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PubMed1973

The contractile and regulatory proteins of insect flight muscle.

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.

Actins↗
PubMed1995

Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells.

Small GTP binding proteins of the rab/YPT family are essential regulators of vectorial transport in the eukaryotic cell. Members of the rab/YPT1 family are found on the cytoplasmic surface of distinct intracellular membrane compartments. Membrane attachment is facilitated by a C-terminal geranylgeranyl moiety. In this report we investigated posttranslational modification and membrane binding of the rab6 protein, a member of the rab/YPT family located on the Golgi apparatus. A set of point mutations, which simulate the GDP or GTP bound conformation, was introduced into the rab6 cDNA. The mutated cDNAs were expressed in insect cells and the ability of the protein products to undergo geranylgeranyl modification and membrane association was assessed by Triton X-114 partition and cell fractionation. We report here that the modification of rab6 in insect cells depends on protein conformation. Only the GDP bound form, but not the GTP bound form is isoprenylated and subsequently membrane bound.

Amino Acid Sequence↗
PubMed1995

Expression of TAR RNA-Binding Protein in Baculovirus and Co-Immunoprecipitation with Insect Cell Protein Kinase.

TAR RNA-binding protein TRBP was originally isolated by its binding affinity for radiolabeled HIV-1 leader RNA. Subsequent studies have suggested that this protein is one member of a family of double-stranded RNA-binding proteins. Recent findings indicate that TRBP might function to antagonize the translational inhibitory effect that can be mediated through cellular protein kinase, PKR. Here, we report on the over-expression of a cDNA coding for TRBP in eukaryotic SF9 cells using baculovirus. We characterized the nuclear localization of TRBP in insect cells, and we demonstrate that TRBP co-immunoprecipitates with a protein in these cells antigenically related to human PKR. Copyright 1995 S. Karger AG, Basel

Journal Article↗
PubMed2004

Membrane binding properties and terminal residues of the mature hepatitis C virus capsid protein in insect cells.

The immature core protein (p23, residues 1 to 191) of hepatitis C virus undergoes posttranslational modifications including intramembranous proteolysis within its C-terminal signal sequence by signal peptide peptidase to generate the mature form (p21). In this study, we analyzed the cleavage site and other amino acid modifications that occur on the core protein. To produce the posttranslationally modified core protein, we used a baculovirus-insect cell expression model system. As previously reported, p23 is processed to form p21 in insect as well as in mammalian cells. p21 was found to be associated with the cytoplasmic membrane, and its significant portion behaved as an integral membrane protein. The protein was purified from the membrane by a simple and unique procedure on the basis of its membrane-binding properties and solubility in detergents. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of purified p21 showed that the average molecular mass (m/z 19,307) of its single-charged ion differs by m/z 1,457 from that calculated for p23. To determine the posttranslational modifications, tryptic p21 peptides were analyzed by MALDI-TOF MS. We found three peptides that did not match the theoretically derived peptides of p23. Analysis of these peptides by MALDI-TOF tandem MS revealed that they correspond to N-terminal peptides (residues 2 to 9 and 2 to 10) starting with alpha-N-acetylserine and C-terminal peptide (residues 150 to 177) ending with phenylalanine. These results suggest that the mature core protein (molecular mass of 19,306 Da) includes residues 2 to 177 and that its N terminus is blocked with an acetyl group.

Amino Acid Sequence↗
PubMed1999

Structural characteristics of tenecin 3, an insect antifungal protein.

Tenecin 3, an antifungal protein, previously isolated from the insect Tenebrio molitor, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 remain unknown. As an initial step to study the mechanism and functions, physical and structural properties of tenecin 3 were examined by circular dichroism (CD) analysis and 2D nuclear overhauser effect spectroscopy. These analyses suggest that tenecin 3 has a propensity of random structure with very loose turn-like elements. The CD results also indicate that this random structural propensity is not significantly affected by temperature, pH, and by the presence of organic solvents or sodium dodecyl sulfate (SDS) micelles. However, the hydrodynamic studies suggest that tenecin 3 is not in extended form in spite of its random structural feature.

Animals↗
PubMed1990

Hemolin: an insect-immune protein belonging to the immunoglobulin superfamily.

Insects have an efficient defense system against infections. Their antibacterial immune proteins have been well characterized. However, the molecular mechanisms by which insects recognize foreignness are not yet known. Data are presented showing that hemolin (previously named P4), a bacteria-inducible hemolymph protein of the giant silk moth Hyalophora cecropia, belongs to the immunoglobulin superfamily. Functional analyses indicate that hemolin is one of the first hemolymph components to bind to the bacterial surface, taking part in a protein complex formation that is likely to initiate the immune response.

