sequences. LobG_β2 mRNA was not detectable in the brain, eye plus eyestalk, leg, dactyl, olfactory organ, or tail muscle. In contrast, lobG_β1 was expressed in all these tissues as a single mRNA species of 6.4 kb and a protein of 37 kD. In the brain and olfactory organ, G_β immunoreactivity was almost exclusively confined to neurites: the neuropil regions of the brain and the outer dendrites of the olfactory receptor neurons. Coimmunoprecipitation revealed that lobster G_β interacted with both G_αs and G_αq. LobG_β1 is likely to be involved in a wide range of signaling events including olfactory transduction and synaptic transmission in the brain. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 525–536     

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.     

Ferritin acts as a target site for the snowdrop lectin (GNA) in the midgut of the cotton leafworm Spodoptera littoralis

The snowdrop lectin GNA (Galanthus nivalis agglutinin) has been shown to possess insecticidal activity to a range of economically important insect pests. However, the precise mechanism of insecticidal action of GNA against insects remains unknown. In this investigation, we attempted to purify and identify receptor(s) responsible for binding of GNA in the larval midgut of a major lepidopteran pest (the cotton leafworm, Spodoptera littoralis) to better understand its mode of action. Therefore, cytoplasmic as well as membrane proteins from 800 larval midguts were chromatographed on a column with immobilized GNA. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis analysis of the proteins eluted from the GNA column followed by sequencing of the GNA‐binding proteins and BLAST analyses revealed that the N‐terminal sequences of a 24 kDa polypeptide purified from the cytoplasmic and membrane protein fraction revealed sequence similarity to sequences encoding heavy chain homologs of ferritin from Manduca sexta (76% sequence identity), Calpodes ethlius (80% sequence identity) and Bombyx mori (61% sequence identity). Furthermore, the N‐terminal sequence of a 31 kDa polypeptide from the membrane protein fraction showed sequence similarity to a light chain homolog of ferritin from Manduca sexta (88% sequence identity).     

Isolation and purification of proteins from the symbiosome membrane of yellow lupine root nodules

A unique feature of the symbiotic association between legume plants and rhizobia is the plant-derived membrane which separates the symbionts within root nodule; this membrane is termed the peribacteroid membrane (PBM). Although this membrane plays a vital role in facilitating transport and other processes in nodules, little is known about the proteins that are associated with and are an integral part of it. The objective of this work was to apply modern methods of protein purification to the characterisation of proteins of peribacteroid membrane from nodules of yellow lupine (Lupines luteus). The 17-kDa protein was isolated from purified peribacteroid membrane using size exclusion and ion exchange chromatography (FPLC). The N-terminal amino acid sequence of this protein was determined; the sequence does not match any of the previously reported lupine and other legume sequences. Following detergent solubilisation of purified peribacteroid membrane, integral proteins of 15 to 20 kDa were purified by size exclusion chromatography.     

Defining the fold space of membrane proteins: the CAMPS database

Recent progress in structure determination techniques has led to a significant growth in the number of known membrane protein structures, and the first structural genomics projects focusing on membrane proteins have been initiated, warranting an investigation of appropriate bioinformatics strategies for optimal structural target selection for these molecules. What determines a membrane protein fold? How many membrane structures need to be solved to provide sufficient structural coverage of the membrane protein sequence space? We present the CAMPS database (Computational Analysis of the Membrane Protein Space) containing almost 45,000 proteins with three or more predicted transmembrane helices (TMH) from 120 bacterial species. This large set of membrane proteins was subjected to single‐linkage clustering using only sequence alignments covering at least 40% of the TMH present in a given family. This process yielded 266 sequence clusters with at least 15 members, roughly corresponding to membrane structural folds, sufficiently structurally homogeneous in terms of the variation of TMH number between individual sequences. These clusters were further subdivided into functionally homogeneous subclusters according to the COG (Clusters of Orthologous Groups) system as well as more stringently defined families sharing at least 30% identity. The CAMPS sequence clusters are thus designed to reflect three main levels of interest for structural genomics: fold, function, and modeling distance. We present a library of Hidden Markov Models (HMM) derived from sequence alignments of TMH at these three levels of sequence similarity. Given that 24 out of 266 clusters corresponding to membrane folds already have associated known structures, we estimate that 242 additional new structures, one for each remaining cluster, would provide structural coverage at the fold level of roughly 70% of prokaryotic membrane proteins belonging to the currently most populated families. Proteins 2006. © 2006 Wiley‐Liss, Inc.     

