Identification and partial characterization of the allergenic proteins of Ricinus communis L. pollen - a new approach

The pollen of Ricinus communis L., a potentially allergenic plant, was extracted to identify the allergenic determinants responsible for causing respiratory disorders. The soluble proteins were extracted and subjected to ammonium sulphate precipitation at 80% saturation and the total protein separated on 12% SDS-Polyacrylamide gel. In order to avoid the time consuming and expensive biochemical methods of column chromatography, each band was directly recovered from the gel by electroelution and the allergenic proteins identified directly by skin tests, without the necessity of Phadezym RAST or ELISA inhibition by reaction with serum IgE, the general procedure to identify the allergens. The fourth and the fifth band in the protein profile of R. communis pollen, RC4 (77 kD) and RC5 (66 kD) were the two major allergenic components. RC3 (91 kD) also induced a considerable amount of reactivity in sensitive patients. Contrary to the earlier reports of protein bands of R. communis ranging from 14 kD to 70 kD, 4 bands above 70 kD i.e. RC1 (123 kD), RC2 (97 kD), RC3 (91 kD) and RC4 (77 kD) are reported here for the first time. Immunodiffusion analysis with pooled sera of patients sensitive to the total extract also revealed similar results.     

Time‐course proteome analysis of developing extrafloral nectaries of Ricinus communis

Floral and extrafloral nectaries are unique organs that secrete energy rich chemical components, but their contribution for nectar production is largely unknown. Here, we present the first comparative proteome dataset of four developmental stages of the extrafloral nectaries from castor plant (Ricinus communis), an important biofuel crop. Respectively, from stage I—IV, we identified 626, 613, 449 and 356 proteins, respectively, summing up 882 nonredundant proteins. Surprisingly, we identified two isoforms of the potent toxin ricin, indicating that ricin expression is not limited to seeds, but it may serve a general defense purpose for the castor plant. To date, this is the most complete dataset of proteins either from floral or extrafloral nectaries, thus contributing to lay the foundations for investigations on their ecological and evolutionary importance.     

Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress

In Central and Southern Italy, where durum wheat represents one of the most widely cultivated crops, grain filling occurs during Spring, a period characterized by sudden increases in temperature. Wheat grain proteins are classified into albumins, globulins, and prolamins. The nonprolamin fractions include proteins with metabolic activity or structural function. In order to investigate the consequences of heat stress on the accumulation of nonprolamin proteins in mature durum wheat kernels, the Italian cultivar Svevo was subjected to two thermal regimes (heat stress versus control). The 2‐D patterns of nonprolamin proteins were monitored to identify polypeptides affected by heat stress during grain fill. This study shows that heat stress alters significantly the durum wheat seed proteome, although the changes range is only between 1.2‐ and 2.2‐fold. This analysis revealed 132 differentially expressed polypeptides, 47 of which were identified by MALDI‐TOF and MALDI‐TOF‐TOF MS and included HSPs, proteins involved in the glycolysis and carbohydrate metabolism, as well as stress‐related proteins. Many of the heat‐induced polypeptides are considered to be allergenic for sensitive individuals.     

城市中的花粉致敏植物及其影响因素

大量致敏花粉飘散于空气中会引发花粉症,严重危害人体健康。由于不当的城市绿地建设使得花粉致敏植物在城市中大量聚集,加之城市热岛效应和交通污染等多方面的因素,使得城市中花粉症患者数量激增。综述了花粉致敏植物的种类、物候特征和影响因素提出了控制花粉致敏植物的建议;总结了目前花粉致敏植物研究中应进一步关注的问题,旨在为改善城市空气质量,建设合理绿化环境提供科学依据。     

Detection and release of allergenic proteins in Parietaria judaica pollen grains

Summary. Rapid diffusion of allergenic proteins in isotonic media has been demonstrated for different pollen grains. Upon contact with stigmatic secretion or with the mucosa of sensitive individuals, pollen grains absorb water and release soluble low-molecular-weight proteins, these proteins enter in the secretory pathway in order to arrive at the cell surface. In this study we located allergenic proteins in mature and hydrated-activated pollen grains of Parietaria judaica L. (Urticaceae) and studied the diffusion of these proteins during the first 20 min of the hydration and activation processes. A combination of transmission electron microscopy and immunocytochemical methods was used to locate these proteins in mature pollen and in pollen grains after different periods of hydration and activation processes. Activated proteins reacting with antibodies in human serum from allergic patients were found in the cytoplasm, wall, and exudates from the pollen grains. The allergenic component of these pollen grains changes according to the pollen state; the presence of these proteins in the exine, the cytoplasm, and especially in the intine and in the material exuded from the pollen grains, is significant in the hydrated-activated studied times, whereas this presence is not significant in mature pollen grains. The rapid activation and release of allergenic proteins of P. judaica pollen appears to be the main cause of the allergenic activity of these pollen grains. Correspondence and reprints: Department of Plant Biology, Faculty of Biology, University of León, Campus de Vegazana, 24071 León, Spain.     

