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PubMed2005

Systematic size study of an insect antifreeze protein and its interaction with ice.

Because of their remarkable ability to depress the freezing point of aqueous solutions, antifreeze proteins (AFPs) play a critical role in helping many organisms survive subzero temperatures. The beta-helical insect AFP structures solved to date, consisting of multiple repeating circular loops or coils, are perhaps the most regular protein structures discovered thus far. Taking an exceptional advantage of the unusually high structural regularity of insect AFPs, we have employed both semiempirical and quantum mechanics computational approaches to systematically investigate the relationship between the number of AFP coils and the AFP-ice interaction energy, an indicator of antifreeze activity. We generated a series of AFP models with varying numbers of 12-residue coils (sequence TCTxSxxCxxAx) and calculated their interaction energies with ice. Using several independent computational methods, we found that the AFP-ice interaction energy increased as the number of coils increased, until an upper bound was reached. The increase of interaction energy was significant for each of the first five coils, and there was a clear synergism that gradually diminished and even decreased with further increase of the number of coils. Our results are in excellent agreement with the recently reported experimental observations.

Animals↗
PubMed1981

Occurrence of antibodies against insect virus proteins in mammals: simple model to differentiate between passive exposure and active virus growth.

Antibodies against an "enterovirus-like" virus of insects, cricket paralysis virus, occur in the sera of domestic animals. When these antibodies were used in combination with the immunoprecipitation of radiolabeled virus proteins from infected Drosophila cells in culture, it could be demonstrated that the animals were exposed to preformed virus.

Animals↗
PubMed1997

Cell adhesion properties of hemolin, an insect immune protein in the Ig superfamily.

The isolation of antibacterial peptides from the giant silkmoth Hyalophora cecropia has opened the area of animal antibiotics [Boman, H. G. (1991) Cell 65, 205-207] and the study of insect immune genes has revealed striking similarities to many immune response genes in mammals [Hultmark, D. (1994) Nature 267, 116-117]. However, the molecules and mechanisms behind primordial immune recognition are not understood. One candidate for one such recognition molecule is hemolin, a 48-kDa immunoglobulin-related protein first isolated from H. cecropia, where it is up-regulated upon infection and secreted into the hemolymph. Hemolin was shown to bind to bacteria and to hemocytes, giving rise to changes in hemocyte adhesiveness and intracellular phosphorylation patterns [Faye, I. & Kanost, M. (1997) in Molecular mechanisms of immune responses in insects (Brey, P. T. & Hultmark, D., eds) Chapman and Hall, London]. In the present publication, we give evidence for the presence of a 52-kDa membrane form of hemolin on hemocytes, based on flow-activated cell sorting and membrane protein extractions. In addition we reveal calcium-dependent homophilic binding properties of hemolin, using hemolin-coated microspheres. When biotinylated recombinant hemolin was allowed to bind to hemocyte membranes, higher molecular-mass complexes were formed. Furthermore, we used immunological methods and Northern-blot analysis to demonstrate the presence of hemolin in embryos and retinal discs, suggesting that hemolin is expressed in several tissues at different developmental stages. These results show novel cell adhesion features of hemolin, corroborating its multifunctional character with putative roles in cellular and humoral immunity and in development.

Amino Acid Sequence↗
PubMed1990

Expression of the Lassa virus nucleocapsid protein in insect cells infected with a recombinant baculovirus: application to diagnostic assays for Lassa virus infection.

The coding region of the gene for the nucleocapsid protein of Lassa virus has been inserted into the genome of Autographa californica nuclear polyhedrosis virus (AcNPV) using the transfer vector pAcYM1, so that expression of the foreign DNA is under the control of the promoter of the AcNPV polyhedrin gene. Infection of cultured Spodoptera frugiperda cells with recombinant virus resulted in the synthesis of high levels of a protein that was indistinguishable from the authentic Lassa virus protein by SDS gel electrophoresis and immunoblotting with a variety of specific immune sera and monoclonal antibodies (MAbs). The kinetics of appearance of the protein were comparable to those of polyhedrin production in wild-type AcNPV-infected cells. The recombinant material was antigenic when used in ELISA for Lassa virus-specific antibodies, reacting well with MAbs specific for the nucleocapsid protein and with sera from experimentally infected guinea-pigs. The recombinant ELISA was able to clearly distinguish sera from human cases of Lassa fever against a panel of known negative sera of African origin. Recombinant-infected insect cells were an effective substitute for mammalian cells infected with Lassa virus itself in the immunofluorescence assay for Lassa virus-specific antibodies. This system offers attractive alternatives to the use of Lassa virus-infected materials as reagents in diagnostic procedures.

