RESUMO
Allergy to freshly consumed apple fruits is often associated to pollinosis and manifested as oral allergy syndrome (OAS). The allergenic properties of apple varieties differ greatly, spanning from low allergenic to high allergenic varieties. The knowledge of the genetic determinants for allergenicity has been of great interest in scientific community for several years, but the molecular mechanisms involved are still little understood. Here, factors putatively involved in allergenicity were investigated at biochemical and molecular level in pollen and in fruits of apple varieties differing in their allergenic potential. Among putative sensitizing factors, transglutaminase (TGase) and phospholipase A2 (PLA2) were considered together with reactive oxygen species (ROS) and known apple allergen genes, with particular attention devoted to the Mal d 1 gene family, the most important one in sensitization. We found that the expression of some allergen genes and the activities of TGase, PLA2 and ROS producing enzyme are lower in the hypo-allergenic variety 'Durello di Forlì' in comparison with the high-allergenic genotypes 'Gala' and 'Florina'. These results highlight correlations among allergen expressions, enzymatic activities and apple cultivars; these data underline the possibility that some of them could be used in the future as markers for allergenicity.
Assuntos
Alérgenos/genética , Hipersensibilidade Alimentar , Malus/genética , Alérgenos/efeitos adversos , Alérgenos/imunologia , Frutas/efeitos adversos , Frutas/genética , Frutas/imunologia , Humanos , Malus/efeitos adversos , Malus/imunologia , Pólen/efeitos adversos , Pólen/genética , Pólen/imunologiaRESUMO
Transglutaminases (TGases) are ubiquitous enzymes that take part in a variety of cellular functions. In the pollen tube, cytoplasmic TGases are likely to be involved in the incorporation of primary amines at selected peptide-bound glutamine residues of cytosolic proteins (including actin and tubulin), while cell wall-associated TGases are believed to regulate pollen tube growth. Using immunological probes, we identified TGases associated with different subcellular compartments (cytosol, membranes, and cell walls). Binding of cytosolic TGase to actin filaments was shown to be Ca(2+) dependent. The membrane TGase is likely associated with both Golgi-derived structures and the plasma membrane, suggesting a Golgi-based exocytotic delivery of TGase. Association of TGase with the plasma membrane was also confirmed by immunogold transmission electron microscopy. Immunolocalization of TGase indicated that the enzyme was present in the growing region of pollen tubes and that the enzyme colocalizes with cell wall markers. Bidimensional electrophoresis indicated that different TGase isoforms were present in distinct subcellular compartments, suggesting either different roles or different regulatory mechanisms of enzyme activity. The application of specific inhibitors showed that the distribution of TGase in different subcellular compartments was regulated by both membrane dynamics and cytoskeleton integrity, suggesting that delivery of TGase to the cell wall requires the transport of membranes along cytoskeleton filaments. Taken together, these data indicate that a cytoplasmic TGase interacts with the cytoskeleton, while a different TGase isoform, probably delivered via a membrane/cytoskeleton-based transport system, is secreted in the cell wall of pear (Pyrus communis) pollen tubes, where it might play a role in the regulation of apical growth.
Assuntos
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Tubo Polínico/citologia , Tubo Polínico/enzimologia , Pyrus/citologia , Pyrus/enzimologia , Transglutaminases/metabolismo , Citoesqueleto de Actina , Actinas/metabolismo , Cálcio/metabolismo , Compartimento Celular , Parede Celular/enzimologia , Eletroforese em Gel de Poliacrilamida , Isoenzimas/metabolismo , Microtúbulos/metabolismo , Modelos Biológicos , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/ultraestrutura , Ligação Proteica , Transporte Proteico , Pyrus/ultraestruturaRESUMO
Allergy to citrus fruits is often associated with pollinosis and sensitization to other plants due to a phenomenon of cross-reactivity. The aims of the present study were to highlight the cross-reactivity among citrus and the major allergenic pollens/fruits, throughout clinical and molecular investigations, and to evaluate the sensitization frequency to citrus fruits in a population of children and adults with pollinosis. We found a relevant percentage of sensitisation (39%) to citrus fruits in the patients recruited and in all of them the IgE-mediated mechanism has been confirmed by the positive response to the prick-to-prick test. RT-PCR experiments showed the expression of Cit s 1, Cit s 3 and a profilin isoform, already described in apple, also in Citrus clementine pollen. Data of multiple sequence alignments demonstrated that Citrus allergens shared high percentage identity values with other clinically relevant species (i.e. Triticum aestivum, Malus domestica), confirming the possible cross-allergenicity citrus/grasses and citrus/apple. Finally, a novelty of the present work has been the expression of two phospholipaseA2 isoforms (PLA2 α and ß) in Citrus as well as in Triticum pollens; being PLA2 able to generate pro-inflammatory factors, this enzyme could participate in the activation of the allergenic inflammatory cascade.