Proteomic characterization of Phragmites communis in ecotypes of swamp and desert dune

Phragmites communis Trin. (common reed) is a recognized model plant for studying its adaptation to contrasting and harsh environments. To understand the inherent molecular basis for its remarkable resistance to combined stresses, we performed a comprehensive proteomic analysis of the leaf proteins from two ecotypes, i.e. swamp and desert dune, naturally growing in the desert region of northwestern China. First, a proteome reference map of Phragmites was established based on the swamp ecotype. Proteins were resolved by 2‐D/SDS‐PAGE and identified by MALDI‐TOF/TOF MS. In total, 177 spots were identified corresponding to 51 proteins. The major proteins identified are proteins involved in photosynthesis, glutathione and ascorbic acid metabolism as well as protein synthesis and quality control. Second, the 2‐DE profiles of the two ecotypes were compared quantitatively via DIGE analysis. Compared with swamp ecotype, 51 proteins spots are higher‐expressed and 58 protein spots are lower‐expressed by twofold or more in desert dune ecotype. Major differences were found for the proteins involved in light reaction of photosynthesis, protein biosynthesis and quality control and antioxidative reactions. The physiological significance of such differences is discussed in the context of a flow of complex events in relation to plant adaptation to combined environmental stresses.     

A modified FCR test to evaluate pollen viability in Juniperus communis L.

Pollen viability of Juniperus communis L. and other gymnosperms with taxoid type pollen cannot be assessed with the more common viability tests because the thick sporoderm prevents reagents from penetrating into the cytoplasm. Here we describe a technique for pre‐hydration of pollen that overcomes this problem so that the common FCR test can be used to assess its viability. Pollen of J. communis must be re‐hydrated by suspending in water. This re‐hydration causes the splitting of the exine and a huge swelling of the intine. The sporoderm becomes permeable to fluoresceine diacetate and the FCR‐viability test can be applied. The FCR result is supported by scoring the germination percentage in vitro.     

Cell wall acyl-lipids, proteins and polysaccharides in mature and germinated olive pollen

Proteins, acyl-lipids and polysaccharides from cell walls of mature and germinated olive pollen were studied. In general, hemicelluloses are the most abundant polysaccharides, arabinose in mature and glucose in germinated pollen being the main components of these macromolecules. Protein content and its amino acid composition are very similar in walls from mature and germinated pollen, these compounds showing a weak acid character. Free-fatty acids are the most abundant lipid molecules in mature and germinated pollen walls and a decrease in acyl-lipids, especially in polar lipids, as well as a higher unsaturation of their fatty acid components are observed after germination.     

Identification of specific IgE binding proteins in Castor bean (Ricinus communis) pollen obtained from different source materials

Allergenic components of Ricinus communis pollen obtained from different stages of inflorescence, different time intervals, different years and places were studied by immunoblot analysis. Proteins separated by SDS-PAGE and transferred to NC were identified using pooled sera from 15 skin and RAST positive patients. The IgE binding components in M.W. range of 14 to 70 kD were identified. The protein fractions of 70, 66, 64, 60, 50, 45, 36, 22 and 14 kD are the most prominent allergenic bands. Six samples collected during same pollination season from the same place showed similar allergenic profile. Of the samples collected from different stages of inflorescence, pollen of immature buds showed only three bands as compared to 18 from mature buds and flowers. Variability was seen in the IgE binding components of pollen stored for different years and obtained from different geographic regions of India. The IgE binding pattern of fifteen sera were heterogenous. The number of bands identified by different sera varied from 3 to 18. Two protein components of 66 and 36 kD were recognised by 14 (93.3%) of the 15 sera studied. The result suggests that there exists variations in the specific IgE binding pattern in pollen samples of Castor Bean, obtained from difference source materials.     