Amino Acid Sequence↗
PubMed1998

Bacterial expression of tenecin 3, an insect antifungal protein isolated from Tenebrio molitor, and its efficient purification.

Tenecin 3, an antifungal protein isolated from the insect Tenebrio molitor larvae, inhibits growth of the fungus Candida albicans. However, the antifungal mechanism and functions of tenecin 3 have not yet been studied due to its very low availability from the natural source. Here we report an expression system of the recombinant tenecin 3 in E. coli, whose amino acid composition is the same with that of the natural tenecin 3. We also devised a simple and easy procedure to isolate the recombinant protein from the bacterial cell extracts. The recombinant tenecin 3 showed an antifungal activity against C. albicans as the natural tenecin 3 did. Therefore large quantities of tenecin 3 can be easily obtained by the expression and purification system of tenecin 3 described in this report.

Amino Acid Sequence↗
PubMed1994

Self-assembly of human papillomavirus type 16 capsids by expression of the L1 protein in insect cells.

The L1 major protein of human papillomavirus type 16 was expressed in Sf-21 insect cells with a recombinant baculovirus vector. Virus-like particles similar in appearance to empty virions were identified by electron microscopy at densities of 1.29-1.30. Purified particles reacted with monoclonal anti-HPV-16-L1 antibody in Western blot and immuno dot blot suggesting that conformational epitopes are present in the recombinant particles. Immunodot blot assays using human sera correlated with the detection of HPV-16 DNA by the polymerase chain reaction. The results suggest that HPV-16-L1 virions produced by the baculovirus system might be useful for developing serologic tests to measure antibodies to conformational epitopes and may offer potential for vaccine development.

Animals↗
PubMed1987

Characterization of the toxicity and cytopathic specificity of a cloned Bacillus thuringiensis crystal protein using insect cell culture.

An insecticidal protein gene from Bacillus thuringiensis var. aizawai was cloned in Escherichia coli. The cloned gene expressed at a high level and the synthesized protein appeared as an insoluble, phase-bright inclusion in the cytoplasm. These inclusions were isolated by density gradient centrifugation, the isolated protein was activated in vitro by different proteolytic regimes and the toxicity of the resulting preparations was studied using insect cells grown in tissue culture. The inclusions consisted of a 130 kDa polypeptide which was processed to a protease-resistant 55 kDa protein by tryptic digestion. This preparation lysed lepidopteran (Choristoneura fumiferana) CF1 cells but not dipteran (Aedes albopictus) cells. When the crystal protein was activated by sequential treatment, first with trypsin and then with Aedes aegypti gut proteases, the resulting 53 kDa polypeptide was now toxic only to the dipteran cells and not to the lepidopteran cells. Thus the dual specificity of this var. aizawai toxin results from differential proteolytic processing of a single protoxin. The trypsin-activated preparation was weakly active against Spodoptera frugiperda cells. Membrane binding studies of the trypsin-activated toxin revealed a 68 kDa protein in the lepidopteran cell membranes, which may be the receptor for this toxin.

Animals↗
PubMed1985

Arthrin: a new actin-like protein in insect flight muscle.

There are one or more proteins of 50,000 to 60,000 Mr in the thin filaments of insect flight muscle. A protein of 55,000 Mr has been isolated from insect fibrillar flight muscle and called arthrin. Despite its higher molecular weight, arthrin is in many ways like actin. The amino acid composition of arthrin was similar to that of actin. There were similarities in the peptides produced by digesting the denatured proteins and mild digestion of polymerized proteins cleaved similar-sized fragments from arthrin and actin. Polymerized arthrin activated the Mg2+ ATPase of myosin to the same extent as actin and the ATPase was regulated by rabbit or Lethocerus troponin and tropomyosin. Arthrin did not itself act as troponin-T. Electron microscopy of negatively stained specimens showed that arthrin and actin filaments were similar in structure and that arthrin could be decorated by rabbit subfragment-1 to form normal-looking arrowheads. Arthrin formed paracrystals at an optimum concentration of MgCl2 (25 mM) that was somewhat lower than the optimum for actin paracrystals. Optical diffraction showed that the structure of the paracrystals was similar to those formed from actin. The mass of arthrin and actin filaments relative to phage fd was measured by scanning transmission electron microscopy; the relative mass of arthrin and actin was 1.33, in agreement with molecular weight estimations. Therefore arthrin has the properties of a heavy form of actin. The proportion of actin, arthrin and troponin-T in Lethocerus myofibrils was six moles of actin to one mole of arthrin and one mole of troponin-T. The function of arthrin is not known.

