RESUMEN

The aggressivity is modulated in honeybee brain through a series of actions in cascade mode, with the participation of the neuropeptides AmAST A (59-76) and AmTRP (254-262). The aggressivity of honeybees was stimulated by injecting both neuropeptides in the hemocoel of the worker honeybees, which were submitted to behavioral assays of aggression. The brain of stinger individuals were removed by dissection and submitted to proteomic analysis; shotgun proteomic approach of honeybee brain revealed that both neuropeptides activate a series of biochemical processes responsible by production of energy, neuronal plasticity and cell protection. In addition to this, AmTRP (254-262) elicited the expression of proteins related to the processing of the potential of action and lipid metabolism; meanwhile AmAST A (59-76) elicited the metabolism of steroids and Juvenile hormone-related metabolism, amongst others. Apparently, the most complex biochemical process seems to be the regulation of ATP production, which occurs at two levels: i) by a subgroup of proteins common to the three experimental groups, which are over-/under-regulated through glycolysis, pyruvate pathway, Krebbs cycle and oxidative phosphorylation; ii) by a subgroup of proteins unique to the each experimental group, which seems to be regulated through Protein-Protein Interactions, where the protein network regulated by AmTRP (254-262) seems to be more complex than the other two experimental groups. SIGNIFICANCE: Recently we reported the effect of the neuropeptides AmAST A (59-76) and AmTRP (254-262) in the modulation of the aggressive behavior of the worker honeybees. Up to now it is known that the simple presence of the allatostatin and tachykinin-related-peptide in bee brain, is enough for inducing the aggressive behavior. However, nothing was known about how these neuropeptides perform their action, inducing the aggressive behavior. The results of the present study elucidated some of the metabolic pathways that were activated or inhibited to support the complex defensive behavior, which includes the aggressivity. These results certainly will impact the behavioral research of honeybees, since we are paving the way for understanding the molecular base of regulation, of individual /nest defense of honeybees.

Asunto(s)

Neuropéptidos , Proteómica , Abejas , Animales , Humanos , Encéfalo/metabolismo

RESUMEN

Salivary glands are omnipresent in termites and occur in all developmental stages and castes. They function to produce, store, and secrete compounds, ranging from a feeding function to defensive mechanisms. Here, we provide a complete morphological overview of the salivary glands in the soldierless species Ruptitermes reconditus and R. xanthochiton, and the first proteomic profile of the salivary glands in a Neotropical Apicotermitinae representative, R. reconditus. Salivary glands from both species were composed of several acini, roughly spherical structures composed of two types of central cells (type I and II) and peripheral parietal cells, as well as transporting ducts and two salivary reservoirs. Central cells were richly supplied with electron-lucent secretory vesicles and rough endoplasmic reticulum, a feature of protein-secreting cells. Parietal cells of Ruptitermes spp. had conspicuous characteristics such as electron-lucent secretory vesicles surrounded by mitochondria and well-developed microvilli. Moreover, different individuals showed variation in the secretory cycle of salivary acini, which may be related to polyethism. Ultrastructural analysis evidenced a high synthesis of secretion and also the occurrence of lysosomes and autophagic structures in central cells. Proteomic analysis of the salivary glands revealed 483 proteins divided into functional groups, highlighting toxins/defensins and compounds related to alarm communication and colony asepsis. Soldierless termites are quite successful, especially due to morphological adaptations of the workers, including unknown modifications of exocrine glands. Thus, according to our morphological and proteomic findings, we discuss the potential roles of the salivary gland secretion in different social aspects of the sampled species.

Asunto(s)

Proteómica/métodos , Glándulas Salivales/anatomía & histología , Animales , Isópteros

RESUMEN

Orb-weaving spiders use a highly strong, sticky and elastic web to catch their prey. These web properties alone would be enough for the entrapment of prey; however, these spiders may be hiding venomous secrets in the web, which current research is revealing. Here, we provide strong proteotranscriptomic evidence for the presence of toxin/neurotoxin-like proteins, defensins, and proteolytic enzymes on the web silk from Nephila clavipes spider. The results from quantitative-based transcriptomic and proteomic approaches showed that silk-producing glands produce an extensive repertoire of toxin/neurotoxin-like proteins, similar to those already reported in spider venoms. Meanwhile, the insect toxicity results demonstrated that these toxic components can be lethal and/or paralytic chemical weapons used for prey capture on the web, and the presence of fatty acids in the web may be a responsible mechanism opening the way to the web toxins for accessing the interior of prey's body, as shown here. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among two spider groups, Araneomorphae and Mygalomorphae, and the findings showed protein sequences similar to toxins found in the taxa Scorpiones and Hymenoptera in addition to Araneae. Overall, these data represent a valuable resource to further investigate other spider web toxin systems and also suggest that N. clavipes web is not a passive mechanical trap for prey capture, but it exerts an active role in prey paralysis/killing using a series of neurotoxins.