Animals↗
PubMed1991

Use of a stationary bed reactor and serum-free medium for the production of recombinant proteins in insect cells.

Insect cells (Spodoptera frugiperda) have been cultured in a stationary bed reactor, packed with a fibrous polyester carrier. When the bioreactor was perfused with serum-supplemented medium, a cell density of 6 x 10(6) cells ml-1 packed carrier was reached. Scanning electron microscopy investigations have shown that the insect cells grew along the three-dimensionally oriented fibers of the Fibra-cel carrier. After infection of the logarithmically growing cells with a recombinant baculovirus (Autographa californica) containing the gene coding for beta-galactosidase, the medium in the bioreactor was changed to serum-free medium. At day 13 postinfection (p.i.), a beta-galactosidase level of 320 microgram ml-1 and, at day 17 p.i., a virus titer of 2.1 x 10(8) TCID50 units ml-1 (day 17 p.i.) were reached. In another bioreactor, operated in a similar way but with serum-containing medium, a beta-galactosidase concentration of 360 microgram ml-1 and a virus titer of 2.3 x 10(8) TCID50 units ml-1 were obtained. These results indicate the potential use of this production system for the production of recombinant protein and baculovirus in insect cells.

Animals↗
PubMed1993

Morphogenic capabilities of human immunodeficiency virus type 1 gag and gag-pol proteins in insect cells.

To determine the interaction between the gag precursor and the viral protease and to confirm the role of gag precursor in formation of human immunodeficiency virus type 1 particles, the gag and protease encoding regions of a proviral genome with mutations at the site between p17 and p24 or p24 and p15 were expressed by recombinant baculoviruses under the transcriptional control of the strong polyhedrin promoter. Western blot analyses of the expressed products of p17-p24 mutated viruses revealed that both 41- and 55-kDa proteins were synthesized. However, free p24, p17, and the other smaller cleavage products (p9, p6) could not be detected in infected insect cells. The second recombinant virus (p24-p15) synthesized not only a 55k-Da protein, but also a number of smaller products including a 40k-Da protein, p24, and p17. Examination of the insect cells infected by either of these two recombinant viruses by electron microscopy failed to detect any gag particle formation, although some irregular membrane protrusions and profound distortions of the cell surface were clearly visible in the cells infected with recombinant mutant p17-p24 virus, but not with recombinant p24-p15 mutants. To investigate the morphogenic capability of the gag-pol fusion protein, a mutant gag-pol gene containing an inactive protease as well as a modified gag-pol gene lacking the frameshifting activity were expressed in insect cells. While the inactive protease mutant was capable of forming immature particles that were secreted, the frameshifting mutant synthesized only an aberrant form of gag particles with a large radius of curvature in lieu of spherical particles. However, when this mutant was expressed in insect cells in the presence of a truncated gag protein with M(r) of 46 kDa (lacking only the p6 domain), normal immature particles containing both antigens were formed.

Amino Acid Sequence↗
PubMed1991

Expression of human Alzheimer amyloid precursor protein in insect cells.

The amyloid beta-peptide is a major constituent of amyloid deposited in the brains of patients with Alzheimer's disease and is derived from a larger precursor protein/s (APP-695, 751, 770). A human cDNA encoding full-length APP-751 was inserted into the genome of Autographa californica nuclear polyhedrosis virus under transcriptional regulation of the viral polyhedrin gene promoter. The recombinant virus was used to infect insect cells, which resulted in the abundant expression of APP-751. Analysis of infected cell proteins indicate that APP-751 is localized in the membrane fraction; however, a significant amount of the protein was cleaved and released into the medium. The NH2-terminal sequence of recombinant APP-751 from the membrane fraction was identical to that of mammalian APP. Immunoblot analysis suggests that the secreted form results from cleavage within the beta-peptide.