Actins↗
PubMed1996

Sequencing analysis of cDNA clones encoding the American cockroach Cr-PI allergens. Homology with insect hemolymph proteins.

A previous article described the isolation of several lambdagt22A cDNA clones expressing the American cockroach (Periplaneta americana) Cr-PI allergens recognized by both human atopic IgE antibodies and anti-Cr-PI monoclonal antibodies (Wu, C. H., Lee, M. F., and Liao, S. C.(1995) J. Allergy Clin. Immunol. 96, 352-359). This article presents the nucleotide and deduced amino acid sequences of two cDNA clones encoding major allergens of P. americana. Clones C12 and C20 encode proteins of 685 and 631 amino acids with two potential N-glycosylation sites each. The predicted molecular weights for C12 and C20 cloned proteins are 79,300 and 75, 500 with isoelectric point values of 6.26 and 6.63, which are compatible with the determined sizes (Mr 78,000 and 72,000) and isoelectric point value (6.2) of the Cr-PI allergens of P. americana. A high degree of identity (69.1%), including several overlapped predicted central antigenic determinant residues, was found between two allergens. The anti-fusion protein antibody-based enzyme-linked immunosorbent assay was able to detect crude American cockroach extract, Cr-PI, recombinant proteins, and commercial cockroach extracts, which provides further evidence that two allergens share common antigen determinants. Recombinant allergens of clones C12 and C20 both showed 47.4% skin reactivities on 19 cockroach-sensitive asthmatic patients. Unexpectedly, although no sequence similarity was found to other known allergens, two aromatic amino acid-rich allergens were found to have a striking sequence identity to insect storage proteins (20.1-33.9%), insect juvenile hormone-suppressible proteins (30.9-36.4%), and arthropod hemocyanins (29.7-34.6%). Results suggested that two prominent allergens of P. americana are ancestrally related to these insect hemolymph proteins and represent a new group of proteins in the hemocyanin superfamily. These data will now facilitate epitope-mapping studies, and the recombinant allergens may be valuable for diagnostic and therapeutic purposes.

Adolescent↗
PubMed1994

Expression of the Sendai virus fusion protein in insect cells and characterization of its post-translational modifications.

The fusion (F) protein of Sendai virus was expressed with a baculovirus system in insect cells. The F protein is synthesized in its uncleaved form F0, which appears in SDS-containing gels as two bands with M(r) values of 66K and 64K. Digestion of the F protein with endoglycosidase H revealed that the 66K species contains high mannose-type carbohydrates, whereas partially processed oligosaccharides are attached to the 64K species. Pulse-chase experiments showed that the F protein is initially synthesized as its 66K form. After 1 h of chase this precursor glycoprotein is partially converted to the 64K species and exposed at the cell surface. Other modifications of the F protein in insect cells are the formation of intramolecular disulphide linkages and oligomerization. However, processing and plasma membrane transport of the F protein in insect cells are incomplete and retarded compared to the F protein synthesized in Sendai virus-infected mammalian cells.

Animals↗
PubMed2001

Matrix proteins from insect pliable cuticles: are they flexible and easily deformed?