Assuntos
Alérgenos/imunologia , Citrus/imunologia , Hipersensibilidade Alimentar/imunologia , Proteínas de Plantas/imunologia , Pólen/imunologia , Rinite Alérgica Sazonal/imunologia , Adolescente , Alérgenos/química , Criança , Citrus/química , Reações Cruzadas , Feminino , Hipersensibilidade Alimentar/patologia , Fosfolipases A2 do Grupo IV/química , Fosfolipases A2 do Grupo IV/imunologia , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Malus/química , Malus/imunologia , Proteínas de Plantas/química , Poaceae/química , Poaceae/imunologia , Profilinas/química , Profilinas/imunologia , Rinite Alérgica Sazonal/patologia , Homologia de Sequência de Aminoácidos , Testes Cutâneos , Triticum/química , Triticum/imunologia , Adulto JovemRESUMO
Immature cells of etiolated apices of sprouts growing from Helianthus tuberosus (H. t.) tubers showed Ca(2+)-dependent transglutaminase (TG, EC 2.3.2.13) activity on fibronectin (more efficiently) and dimethylcasein as substrates. Three main TG bands of about 85, 75 and 58 kDa were isolated from the 100,000×g apices supernatant through a DEAE-cellulose column at increasing NaCl concentrations and immuno-identified by anti-TG K and anti-rat prostate gland TG antibodies. These three fractions had catalytic activity as catalyzed polyamine conjugation to N-benzyloxycarbonyl-L-γ-glutaminyl-L-leucine (Z-L-Gln-L-Leu) and the corresponding glutamyl-derivatives were identified. The amino acid composition of these TG proteins was compared with those of several sequenced TGs of different origin. The composition of the two larger bands presented great similarities with annotated TGs; in particular, the 75 kDa form was very similar to mammalian inactive EPB42. The 58 kDa form shared a low similarity with other TGs, including a maize sequence of similar molecular mass, which, however, did not present the catalytic triad in the position of all annotated TGs. A 3D model of the H. t. TGs was built adopting TG2 as template. These novel plant TGs are hypothesized to be constitutive and discussed in relation to their possible roles in immature cells. These data suggest that in plants, multiple TG forms are active in the same organ and that plant and animal enzymes probably are very close not only for their catalytic activity but also structurally.