Asunto(s)

Proteómica , Arañas , Secuencia de Aminoácidos , Animales , Evolución Biológica , Seda/genética , Arañas/genética , Ponzoñas

RESUMEN

Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.

RESUMEN

Celiac disease (CD) is a chronic illness characterized by an inflammatory process triggered by gluten protein intake. Recent evidence has suggested that the lower relative abundance of bifidobacteria in the intestinal lumen may be associated with CD. Herein, we assessed the effect of the Bifidobacterium species Bifidobacterium bifidum, Bifidobacterium longum, Bembidion breve, Bifidobacterium animalis alone, and also a Bifidobacterium consortium on the digestion of intact gluten proteins (gliadins and glutenins) and the associated immunomodulatory responses elicited by the resulting peptides. The cytotoxicity and proinflammatory responses were evaluated through the activation of NF-kB p65 and the expression of cytokines TNF-α and IL-1ß in Caco-2 cell cultures exposed to gluten-derived peptides. The peptides induced a clear reduction in cytotoxic responses and proinflammatory marker levels compared to the gluten fragments generated during noninoculated gastrointestinal digestion. These results highlight the possible use of probiotics based on bifidobacteria as a prospective treatment for CD.

Asunto(s)

Bifidobacterium/metabolismo , Gliadina/metabolismo , Glútenes/metabolismo , Biotransformación , Células CACO-2 , Enfermedad Celíaca/tratamiento farmacológico , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Gliadina/química , Gliadina/inmunología , Glútenes/inmunología , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Probióticos/administración & dosificación , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología

RESUMEN

Termite societies are abundant in the tropics, and are therefore exposed to multiple enemies and predators, especially during foraging activity. Soldiers constitute a specialized defensive caste, although workers also participate in this process, and even display suicidal behavior, which is the case with the species Neocapritermes braziliensis. Here we describe the morphology, mechanisms of action, and proteomics of the salivary weapon in workers of this species, which due to the autothysis of the salivary glands causes their body rupture, in turn releasing a defensive secretion, observed during aggressiveness bioassays. Salivary glands are paired, composed of two translucent reservoirs, ducts and a set of multicellular acini. Histological and ultrastructural techniques showed that acini are composed of two types of central cells, and small parietal cells located in the acinar periphery. Type I central cells were abundant and filled with a large amount of secretion, while type II central cells were scarce and presented smaller secretion. Parietal cells were often paired and devoid of secretion. The gel-free proteomic approach (shotgun) followed by mass spectrometry revealed 235 proteins in the defensive secretion, which were classified into functional groups: (i) toxins and defensins, (ii) folding/conformation and post-translational modifications, (iii) salivary gland detoxification, (iv) housekeeping proteins and (v) uncharacterized and hypothetical proteins. We highlight the occurrence of neurotoxins previously identified in arachnid venoms, which are novelties for termite biology, and contribute to the knowledge regarding the defense strategies developed by termite species from the Neotropical region.

Asunto(s)

Conducta Animal/fisiología , Isópteros/fisiología , Toxinas Biológicas/química , Animales , Bases de Datos de Proteínas , Proteómica , Saliva/química , Toxinas Biológicas/biosíntesis

RESUMEN

Orb-weaving spiders can produce different silk fibers, which constitute outstanding materials characterized by their high strength and elasticity. Researchers have tried to reproduce the fibers of these proteins synthetically and/or by using recombinant DNA technology, but only a few of the natural physicochemical and biophysical properties have been obtained to date. Female orb-web-spiders present seven silk-glands, which synthesize the spidroins and a series of other proteins, which interact with the spidroins, resulting in silk fibers with notable physicochemical properties. Despite the recognized importance of the silk-glands for understanding how the fibers are produced and processed, the investigation of these glands is at a nascent stage. In the current study we present the assembled transcriptome of silk-producing glands from the orb-weaving spider Nephila clavipes, as well as develop a large-scale proteomic approach for in-depth analyses of silk-producing glands. The present investigation revealed an extensive repertoire of hitherto undescribed proteins involved in silk secretion and processing, such as prevention of degradation during the silk spinning process, transportation, protection against proteolytic autolysis and against oxidative stress, molecular folding and stabilization, and post-translational modifications. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among three groups of silk-producing organisms - (i) Araneomorphae spiders, (ii) Mygalomorphae spiders, and (iii) silk-producing insects. A common orthologous gene, which was annotated as silk gland factor-3 is present among all species analysed. This protein belongs to a transcription factor family, that is important and related to the development of the silk apparatus synthesis in the silk glands of silk-producing arthropods.