Proteins from pliable cuticle of locusts, Schistocerca gregaria, and silk moth larvae, Hyalophora cecropia, were studied in solution by means of a fluorescent probe, 8-anilinonaphthalene-1-sulphonic acid (ANS), which is much more fluorescent in non-polar media than in polar media. An intense ANS-fluorescence was observed in the presence of the cuticular proteins at pH-values close to their acidic isoelectric points, and the fluorescence decreased markedly when pH was increased to neutrality or when small amounts of denaturants were added. Aggregation and eventual precipitation of both H. cecropia and locust proteins were obtained by addition of neutral salts, and the aggregation was accompanied by an increased ANS-fluorescence intensity. A decreased ANS-fluorescence was observed at salt concentrations too low to cause visible aggregation of the H. cecropia proteins, probably due to weakened electrostatic interactions between chain segments, but such a decrease was not observed for the locust proteins. The changes in intensity of ANS-fluorescence induced by addition of small amounts of denaturants or salts to solutions of the proteins indicate that more hydrophobic residues are exposed to the solvent, when either hydrophobic interactions or electrostatic attractions between chain segments are weakened. The result is a less compact protein structure, where fewer and smaller hydrophobic clusters are available for protecting ANS-molecules from the quenching effects of water. The effects of denaturants on ANS-fluorescence in the presence of the cuticular proteins are different from those observed for globular proteins, such as hen egg albumen, and the differences can be explained by the suggestion that the cuticular proteins do not have a precisely folded and densely packed hydrophobic core comparable to that present in native globular proteins, and that accordingly they do not undergo a process of denaturation corresponding to that of globular proteins. The behaviour of the cuticular proteins resembles that described for unordered, randomly coiled, thermally agitated polymer chains, whose hydrodynamic volumes depend upon the composition of the medium. It is proposed that the major part of the peptide chains of the cuticular proteins are in an unordered, random structure both when the proteins are in solution and when present in the intact cuticle; probably only the chain regions involved in binding the proteins to chitin will have a well-defined spatial organisation.

Anilino Naphthalenesulfonates↗
PubMedDate not supplied

Insect storage proteins: gene families and receptors.

The accumulation and utilization of storage proteins are prominent events linked to the metamorphosis of holometabolous insects. Storage proteins are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. Within the pupal fat body, these proteins are initially stored in protein granules, and later proteolytically broken down to supply amino acid resources necessary for the completion of adult development. Most, but not all storage proteins belong to a superfamily of hexameric larval serum proteins that are evolutionarily related to hemocyanin. This article reviews the classification of these proteins, based on their amino acid sequences, and the current knowledge of the receptors that mediate their selective uptake into pupal fat body.

Animals↗
PubMed2000

Endogenous glutathione-binding proteins of insect cell lines: characterization and removal from glutathione S-transferase (GST) fusion proteins.

After affinity purification on immobilized glutathione, insect-cell-derived glutathione S-transferase (GST) fusion proteins contain variable amounts of protein contaminants of about 23-24 kDa. We have isolated these glutathione-binding proteins from the widely used Sf9 and Hi5 insect cell lines and characterized them by LC-MS and N-terminal sequencing. Based on the observation that these proteins have higher affinity for glutathione than GST fusions, we have found that by using differential elution conditions the amount of such contaminants in GST fusion preparations can be strongly reduced directly during the affinity purification step. The main interest of these results is that they are not restricted to a specific construct, but rather they seem to apply to various insect-cell-derived GST fusions.

Animals↗
PubMed1995

[Antimicrobial proteins of insect and their clinical application].

We isolated three antimicrobial proteins from the hemolymph of immunized Sarcophaga peregrina (flesh fly) larvae. These are sarcotoxin I, sapecin and antifungal protein (AFP). Sarcotoxin I and sapecin are highly specific for Gram-negative and Gram-positive bacteria, respectively. AFP was found to have fungicidal activity. Primary target for these antimicrobial proteins was assumed to be microbial membrane. We were able to identify a core undecapeptide of sapecin B. We modified this peptide and finally obtained KLKLLLLLKLK-NH2 and its D-enantiomer. These peptides were effective against various bacteria including MRSA. We found that MRSA-infected mine are cured by treating with these peptides.

Animals↗
PubMed2000

The structure and organization of lamprin genes: multiple-copy genes with alternative splicing and convergent evolution with insect structural proteins.

Lamprin is a unique structural protein which forms the extracellular matrix of several cartilaginous structures found in the lamprey. Lamprin is noncollagenous in nature but shows sequence similarities to elastins and to insect structural proteins. Here, we characterize the structure and organization of lamprin genes, demonstrating the presence of multiple similar but not identical copies of the lamprin gene in the genome of the lamprey. In at least one species of lamprey, Lampetra richardsoni, the multiple gene copies are arranged in tandem in the genome in a head-to-tail orientation. Lamprin genes from Petromyzon marinus contain either seven or eight exons, with exon 4 being alternatively spliced in all genes, resulting in a total of six different lamprin transcripts. All exon junctions are of class 1,1. An unusual feature of the lamprin gene structure is the distribution of the 3' untranslated region sequence among multiple exons. A TATA box and cap sequence have been identified in upstream sequences in close proximity to the transcription start site, but no CAAT box could be identified. Sequence and gene structure comparisons between lamprins, elastins, and insect structural proteins suggest that the regions of sequence similarity are the result of a process of convergent evolution.

Alternative Splicing↗