Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) spike (S) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. To investigate SCoV S protein, full-length and individual domains of S protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of SARS. S protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. Reactivity was evident by both flow cytometry and Western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. The antibody response to SCoV S protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. Recombinant SCoV S protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for SARS, but our data suggest that assay format and choice of S antigen are important considerations.

Control of human immunodeficiency virus type-1 protease activity in insect cells expressing Gag-Pol rescues assembly of immature but not mature virus-like particles

Expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in insect cells using baculovirus vectors leads to the abundant production of virus-like particles (VLPs) that represent the immature form of the virus. When Gag-Pol is included, however, VLP production is abolished, a result attributed to premature protease activation degrading the intracellular pool of Gag precursor before particle assembly can occur. As large-scale synthesis of mature noninfectious VLPs would be useful, we have sought to control HIV protease activity in insect cells to give a balance of Gag and Gag-Pol that is compatible with mature particle formation. We show here that intermediate levels of protease activity in insect cells can be attained through site-directed mutagenesis of the protease and through antiprotease drug treatment. However, despite Gag cleavage patterns that mimicked those seen in mammalian cells, VLP synthesis exhibited an essentially all-or-none response in which VLP synthesis occurred but was immature or failed completely. Our data are consistent with a requirement for specific cellular factors in addition to the correct ratio of Gag and Gag-Pol for assembly of mature retrovirus particles in heterologous cell types. (C) 2003 Elsevier Science (USA). All rights reserved.

Interaction of the D-2short dopamine receptor with G proteins: analysis of receptor G protein selectivity

The human D-2short (D-2S) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [H-3]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [S-35]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [S-35]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D-2S receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([H-3]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [H-3]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [H-3]NPA binding with greater potency (similar to6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D-2S/Go preparation. [H-3]Spiperone binding occurred with an increased B-max in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D-2S receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein. (C) 2003 Elsevier Science Inc. All rights reserved.

Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.

The DNA puff 4C expresses a salivary secretion protein in Trichosia pubescens (Diptera; Sciaridae)

DNA puffs are genomic regions of polytene chromosomes that undergo developmentally controlled DNA amplification and transcription in salivary glands of sciarid flies. Here, we tested the hypothesis that DNA puff genes code for salivary proteins in Trichosia pubescens. To do that, we generated antibodies against saliva and immunoscreened a cDNA library made from salivary glands. We isolated clones corresponding to DNA puff regions, including clone D-50 that contained the entire coding sequence of the previously isolated C4B1 gene from puff 4C. Indeed, we showed that puff 4C is a DNA puff region detecting its local transcription and its extra rounds of DNA incorporation compared to neighboring regions. We further confirmed D-50 clone identity in Western blots reacted with the anti-saliva anitiserum. We detected a recombinant protein expressed by this clone that had the expected size for a full-length product of the gene. We end with a discussion of the relationship between DNA puff genes and their products.

Protein identification pipeline for the homology-driven proteomics

Homology-driven proteomics is a major tool to characterize proteomes of organisms with unsequenced genomes. This paper addresses practical aspects of automated homology-driven protein identifications by LC-MS/MS on a hybrid LTQ orbitrap mass spectrometer. All essential software elements supporting the presented pipeline are either hosted at the publicly accessible web server, or are available for free download. (C) 2008 Elsevier B.V. All rights reserved.

Molecular analysis of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) shows an interruption of an inhibitor of apoptosis gene (iap-3) by a new class-II piggyBac-related insect transposon

A new piggyBac-related transposable element (TE) was found in the genome of a mutant Anticarsia gemmatalis multiple nucleopolyhedrovirus interrupting an inhibitor of apoptosis gene. This mutant virus induces apoptosis upon infection of an Anticarsia gemmatalis cell line, but not in a Trichoplusia ni cell line. The sequence of the new TE (which was named IDT for iap disruptor transposon) has 2531 bp with two DNA sequences flanking a putative Transposase (Tpase) ORF of 1719 bp coding for a protein with 572 amino acids. These structural features are similar to the piggyBac TE, also reported for the first time in the genome of a baculovirus. We have also isolated variants of this new TE from different lepidopteran insect cells and compared their Tpase sequences.