Asunto(s)

Fibroínas/genética , Seda/genética , Arañas/genética , Transcriptoma/genética , Animales , Evolución Biológica , Femenino , Fibroínas/metabolismo , Ontología de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Filogenia , Proteómica , Seda/biosíntesis , Arañas/metabolismo

RESUMEN

The partial proteome of Polybia paulista wasp venom was previously reported elsewhere using a gel-dependent approach and resulted in the identification of a limited number of venom toxins. Here, we reinvestigated the P. paulista venom using a gel-free shotgun proteomic approach; the highly dynamic range of this approach facilitated the detection and identification of 1673 proteins, of which 23 venom proteins presented N-linked glycosylation as a posttranslational modification. Three different molecular forms of PLA1 were identified as allergenic proteins, and two of these forms were modified by N-linked glycosylation. This study reveals an extensive repertoire of hitherto undescribed proteins that were classified into the following six different functional groups: (i) typical venom proteins; (ii) proteins related to the folding/conformation and PTMs of toxins; (iii) proteins that protect toxins from oxidative stress; (iv) proteins involved in chemical communication; (v) housekeeping proteins; and (vi) uncharacterized proteins. It was possible to identify venom toxin-like proteins that are commonly reported in other animal venoms, including arthropods such as spiders and scorpions. Thus, the findings reported here may contribute to improving our understanding of the composition of P. paulista venom, its envenoming mechanism and the pathologies experienced by the victim after the wasp stinging accident. BIOLOGICAL SIGNIFICANCE: The present study significantly expanded the number of proteins identified in P. paulista venom, contributing to improvements in our understanding of the envenoming mechanism produced by sting accidents caused by this wasp. For example, novel wasp venom neurotoxins have been identified, but no studies have assessed the presence of this type of toxin in social wasp venoms. In addition, 23 N-linked glycosylated venom proteins were identified in the P. paulista venom proteome, and some of these proteins might be relevant allergens that are immunoreactive to human IgE.

Asunto(s)

Proteínas de Insectos/metabolismo , Proteómica , Venenos de Avispas/metabolismo , Avispas/metabolismo , Animales

RESUMEN

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

Asunto(s)

Venenos de Artrópodos/enzimología , Himenópteros/enzimología , Mordeduras y Picaduras de Insectos/inmunología , Fosfolipasas A1/inmunología , Fosfolipasas A2/inmunología , Secuencia de Aminoácidos , Animales , Venenos de Artrópodos/inmunología , Humanos , Mordeduras y Picaduras de Insectos/enzimología , Fosfolipasas A1/química , Fosfolipasas A1/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo

RESUMEN

The capture spiral of web from N. clavipes spider consists of a single type of spidroin - the flagelliform silk protein, a natural material representing a combination of strength and high elasticity. Flagelliform spider silk is the most extensible silk fibre produced by orb weaver spiders and the structure of this remarkable material is still largely unknown. In the present study we used a proteomic approach to elucidate the complete sequence and the post-translational modifications of flagelliform silk proteins. The long sequence of flagelliform silk protein presents 45 hydroxylated proline residues, which may contribute to explain the mechanoelastic property of these fibres, since they are located in the GPGGX motif. The 3D-structure of the protein was modelled considering the three domains together, i.e., the N- and C-terminal non-repetitive domains, and the central repetitive domain. In the resulting molecular model there is a predominance of random structures in the solid fibres of the silk protein. The N-terminal domain is composed of three α-helices and the C-terminal domain is composed of one small helical section. Proteomic data reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based spider polymers.