Characterization of SSRs from the American lobster,Homarus americanus

The American lobster (Homarus americanus), a commercially important benthic marine crustacean, is widely distributed along the continental shelf of the western North Atlantic. The population substructure of this species remains poorly understood despite its economic value. Informative markers are required to clarify relationships between local populations. To this end, we developed eight polymorphic short sequence repeats (SSR) for the American lobster, which were derived from expressed sequence tags. Additionally, we tested four SSRs previously identified for the Norway lobster (Nephrops norvegicus L.) for cross‐species utility; only one of these showed polymorphism.     

Modeling repetitive,non‐globular proteins

While ab initio modeling of protein structures is not routine, certain types of proteins are more straightforward to model than others. Proteins with short repetitive sequences typically exhibit repetitive structures. These repetitive sequences can be more amenable to modeling if some information is known about the predominant secondary structure or other key features of the protein sequence. We have successfully built models of a number of repetitive structures with novel folds using knowledge of the consensus sequence within the sequence repeat and an understanding of the likely secondary structures that these may adopt. Our methods for achieving this success are reviewed here.     

Proteolytic processing of the vitellogenin precursor in the boll weevil,Anthonomus grandis

The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M_rs of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M_r honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M_r boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10–15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein. © 1993 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

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.     

Protein Sequences of Two Keto Ester Reductases: Possible Identity as Hypothetical Proteins

We determined the amino acid sequences of two keto ester reductases (YKER-V and -VI) purified from a cell-free extract of Saccharomyces cerevisiae. The N-terminal and internal amino acid sequences of YKER-VI (AcKR) were in agreement with the sequence of hypothetical 36.4-kDa protein (S. cerevisiae chromosome X reading frame ORF YJR105w) in yeast. The N-terminal amino acid sequence of YKER-V was also identical with that of the hypothetical protein coded by yeast chromosome XIV or II. These results suggested that two hypothetical proteins were expressed as keto ester reductases in yeast cells.     

A conserved domain in arthropod cuticular proteins binds chitin

Many insect cuticular proteins include a 35-36 amino acid motif known as the R&R consensus. The extensive conservation of this region led to the suggestion that it functions to bind chitin. Provocatively, it has no sequence similarity to the well-known cysteine-containing chitin-binding domain found in chitinases and some peritrophic membrane proteins. Using fusion proteins expressed in E. coli, we show that an extended form of the R&R consensus from proteins of hard cuticles is necessary and sufficient for chitin binding. Recombinant AGCP2b, a putative cuticular protein from the mosquito Anopheles gambiae, was expressed in E. coli and the purified protein shown to bind to chitin beads. A stretch of 65 amino acids from AGCP2b, including the R&R consensus, conferred chitin binding to glutathione-S-transferase (GST). Directed mutagenesis of some conserved amino acids within this extended R&R consensus from hard cuticle eliminated chitin binding. Thus arthropods have two distinct classes of chitin binding proteins, those with the chitin-binding domain found in lectins, chitinases and peritrophic membranes (cysCBD) and those with the cuticular protein chitin-binding domain (non-cysCBD).     

Chlorinated tyrosine derivatives in insect cuticle

A method for quantitative measurement of 3-monochlorotyrosine and 3,5-dichlorotyrosine in insect cuticles is described, and it is used for determination of their distribution in various cuticular regions in nymphs and adults of the desert locust, Schistocerca gregaria. The two chlorinated tyrosine derivatives were present in all analyzed regions in mature adult locusts, the highest concentrations were found in the sclerotized cuticle of femur and tibia, but significant amounts were also present in the unsclerotized arthrodial membranes. Small amounts of the two amino acids were obtained from pharate, not-yet sclerotized cuticle of adult femur and tibia, the amounts increased rapidly during the first 24 h after ecdysis and more slowly during the next two weeks. Control analyses using stable isotope dilution mass spectrometry have confirmed that the chlorinated tyrosines are not artifacts formed during sample hydrolysis. Mono- and dichlorotyrosine are also present in cuticular samples from other insect species, such as the beetle, Tenebrio molitor, the moth Hyalophora cecropia, the cockroach Blaberus craniifer, and the bug Rhodnius prolixus, but not in the sclerotized puparial cuticle of the blowfly, Calliphora vicina, or in sclerotized ootheca from the cockroach, Periplaneta americana. Cuticular sclerotization and formation of chlorotyrosines occur simultaneously in locust legs; sclerotized cuticles tend to have a higher content of chlorotyrosines than unsclerotized cuticles, but it is concluded that the chlorotyrosines are not just a by-product from the sclerotization process.     

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.