The defensive functions of plant inhibitors are not restricted to insect enzyme inhibition

Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bouhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH(2) (RGE) and IVYYPDRGETGL-NH(2) (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein. (C) 2009 Elsevier Ltd. All rights reserved.

INSECT CHYMOTRYPSINS: CHLOROMETHYL KETONE INACTIVATION AND SUBSTRATE SPECIFICITY RELATIVE TO POSSIBLE COEVOLUTIONAL ADAPTATION OF INSECTS AND PLANTS

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Ewer insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization. using different, colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from, bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe P-nitroanilide). Chloromethyl ketones (TPCK, N-alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N-carbobenzoxy-Gly-Gly-phe-CK) inactivated all chymotrypsins legated. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1)s(-1); Z-GGF-CK, 150 to 450 M(-1)s(-1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1)s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that. in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa, value. This is Proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed. to be an adaptation to the presence of dietary ketones. (C) 2009 Wiley Periodicals, Inc.

Subsite substrate specificity of midgut insect chymotrypsins

Insect chymotrypsins are distinctively sensitive to plant protein inhibitors, suggesting that they differ in subsite architecture and hence in substrate specificities. Purified digestive chymotrypsins from insects of three different orders were assayed with internally quenched fluorescent oligopeptides with three different amino acids at P1 (Tyr, Phe, and Leu) and 13 amino acid replacements in positions P1`, P2, and P3. The binding energy (Delta G(s), calculated from Km values) and the activation energy (Delta G(T)(double dagger), determined from k(cat)/K-m values) were calculated. The hydrophobicities of each subsite were calculated from the efficiency of hydrolysis of the different amino acid replacements at that subsite. The results showed that except for S1, the other subsites (S2, S3, and S1`) vary among chymotrypsins. This result contrasts with insect trypsin data that revealed a trend along evolution, putatively associated with resistance to plant inhibitors. In spite of those differences, the data suggested that in lepidopteran chymotrypsins S2 and S1` bind the substrate ground state, whereas only S1` binds the transition state, supporting aspects of the present accepted mechanism of catalysis. 2008 Elsevier Ltd. All rights reserved.

A short proregion of trialysin, a pore-forming protein of Triatoma infestans salivary glands, controls activity by folding the N-terminal lytic motif

Triatoma infestans (Hemiptera: Reduviidae) is a hematophagous insect that transmits the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas` disease. Its saliva contains trialysin, a protein that forms pores in membranes. Peptides based on the N-terminus of trialysin lyse cells and fold into alpha-helical amphipathic segments resembling antimicrobial peptides. Using a specific antiserum against trialysin, we show here that trialysin is synthesized as a precursor that is less active than the protein released after saliva secretion. A synthetic peptide flanked by a fluorophore and a quencher including the acidic proregion and the lytic N-terminus of the protein is also less active against cells and liposomes, increasing activity upon proteolysis. Activation changes the peptide conformation as observed by fluorescence increase and CD spectroscopy. This mechanism of activation could provide a way to impair the toxic effects of trialysin inside the salivary glands, thus restricting damaging lytic activity to the bite site.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64 kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22 kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. (C) 2007 Elsevier Ltd. All rights reserved.