Asunto(s)

Fibroínas/química , Seda/química , Arañas/química , Animales , Fenómenos Biomecánicos , Fibroínas/metabolismo , Fibroínas/ultraestructura , Microscopía Electrónica de Rastreo , Modelos Moleculares , Conformación Proteica , Procesamiento Proteico-Postraduccional

RESUMEN

Among venomous animals, Hymenoptera have been suggested as a rich source of natural toxins. Due to their broad ecological diversity, venom from Hymenoptera insects (bees, wasps and ants) have evolved differentially thus widening the types and biological functions of their components. To date, insect toxinology analysis have scarcely uncovered the complex composition of bee, wasp and ant venoms which include low molecular weight compounds, highly abundant peptides and proteins, including several allergens. In Hymenoptera, these complex mixtures of toxins represent a potent arsenal of biological weapons that are used for self-defense, to repel intruders and to capture prey. Consequently, Hymenoptera venom components have a broad range of pharmacological targets and have been extensively studied, as promising sources of new drugs and biopesticides. In addition, the identification and molecular characterization of Hymenoptera venom allergens have allowed for the rational design of component-resolved diagnosis of allergy, finally improving the outcome of venom immunotherapy (VIT). Until recently, a limited number of Hymenoptera venoms had been unveiled due to the technical limitations of the approaches used to date. Nevertheless, the application of novel techniques with high dynamic range has significantly increased the number of identified peptidic and proteinaceous toxins. Considering this, the present review summarizes the current knowledge about the most representative Hymenoptera venom peptides and proteins which are under study for a better understanding of the insect-caused envenoming process and the development of new drugs and biopesticides.

Asunto(s)

Venenos de Artrópodos/química , Venenos de Artrópodos/toxicidad , Himenópteros/química , Animales , Venenos de Artrópodos/farmacología , Proteínas de Insectos/química , Proteínas de Insectos/farmacología , Proteínas de Insectos/toxicidad , Péptidos/química , Péptidos/farmacología , Péptidos/toxicidad

RESUMEN

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

Asunto(s)

Venenos de Hormiga/inmunología , Venenos de Abeja/inmunología , Hipersensibilidad/inmunología , Proteínas de Insectos/inmunología , Fosfolipasas A1/inmunología , Venenos de Avispas/inmunología , Alérgenos/inmunología , Animales , Hormigas/enzimología , Hormigas/inmunología , Abejas/enzimología , Abejas/inmunología , Brasil , Reacciones Cruzadas , Europa (Continente) , Femenino , Humanos , Hipersensibilidad/sangre , Hipersensibilidad/etiología , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Pruebas Intradérmicas , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes/inmunología , Avispas/enzimología , Avispas/inmunología

RESUMEN

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.

Asunto(s)

Alérgenos , Venenos de Avispas/química , Alérgenos/química , Alérgenos/genética , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Humanos , Hipersensibilidad/diagnóstico , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Modelos Moleculares , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Venenos de Avispas/genética , Venenos de Avispas/inmunología , Venenos de Avispas/aislamiento & purificación

RESUMEN

The proteins from the silk-producing glands were identified using both a bottom-up gel-based proteomic approach as well as from a shotgun proteomic approach. Additionally, the relationship between the functions of identified proteins and the spinning process was studied. A total of 125 proteins were identified in the major ampullate, 101 in the flagelliform, 77 in the aggregate, 75 in the tubuliform, 68 in the minor ampullate, and 23 in aciniform glands. On the basis of the functional classification using Gene Ontology, these proteins were organized into seven different groups according to their general function: (i) web silk proteins-spidroins, (ii) proteins related to the folding/conformation of spidroins, (iii) proteins that protect silk proteins from oxidative stress, (iv) proteins involved in fibrillar preservation of silks in the web, (v) proteins related to ion transport into and out of the glands during silk fiber spinning, (vi) proteins involved in prey capture and pre-digestion, and (vii) housekeeping proteins from all of the glands. Thus, a general mechanism of action for the identified proteins in the silk-producing glands from the Nephila clavipes spider was proposed; the current results also indicate that the webs play an active role in prey capture.

Asunto(s)

Estructuras Animales/química , Proteínas de Insectos/aislamiento & purificación , Proteómica , Seda/química , Arañas/fisiología , Secuencia de Aminoácidos , Estructuras Animales/metabolismo , Estructuras Animales/ultraestructura , Animales , Expresión Génica , Ontología de Genes , Proteínas de Insectos/clasificación , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo , Conformación Molecular , Anotación de Secuencia Molecular , Seda/metabolismo