Strategy for surveying the proteome using affinity proteomics and mass spectrometry

Antibody‐based microarrays is a rapidly evolving technology that has gone from the first proof‐of‐concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high‐ as well as low‐abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in‐depth global proteome surveys, we propose to interface affinity proteomics with MS‐based read‐out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5–100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10⁴ individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif‐specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif‐containing peptides are specifically captured, enriched and subsequently detected and identified using MS.     

Mountain cedar pollen induces IgE-independent mast cell degranulation, IL-4 production, and intracellular reactive oxygen species generation

Cedar pollens cause severe allergic disease throughout the world. We have previously characterized allergenic pollen glycoproteins from mountain cedar (Juniperus ashei) that bind to allergen-specific immunoglobulin E (IgE). In the present report, we investigated an alternative pathway of mast cell activation by mountain cedar pollen extract through IgE-independent mechanisms. We show that mountain cedar pollen directly induces mast cell serotonin and IL-4 release and enhances release induced by IgE cross-linking. Concomitant with mediator release, high levels of intracellular reactive oxygen species (ROS) were generated, and both ROS and serotonin release were inhibited by anti-oxidants. These findings suggest that alternative mechanisms exist whereby pollen exposure enhances allergic inflammatory mediator release through mechanisms that involve ROS. These mechanisms have the potential for enhancing the allergenic potency of pollens.     

Tubers from potato lines expressing a tomato Kunitz protease inhibitor are substantially equivalent to parental and transgenic controls

Recombinant protease inhibitors represent useful tools for the development of insect‐resistant transgenic crops, but questions have been raised in recent years about the impact of these proteins on endogenous proteases and chemical composition of derived food products. In this study, we performed a detailed compositional analysis of tubers from potato lines expressing the broad‐spectrum inhibitor of Ser and Asp proteases, tomato cathepsin D inhibitor (SlCDI), to detect possible unintended effects on tuber composition. A compositional analysis of key nutrients and toxic chemicals was carried out with tubers of SlCDI‐expressing and control (comparator) lines, followed by a two‐dimensional gel electrophoresis (2‐DE) proteomic profiling of total and allergenic proteins to detect eventual effects at the proteome level. No significant differences were observed among control and SlCDI‐expressing lines for most chemicals assayed, in line with the very low abundance of SlCDI in tubers. Likewise, proteins detected after 2‐DE showed no quantitative variation among the lines, except for a few proteins in some control and test lines, independent of slcdi transgene expression. Components of the patatin storage protein complex and Kunitz protease inhibitors immunodetected after 2‐DE showed unaltered deposition patterns in SlCDI‐expressing lines, clearly suggesting a null impact of slcdi on the intrinsic allergenic potential of potato tubers. These data suggest, overall, a null impact of slcdi expression on tuber composition and substantial equivalence between comparator and SlCDI‐expressing tubers despite reported effects on leaf protein catabolism. They also illustrate the usefulness of proteomics as a tool to assess the authenticity of foods derived from novel‐generation transgenic plants.     

Comparative proteome analysis of porcine follicular fluid and serum reveals that excessive α2‐macroglobulin in serum hampers successful expansion of cumulus‐oocyte complexes

Porcine follicular fluid (pFF) constitutes the micro‐environment of the maturing oocyte. Although pFF is a transudate of serum, in pigs, it is superior to serum in promoting in vitro expansion of the cumulus cells, a specialized cell population surrounding the oocyte. A comparative proteome analysis of autologous serum and pFF was performed to investigate proteins involved in successful cumulus expansion of porcine oocytes. iTRAQ labeling followed by 2‐D LC ESI‐Q‐TOF MS/MS revealed 63 proteins common to both fluids of which the abundance of 13 proteins was significantly different (p<0.05). Seven proteins were more concentrated in serum whereas six proteins were more abundant in pFF. To investigate the importance of these proteins, the cumulus matrices of COCs were collected after in vitro maturation in media supplemented with either of both biologically fluids and then subjected to 2‐D PAGE analysis. α₂‐Macroglobulin and CH4 and secrete domains of swine IgM, which were both less abundant in pFF, were absent from cumulus matrix extracts after in vitro maturation in pFF. Although both proteins were incorporated in the matrices of cumulus‐oocyte complexes matured in serum, depletion of α₂‐macroglobulin from serum could significantly compensate for the impaired cumulus expansion of oocytes matured in serum.     