Insect Venom Peptides

The insects of the order Hymenoptera ( bees, wasps, and ants) are classified in two groups, based on their life history: social and solitary. The venoms of the social Hymenoptera evolved to be used as defensive tools to protect the colonies of these insects from the attacks of predators. Generally they do not cause lethal effects but cause mainly inflammatory and/or immunological reactions in the victims of their stings. However, sometimes it is also possible to observe the occurrence of systemic effects like respiratory and/or kidney failure. Meanwhile, the venoms of solitary Hymenoptera evolved mainly to cause paralysis of the preys in order to permit egg laying on/within the prey's body; thus, some components of these venoms cause permanent/transient paralysis in the preys, while other components seem to act preventing infections of the food and future progenies. The peptide components of venoms from Hymenoptera are spread over the molar mass range of 1400 to 7000 da and together comprise up to 70% of the weight of freeze-dried venoms. Most of these toxins are linear polycationic amphipatic peptides with a high content of alpha-helices in their secondary structures. These peptides generally account for cell lysis, hemolysis, antibiosis, and sometimes promote the delivery of cellular activators/mediators through interaction with the G-protein receptor, and perhaps some of them are even immunogenic components. In addition to these peptides, the Hymenopteran venoms also may contain a few neurotoxins that target Na+ and/or Ca+2 channels or even the nicotinic ACh receptor. This review summarizes current knowledge of the biologically active Hymenoptera venoms.

Female reproductive system of the sugarcane spittlebug Mahanarva fimbriolata (Auchenorrhyncha): Vitellogenesis dynamics and protein quantification

The present study aimed describing the ovaries of the sugarcane spittlebug Mahanarva fimbriolata which are meroistic telotrophic with nurse cells and oocytes located in the tropharium. SEM revealed paired ovaries located dorsolaterally around the intestine, and oocytes exhibiting shapes ranging from round (less developed) to elliptic (more developed), suggesting a simultaneous, although, asynchronous development. Based on histological data we classified the oocytes in stages from I to V. Stage I oocytes exhibit follicular epithelium with cubic and/or prismatic cells, fine cytoplasmic granules. Stage II oocytes present intercellular spaces in the follicular epithelium due to the incorporation of yolk elements from the hemolymph. Small granules are present in the periphery of oocytes while larger granules are observed in the center. Stage III oocytes are larger and intercellular spaces in the follicular epithelium are evident, as well as the interface between follicular epithelium and oocyte. Yolk granules of different sizes are present in the cytoplasm. During this stage, chorion deposition initiates. Stage IV oocytes exhibit squamous follicular cells and larger intercellular spaces when compared to those observed in the previous stage. The oocyte cytoplasm present granular and viscous yolk, the latter is the result of the breakdown of granules. Stage V oocytes exhibit a follicular epithelium almost completely degenerated, smaller quantities of granular yolk and large amounts of viscous yolk. Based on our findings we established the sequence of yolk deposition in M. fimbriolata oocyte as follows: proteins and lipids, which are first produced by endogenous processes in stages I and II oocytes. Exogenous incorporation begins in stage III. In stages I and II oocytes, lipids are also produced by follicular epithelial cells. The third element to be deposited is polysaccharides, mainly found as complexes. Therefore, the yolk present in the oocytes of this species consists of glycolipoproteins. Molecular weights of proteins present in M. fimbriolata oocytes ranged from 10 to 92 KDa, differently from vitellogenin, the most common protein present in insect oocytes, weighing approximately 180 KDa. (c) 2006 Elsevier Ltd. All rights reserved.

Histochemistry and protein profile of the mandibular glands of workers of the ant Atta sexdens rubropilosa (Hymenoptera: Formicidae)

Insect mandibular glands are always associated to the mandibles; they are part of the salivary glandular system. The mandibular glands are composed by a reservoir associated to the secretory cells, with each secretory cell connected to the reservoir by means of individual canaliculi. These glands play an important role in the production of pheromones, which are compounds involved in defense, communication, and reproduction of the colony. Mandibular glands of soldiers and major and minor workers of the ant Atta sexdens rubropilosa were processed for different histochemical tests, total protein content, and protein electrophoretic profile determination. The histochemical tests detected the presence of lipids, DNA/RNA, polysaccharides, and proteins at different regions of the gland. The protein electrophoretic profiles showed that the total protein content as well as the number of peptides of each caste follow a progressive order in relation to the size of the individual. Thus, we suggest that the production of secretion is directly linked to the task that the individual performs in the colony.