RESUMEN

Most reports about the 3-D structure of spidroin-1 have been proposed for the protein in solid state or for individual domains of these proteins. A gel-based mass spectrometry strategy using collision-induced dissociation (CID) and electron-transfer dissociation (ETD) fragmentation methods was used to completely sequence spidroins-1A and -1B and to assign a series of post-translational modifications (PTMs) on to the spidroin sequences. A total of 15 and 16 phosphorylation sites were detected on spidroin-1A and -1B, respectively. In this work, we present the nearly complete amino acid sequence of spidroin-1A and -1B, including the nonrepetitive N- and C-terminal domains and a highly repetitive central core. We also described a fatty acid layer surrounding the protein fibers and PTMs in the sequences of spidroin-1A and -1B, including phosphorylation. Thus, molecular models for phosphorylated spidroins were proposed in the presence of a mixture fatty acids/water (1:1) and submitted to molecular dynamics simulation. The resulting models presented high content of coils, a higher percentage of α-helix, and an almost neglected content of 310-helix than the previous models. Knowledge of the complete structure of spidroins-1A and -1B would help to explain the mechanical features of silk fibers. The results of the current investigation provide a foundation for biophysical studies of the mechanoelastic properties of web-silk proteins.

Asunto(s)

Fibroínas/química , Modelos Moleculares , Seda/química , Arañas/química , Secuencia de Aminoácidos , Animales , Microscopía Electrónica de Rastreo , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido

Asunto(s)

Alérgenos/química , Epítopos de Linfocito B/química , Venenos de Avispas/inmunología , Adulto , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Epítopos de Linfocito B/inmunología , Femenino , Humanos , Inmunoglobulina E/inmunología , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Péptidos/inmunología , Avispas/inmunología , Adulto Joven

RESUMEN

Antigen-5 is one of the major allergens identified in wasp venoms, and despite the fact that its biological function is still unknown, many studies have demonstrated its allergenicity. In this study, the biochemical and structural characterization of antigen-5 from the venom of the social wasp Polybia paulista are reported. A gel-based mass spectrometry strategy with CID fragmentation methods and classical protocols of protein chemistry, which included N- and C-terminal sequencing, were used to assign the complete sequence and determine the presence/location of the post-translational modifications (PTMs) of this protein. Six different isoforms of antigen-5 were identified in the crude venom of P. paulista ; the most abundant, which corresponds to the intact form of this protein, was recognized by the pool of human specific-IgE. This protein was extensively sequenced through CID mass spectrometry, and a series of PTMs were observed such as hydroxylation, phosphorylation, and glycosylation. Sequence data revealed that this protein has 59.3-93.7% identity with antigen-5 proteins from other known vespid venoms. The molecular model of P. paulista antigen-5 shows that this protein has three α-helices, one 310 helix, and four ß-sheets covering 28 and 17.9% of the sequence, respectively. The identification and characterization of allergenic compounds is essential for the development of advanced component-resolved allergy diagnostics and treatment.

Asunto(s)

Alérgenos/inmunología , Procesamiento Proteico-Postraduccional , Proteómica , Venenos de Avispas/inmunología , Alérgenos/química , Secuencia de Aminoácidos , Animales , Electroforesis en Gel Bidimensional , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Avispas

RESUMEN

The phospholipases A(1) (PLA(1) s) from the venom of the social wasp Polybia paulista occur as a mixture of different molecular forms. To characterize the molecular origin of these structural differences, an experimental strategy was planned combining the isolation of the pool of PLAs from the wasp venom with proteomic approaches by using 2-D, MALDI-TOF-TOF MS and classical protocols of protein chemistry, which included N- and C-terminal sequencing. The existence of an intact form of PLA(1) and seven truncated forms was identified, apparently originating from controlled proteolysis of the intact protein; in addition to this, four of these truncated forms also presented carbohydrates attached to their molecules. Some of these forms are immunoreactive to specific-IgE, while others are not. These observations permit to raise the hypothesis that naturally occurring proteolysis of PLA(1) , combined with protein glycosylation may create a series of different molecular forms of these proteins, with different levels of allergenicity. Two forms of PLA(2) s, apparently related to each other, were also identified; however, it was not possible to determine the molecular origin of the differences between both forms, except that one of them was glycosylated. None of these forms were immunoreactive to human specific IgE.

Asunto(s)

Fosfolipasas A1/análisis , Venenos de Avispas/análisis , Avispas/química , Animales , Glicosilación , Inmunoglobulina E/inmunología , Isoenzimas/análisis , Isoenzimas/química , Isoenzimas/inmunología , Espectrometría de Masas , Fosfolipasas A1/química , Fosfolipasas A1/inmunología , Proteómica , Análisis de Secuencia de Proteína , Venenos de Avispas/inmunología