Alzheimer Disease↗
PubMed1994

Production of a functional full-length Xenopus laevis c-Myc protein in insect cells.

C-Myc is a nuclear phosphoprotein whose normal cellular function has not yet been clearly defined. Studies with this protein have always been constrained by the difficulty of obtaining full-length c-Myc in an active form, whatever the expression system used. We report here experimental conditions optimized to increase the solubility and the purification of c-Myc in a baculovirus expression system. Such conditions allow the production of both soluble and active full-length c-Myc. Interestingly, soluble c-Myc is found associated with a 500-kDa high-molecular-mass complex comparable to that found in human and Xenopus laevis embryos, and which may be required for its function in vivo.

Animals↗
PubMed1990

Isolation and molecular cloning of transferrin from the tobacco hornworm, Manduca sexta. Sequence similarity to the vertebrate transferrins.

An iron-binding glycoprotein of Mr = 77,000 has been isolated from hemolymph of the adult sphinx moth Manduca sexta. Since this protein binds ferric ion both in vivo and in vitro and has a secondary structure similar to that of human serum transferrin and human lactoferrin as judged by CD spectra, we decided to clone its cDNA in order to determine its relationship to the vertebrate transferrins. Antiserum generated against this protein was used to screen a larval fat body cDNA library. A 2.0 kilobase clone was isolated that selects an mRNA which, when translated in vitro, produces an immunoprecipitable 77-kDa protein. When the library was rescreened using the 2.0-kilobase clone as a probe, three full-length clones were isolated, and the complete nucleotide sequence of one 2,183-base pair insert was determined. The deduced protein sequence contains an 18-amino acid signal sequence and a mature protein sequence of 663 amino acids with a calculated Mr of 73,436. The sequence was used to search the National Biomedical Research Foundation (NBRF) protein database, revealing significant similarity to the vertebrate transferrins, a family of 80-kDa glycoproteins which transport and sequester iron in the blood and other body fluids. A multiple sequence alignament shows the greatest areas of similarity to be around the two iron binding sites, although the insect protein seems to contain only one such functional site. Moreover, 23 of the 24 cysteine residues in the insect protein occupy identical positions as compared with the other transferrins, indicating a similar overall tertiary structure. Comparison of the two halves of the insect sequence indicates that the protein may have arisen as a result of gene duplication. The similarity of the M. sexta sequence to the vertebrate transferrins may provide important clues to transferrin evolution.

Amino Acid Sequence↗
PubMed2006

Periplaneta americana arginine kinase as a major cockroach allergen among Thai patients with major cockroach allergies.

Periplaneta americana is the predominant cockroach (CR) species and a major source of indoor allergens in Thailand. Nevertheless, data on the nature and molecular characteristics of its allergenic components are rare. We conducted this study to identify and characterize the P. americana allergenic protein. A random heptapeptide phage display library and monoclonal antibody (MAb) specific to a the P. americana component previously shown to be an allergenic molecule were used to identify the MAb-bound mimotope and its phylogenic distribution. Two-dimensional gel electrophoresis, liquid chromatography, mass spectrometry, peptide mass fingerprinting, and BLAST search were used to identify the P. americana protein containing the MAb-specific epitope. We studied the allergenicity of the native protein using sera of CR-allergic Thai patients in immunoassays. The mimotope peptide that bound to the MAb specific to P. americana was LTPCRNK. The peptide has an 83-100% identity with proteins of Anopheles gambiae, notch homolog scalloped wings of Lucilia cuprina, delta protein of Apis mellifera; neu5Ac synthase and tyrosine phosphatase of Drosophila melanogaster, and a putative protein of Drosophila pseudoobscura. This finding implies that the mimotope-containing molecule of P. americana is a pan-insect protein. The MAb-bound protein of P. americana was shown to be arginine kinase that reacted to IgE in the sera of all of the CR-allergic Thai patients by immunoblotting, implying its high allergenicity. In conclusion, our results revealed that P. americana arginine kinase is a pan-insect protein and a major CR allergen for CR-allergic Thai patients.