Assuntos
Helianthus/enzimologia , Proteínas de Plantas/metabolismo , Plântula/enzimologia , Transglutaminases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Expressão Gênica , Helianthus/citologia , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteína 2 Glutamina gama-Glutamiltransferase , Estrutura Secundária de Proteína , Plântula/citologia , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Transglutaminases/química , Transglutaminases/genéticaRESUMO
Increases in temperature and air pollution influence pollen allergenicity, which is responsible for the dramatic raise in respiratory allergies. To clarify possible underlying mechanisms, an anemophilous pollen (hazel, Corylus avellana), known to be allergenic, and an entomophilous one (apple, Malus domestica), the allergenicity of which was not known, were analysed. The presence also in apple pollen of known fruit allergens and their immunorecognition by serum of an allergic patient were preliminary ascertained, resulting also apple pollen potentially allergenic. Pollens were subjected to simulated stressful conditions, provided by changes in temperature, humidity, and copper and acid rain pollution. In the two pollens exposed to environmental criticalities, viability and germination were negatively affected and different transglutaminase (TGase) gel bands were differently immunodetected with the polyclonal antibody AtPng1p. The enzyme activity increased under stressful treatments and, along with its products, was found to be released outside the pollen with externalisation of TGase being predominant in C. avellana, whose grain presents a different cell wall composition with respect to that of M. domestica. A recombinant plant TGase (AtPng1p) stimulated the secreted phospholipase A(2) (sPLA(2)) activity, that in vivo is present in human mucosa and is involved in inflammation. Similarly, stressed pollen, hazel pollen being the most efficient, stimulated to very different extent sPLA(2) activity and putrescine conjugation to sPLA(2). We propose that externalised pollen TGase could be one of the mediators of pollen allergenicity, especially under environmental stress induced by climate changes.
Assuntos
Alérgenos/imunologia , Corylus/enzimologia , Hipersensibilidade/imunologia , Malus/enzimologia , Pólen/enzimologia , Transglutaminases/metabolismo , Sequência de Bases , Western Blotting , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Humanos , Reação em Cadeia da PolimeraseRESUMO
Pollination of pummelo (Citrus grandis L. Osbeck) pistils has been studied in planta by adding compatible and self-incompatible (SI) pollen to the stigma surface. The pollen germination has been monitored inside the pistil by fluorescent microscopy showing SI altered morphologies with irregular depositions of callose in the tube walls, and heavy callose depositions in enlarged tips. The polyamine (PA) content as free, perchloric acid (PCA)-soluble and -insoluble fractions and transglutaminase (TGase) activity have been analyzed in order to deepen their possible involvement in the progamic phase of plant reproduction. The conjugated PAs in PCA-soluble fraction were definitely higher than the free and the PCA-insoluble forms, in both compatible and SI pollinated pistils. In pistils, pollination caused an early decrease of free PAs and increase of the bound forms. The SI pollination, showed highest values of PCA-soluble and -insoluble PAs with a maximum in concomitance with the pollen tube arrest. As TGase mediates some of the effects of PAs by covalently binding them to proteins, its activity, never checked before in Citrus, was examined with two different assays. In addition, the presence of glutamyl-PAs confirmed the enzyme assay data and excluded the possibility of a misinterpretation. The SI pollination caused an increase in TGase activity, whereas the compatible pollination caused its decrease. Similarly to bound PAs, the glutamyl-PAs and the enzyme activity peaked in the SI pollinated pistils in concomitance with the observed block of the pollen tube growth, suggesting an involvement of TGase in SI response.
Assuntos
Citrus/fisiologia , Polinização , Poliaminas/metabolismo , Transglutaminases/metabolismo , Cromatografia Líquida de Alta Pressão , Citrus/enzimologia , Microscopia de Fluorescência , Percloratos/químicaRESUMO
An extracellular form of the calcium-dependent protein-cross-linking enzyme TGase (transglutaminase) was demonstrated to be involved in the apical growth of Malus domestica pollen tube. Apple pollen TGase and its substrates were co-localized within aggregates on the pollen tube surface, as determined by indirect immunofluorescence staining and the in situ cross-linking of fluorescently labelled substrates. TGase-specific inhibitors and an anti-TGase monoclonal antibody blocked pollen tube growth, whereas incorporation of a recombinant fluorescent mammalian TGase substrate (histidine-tagged green fluorescent protein: His6-Xpr-GFP) into the growing tube wall enhanced tube length and germination, consistent with a role of TGase as a modulator of cell wall building and strengthening. The secreted pollen TGase catalysed the cross-linking of both PAs (polyamines) into proteins (released by the pollen tube) and His6-Xpr-GFP into endogenous or exogenously added substrates. A similar distribution of TGase activity was observed in planta on pollen tubes germinating inside the style, consistent with a possible additional role for TGase in the interaction between the pollen tube and the style during fertilization.