Purification and identification of globulin-1 S allele as a novel allergen with N-glycans in wheat (Triticum aestivum).

Wheat (Triticum aestivum) contains various allergenic proteins and induces diverse allergic reactions. Although many allergens are found to be glycoproteins, research on glycosylated allergens in wheat is rare. In this study, the main glycoprotein in wheat soluble protein was purified by sequential multi-column chromatography including affinity chromatography, gel filtration chromatography, and ion-exchange chromatography. The purified proteins were identified as globulin-1 S allele by liquid chromatography-tandem mass spectrometry and characterized by circular dichroism spectra. The strong IgE-binding capacity of the purified fractions was further demonstrated, suggesting globulin-1 S allele to be a novel allergen in wheat. Further de-N-glycosylated treatment by N-Glycosidase F showed that N-glycosylation enhanced the IgE-binding of globulin-1 S allele. These results identified globulin-1 S allele as a novel allergen in wheat, demonstrated the role of glycosylation in IgE-binding, and provided new insights into the prevention and treatment of wheat allergy.

Allergenicity assessment and allergen profile analysis of different Chinese wheat cultivars.

BACKGROUNDS: As one of the most important cereals, wheat (Triticum aestivum) can cause severe allergic reactions, such as baker's asthma, allergic rhinitis, and atopic dermatitis. A growing number of people are developing allergies to Chinese wheat; however, only a few wheat cultivars have been screened on allergenicity in China. OBJECTIVE: The aim of the present study was to assess the allergenicity of different Chinese wheat cultivars and characterize wheat allergen profiles of patients with allergic rhinitis. METHODS: We determined protein (soluble protein, gliadin, and glutenin) composition in Chinese wheat by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the immunoglobulin E (IgE) binding capacity by enzyme-linked immunosorbent assay (ELISA) and Western blot using 10 positive sera from wheat allergy patients. We identified 5 gel bands with significant IgE binding capacity using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Soluble protein, albumin, and globulin, showed the highest allergenicity, followed by gliadin, while glutenin only had slight allergenicity. In soluble protein, 5 protein bands with molecular weights of 27, 28, 53, 58, and 62 kDa showed very significant allergenicity. Meanwhile, the relative abundances of 28 kDa-protein and 58 kDa-protein were significantly positively correlated with the IgE-binding capacity of Chinese wheat cultivars, which were identified as rRNA N-glycosidase and ß-amylase, respectively, among other proteins in those highly complex gel bands. CONCLUSION AND CLINICAL RELEVANCE: 28 kDa-protein (rRNA N-glycosidase) and 58 kDa-protein (ß-amylase) were speculated to be the main allergens of Chinese wheat causing baker's asthma and allergic rhinitis. These results provide new insights into the prevention and treatment of wheat allergy and the development of hypoallergenic wheat products, whose clinical significance is worth further evaluation.

Proteomic Analysis of ISGylation in Immortalized Porcine Alveolar Macrophage Cell Lines Induced by Type I Interferon.

Interferon-stimulated gene product 15 (ISG15), a ubiquitin-like molecule, can be conjugated to protein substrates through a reversible process known as ISGylation. ISG15 and ISGylation are both strongly upregulated by type I interferons and play putative key roles in host innate immunity against viral infection. However, the function of ISGylation and identities of ISGylation substrates are largely unknown. Here, a novel monoclonal antibody (Mab) that specifically recognizes porcine ISG15 (pISG15) was employed to capture ISG15-conjugated proteins from IFNs-stimulated porcine cell lysates. Next, Mab-captured conjugates were analyzed using proteomics-based tools to identify potential ISGylation protein targets in order to elucidate the roles of ISG15 and ISGylation in porcine cells. Subsequently, 190 putative ISGylation sites were detected within 98 identified ISGylation candidates; several candidates contained more than one ISGylation-modifiable lysine residue, including pISG15 itself. Motif enrichment analysis of confirmed ISGylation sites demonstrated a moderate bias towards certain sites with specific upstream amino acid residues. Meanwhile, results of Gene Ontology (GO)-based annotation and functional enrichment and protein-protein interaction (PPI) network analyses of porcine ISG15-conjugated substrate proteins indicated that these substrates were mainly associated with the host metabolism, especially nucleotide metabolic pathways that ultimately may participate in cellular antiviral defenses. Notably, several ISGs (MX1, IFIT1, OAS1, ISG15 and putative ISG15 E3 ligase Herc6) were also identified as putative ISGylation substrates within a regulatory loop involving ISGylation of ISGs themselves. Taken together, proteomics analysis of porcine ISGylation substrates revealed putative functional roles of ISG15 and novel host ISGylation targets that may ultimately be involved in cellular antiviral responses.

Tuning the conductance of H2O@C60 by position of the encapsulated H2O.

The change of conductance of single-molecule junction in response to various external stimuli is the fundamental mechanism for the single-molecule electronic devices with multiple functionalities. We propose the concept that the conductance of molecular systems can be tuned from inside. The conductance is varied in C60 with encapsulated H2O, H2O@C60. The transport properties of the H2O@C60-based nanostructure sandwiched between electrodes are studied using first-principles calculations combined with the non-equilibrium Green's function formalism. Our results show that the conductance of the H2O@C60 is sensitive to the position of the H2O and its dipole direction inside the cage with changes in conductance up to 20%. Our study paves a way for the H2O@C60 molecule to be a new platform for novel molecule-based electronics and sensors.

Transport properties of the H2O@C60-dimer-based junction.

Theoretical predictions play an important role in finding potential applications in molecular electronics. Fullerenes have a number of potential applications, and the charge flow from a single C60 molecule to another becomes more versatile and more interesting after doping. Here, we report the conductance of two H2O@C60 molecules in series order and how the number of encapsulated water molecules influences the transport properties of the junction. Encapsulating an H2O molecule into one of the C60 cages increases the conductance of the dimer. Negative differential resistance is found in the dimer systems, and its peak-to-valley current ratio depends on the number of encapsulated H2O molecules. The conductance of the C60 dimer and the H2O@C60 dimer is two orders of magnitude smaller than that of the C60 monomer. Furthermore, we demonstrate that the conductance of the molecular junctions based on the H2O@C60 dimer can be tuned by moving the encapsulated H2O molecules. The conductance is H2O-position dependent. Our findings indicate that H2O@C60 can be used as a building block in C60-based molecular electronic devices and sensors.