Proteomic profiling and characterization of differential allergens in the nematodes Anisakis simplex sensu stricto and A. pegreffii

The parasite species complex Anisakis simplex sensu lato (Anisakis simplex sensu stricto; (A. simplex s.s.), A. pegreffii, A. simplex C) is the main cause of severe anisakiasis (allergy) worldwide and is now an important health matter. In this study, the relationship of this Anisakis species complex and their allergenic capacities is assessed by studying the differences between the two most frequent species (A. simplex s.s., A. pegreffii) and their hybrid haplotype by studying active L3 larvae parasiting Merluccius merluccius. They were compared by 2D gel electrophoresis and parallel Western blot (2DE gels were hybridized with pools of sera from Anisakis allergenic patients). Unambiguous spot differences were detected and protein assignation was made by MALDI‐TOF/TOF analysis or de novo sequencing. Seventy‐five gel spots were detected and the corresponding proteins were identified. Differentially expressed proteins for A. simplex s.s., A. pegreffii, and their hybrid are described and results are statistically supported. Twenty‐eight different allergenic proteins are classified according to different families belonging to different biological functions. These proteins are described for the first time as antigenic and potentially new allergens in Anisakis. Comparative proteomic analyses of allergenic capacities are useful for diagnosis, epidemiological surveys, and clinical research. All MS data have been deposited in the ProteomeXchange with identifier PXD000662 ( http://proteomecentral.proteomexchange.org/dataset/PXD000662 ).     

The identification of a pollen-specific LEA-like protein in Lilium longiflorum

A novel pollen‐specific LEA‐like protein, LP28, was detected in Lilium longiflorum using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Immunoblot analysis using antiserum raised against LP28 revealed that the protein was not found in somatic tissues or uninucleate microspores, but accumulated gradually in developing pollen following microspore mitosis. Furthermore, LP28 was abundant in germinated pollen after hydration. The cDNA clone corresponding to LP28 encoded a putative protein of 238 amino acids with a calculated molecular mass of 24·2 kDa and a pI of 4·7. The amino acid sequence is highly hydrophilic except for the N‐terminal hydrophobic signal peptide. The sequence has similarities with group 3 LEA (late embryogenesis abundant) proteins. Immunocytochemical analyses demonstrated that LP28 was mainly found in cytoplasmic granules of the vegetative cell until pollen maturation, but after hydration it appeared in the elongating pollen tube wall. LP28 might be a unique pollen‐specific protein that is transported to the pollen tube wall after germination. Therefore, it is assumed that LP28 plays a role not only in pollen maturation, but also in the growth of the pollen tube, which penetrates the stylar matrix.     

Identification and characterisation of differentially expressed leaf proteins among Vitis species

Grapes are commercially grown worldwide for fresh fruit and wine. They are mainly classified into bunch and muscadine grapes. These species differ in their sugar content and composition, photosynthetic efficiency and tolerance to abiotic and biotic stresses. Grape berry relies on carbohydrates produced during photosynthesis to support its development and composition. In view of the unique physiology and genetic make‐up of muscadine grape, a proteomics study was performed to increase our knowledge of Vitis leaf proteome in order to improve enological and disease tolerance characteristics of grape species. A high throughput two‐dimensional gel electrophoresis (2‐DE) was conducted on muscadine, bunch and hybrid bunch leaf proteins. The differentially expressed proteins were excised from 2‐DE gels, subjected to in‐gel trypsin digestion, and analysed in MALDI/TOF mass spectrometer. The mass spectra were collected and protein identification was performed by searching against Viridiplantae database using Matrix Science algorithm. Proteins were mapped to universal protein resource to study gene ontology. We have discovered >255 proteins with pIs between 3.5 and 8.0 and molecular weight between 12 and 100 kDa among the Vitis species. Comparative analysis of leaf proteome showed that 54 polypeptides varied qualitatively and quantitatively among the three Vitis species studied. Of these, seven proteins were unique to muscadine, two proteins were present in both muscadine and bunch, while 28 proteins were common to all the three species. Bioinformatic analysis of these proteins showed that they are involved in signal transduction pathway, transport of metabolites, energy metabolism, protein trafficking, photosynthesis and defence. We have also identified proteins unique to muscadine grape that are involved in defence and stress tolerance. In addition, photosynthesis‐related proteins were found to be more abundant in Vitis vinifera grape compared to other Vitis species.