Adolescent↗
PubMed1997

Expression, assembly, and proteolytic processing of Helminthosporium victoriae 190S totivirus capsid protein in insect cells.

The dsRNA genome (5.2 kbp) of Helminthosporium victoriae 190S totivirus (Hv190SV) consists of two large overlapping open reading frames (ORFs). The 5' proximal ORF codes for the capsid protein (CP) and the 3' ORF codes for an RNA-dependent RNA polymerase. Although the capsid of Hv190SV is encoded by a single gene, it is composed of two major closely related polypeptides, either p88 and p83 or p88 and p78. Whereas p88 and p83 are phosphoproteins, p78 is nonphosphorylated. Expression of the CP ORF in insect cells generated both p78 and p88 which assembled into virus-like particles. The finding that p78, p83, and p88 share a common N-terminal amino acid sequence is consistent with the determination that N-terminal, but not C-terminal, CP deletions were incompetent for assembly. Evidence was obtained that p78 is derived from p88 via proteolytic cleavage at the C-terminus. Proteolytic processing may play a regulatory role in the virus life cycle since it leads to dephosphorylation of CP and a subsequent decrease in virion transcriptional activity.

Animals↗
PubMed2004

The role of lipoxygenase products on the endocytosis of yolk proteins in insects: participation of cAMP.

The participation of eicosanoids and second messengers in the regulation of endocytosis by the ovaries was investigated using the uptake of Rhodnius heme binding protein (RHBP) as an experimental model. The rate of RHBP uptake decreased up to 40% in the presence of BWA4C and NDGA, 5 and 12-lipoxygenase inhibitors, respectively, suggesting the involvement of lipoxygenase products in endocytosis regulation. Addition of Leukotriene B4 (LTB(4); one product of the 5 lipoxygenase pathway) increased in vitro the uptake of RHBP by 30%. The content of cAMP in the Rhodnius' ovaries were monitored after treatment with different eicosanoids and inhibitors of eicosanoids synthesis. The amount of cAMP decreased in the presence of indomethacin (by 50%), while treatment with PGE(2) induced an increase of 85% of this messenger in the ovaries. The presence of LTB(4) in the medium inhibited in 60% the content of cAMP in the ovaries, while BWA4C induced a 100% increase of this messenger in the ovaries. Addition of 1 microM DBcAMP in the medium resulted in a 30% decrease in the rate of RHBP uptake. Taken together, these data show that cyclooxygenase and lipoxygenase products participate in the control of protein internalization by modulation of cAMP levels.

Animals↗
PubMed1990

Synthesis of foot-and-mouth disease virus capsid proteins in insect cells using baculovirus expression vectors.

Foot-and-mouth disease virus (FMDV) cDNA cassettes containing sequences encoding the capsid precursor P1-2A with and without those encoding the proteases L and 3C were introduced into Autographa californica nuclear polyhedrosis virus (AcMNPV) expression vectors. Procapsid proteins 1AB, 1C and 1D were produced in cells infected with recombinant baculoviruses, when L and 3C were present in the constructs, indicating that these FMDV proteases were active in insect cells. Unlike P1 processing in poliovirus, which has been shown to be catalysed mainly by the 3CD gene product, the 3C protease of FMDV was able to process P1 independently of 3D. Cytotoxicity of the L protease for insect cells prevented the use of the optimized transfer vector, pAcRP23, for inserting L-containing cassettes into AcMNPV. By contrast, viable AcMNPV-FMDV recombinants could be made without restriction on choice of the transfer vector when the L gene was either not expressed or inactivated by an inframe deletion. In the latter case, normal cleavage at the L-P1 junction no longer occurred in cis, and a new processing event, probably catalysed by 3C, was observed within the C-terminal region of the residual L protein. Analysis of baculovirus-expressed products in sucrose gradients showed that a fraction of the capsid proteins is present in an aggregated form, migrating at 70S and possibly resembling FMDV empty capsid particles.

Animals↗