Dynamics Rationalize Proteolytic Susceptibility of the Major Birch Pollen Allergen Bet v 1.

Proteolytic susceptibility during endolysosomal degradation is decisive for allergic sensitization. In the major birch pollen allergen Bet v 1 most protease cleavage sites are located within its secondary structure elements, which are inherently inaccessible to proteases. The allergen thus must unfold locally, exposing the cleavage sites to become susceptible to proteolysis. Hence, allergen cleavage rates are presumed to be linked to their fold stability, i.e., unfolding probability. Yet, these locally unfolded structures have neither been captured in experiment nor simulation due to limitations in resolution and sampling time, respectively. Here, we perform classic and enhanced molecular dynamics (MD) simulations to quantify fold dynamics on extended timescales of Bet v 1a and two variants with higher and lower cleavage rates. Already at the nanosecond-timescale we observe a significantly higher flexibility for the destabilized variant compared to Bet v 1a and the proteolytically stabilized mutant. Estimating the thermodynamics and kinetics of local unfolding around an initial cleavage site, we find that the Bet v 1 variant with the highest cleavage rate also shows the highest probability for local unfolding. For the stabilized mutant on the other hand we only find minimal unfolding probability. These results strengthen the link between the conformational dynamics of allergen proteins and their stability during endolysosomal degradation. The presented approach further allows atomistic insights in the conformational ensemble of allergen proteins and provides probability estimates below experimental detection limits.

Aberrant endosperm formation caused by reduced production of major allergen proteins in a rice flo2 mutant that confers low-protein accumulation in grains.

Rice flo2 mutation produces grains showing a reduced amount of storage proteins. Using Nipponbare and the flo2 mutant, we created rice transformants that showed defective production of major allergen proteins RA14 and RA33 (14-16 kDa and 33 kDa allergen proteins, respectively) by RNAi introduction. The knock-down transformant generated using Nipponbare showed greatly reduced accumulation of both allergen proteins, normal growth, and production of a sufficient amount of normal-shaped seeds. F1 seeds were obtained by crossing between the transformants containing RNAi genes to RA14 and RA33, and showed decreased accumulation of both proteins. However, a peculiar phenotype was observed in the flo2 transformants that lacked accumulation of RA14 or RA33. They showed significantly reduced fertility. A wrinkled grain feature was found on the transformant lacking accumulation of RA14. F1 seeds obtained by crossing these transformants showed significantly lower fertility. F2 seeds showed decreases in both allergen proteins but morphological abnormality with small and severely wrinkled features. These results indicated that it is hard to obtain any transformant lacking accumulation of these allergen proteins using the flo2 mutant, whereas a knock-down transformant of both allergen protein genes was obtained when a wild-type Nipponbare was used as a host. These facts strongly suggest that RA14 and RA33 have some roles in rice seeds.

Effects of High-Pressure Treatment on the Muscle Proteome of Hake by Bottom-Up Proteomics.

A bottom-up proteomics approach was applied for the study of the effects of high-pressure (HP) treatment on the muscle proteome of fish. The performance of the approach was established for a previous HP treatment (150-450 MPa for 2 min) on frozen (up to 5 months at -10 °C) European hake ( Merluccius merluccius). Concerning possible protein biomarkers of quality changes, a significant degradation after applying a pressure ≥430 MPa could be observed for phosphoglycerate mutase-1, enolase, creatine kinase, fructose bisphosphate aldolase, triosephosphate isomerase, and nucleoside diphosphate kinase; contrary, electrophoretic bands assigned to tropomyosin, glyceraldehyde-3-phosphate dehydrogenase, and beta parvalbumin increased their intensity after applying a pressure ≥430 MPa. This repository of potential protein biomarkers may be very useful for further HP investigations related to fish quality.

Isolation and identification of allergens and biogenic amines of Prosopis juliflora genotypes

Background: Prosopis, or mesquite (Prosopis juliflora (Sw.) DC.), was introduced in Saudi Arabia several decades ago and is heavily used in street, roadside, and park plantations. It shows great adaptation to the prevailing climatic conditions such as high temperature, severe drought, and salinity and spreads naturally in many parts of the Kingdom. This research was conducted to isolate allergen proteins and biogenic amines from the pollen grains of P. juliflora genotypes in Saudi Arabia from two regions, namely Al-Qassim and Eastern regions. Results: The results showed that 18 different allergen proteins were detected in P. juliflora genotypes, with molecular weight ranging from 14 to 97 kDa. Moreover, P. juliflora genotypes from the two studied regions contained eight biogenic amines, namely histamine, tyramine, tryptamine, ß-phenylethylamine, butricine, codapherine, spermidine, and spermine. All genotypes from the Al-Qassim region were found to contain all eight amines, while in the Eastern region, histamine was absent in three genotypes, spermine was absent in six genotypes, and spermidine was absent in three genotypes. Genotypes B23, E20, and E21 had the lowest biogenic amine quantity. Conclusions: All identified proteins from mesquite trees from both regions (Eastern and Al-Qassim) cause allergies in patients who are sensitive to pollen grains. Bioamines, except histamine and tyramine, were recorded at varying concentrations in different genotypes.

SoyProDB: A database for the identification of soybean seed proteins.

UNLABELLED: Soybean continues to serve as a rich and inexpensive source of protein for humans and animals. A substantial amount of information has been reported on the genotypic variation and beneficial genetic manipulation of soybeans. For better understanding of the consequences of genetic manipulation, elucidation of soybean protein composition is necessary, because of its direct relationship to phenotype. We have conducted studies to determine the composition of storage, allergen and anti-nutritional proteins in cultivated soybean using a combined proteomics approach. Two-dimensional polyacrylamide gel electrophoresis (2DPAGE) was implemented for the separation of proteins along with matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography mass spectrometry (LC-MS/MS) for the identification of proteins. Our analysis resulted in the identification of several proteins, and a web based database named soybean protein database (SoyProDB) was subsequently built to house and allow scientists to search the data. This database will be useful to scientists who wish to genetically alter soybean with higher quality storage proteins, and also helpful for consumers to get a greater understanding about proteins that compose soy products available in the market. The database is freely accessible. AVAILABILITY: http://bioinformatics.towson.edu/Soybean_Seed_Proteins_2D_Gel_DB/Home.aspx.

Purification, identification and characterization of the major allergen from Linnaeus / 中国免疫学杂志

Objective:To purify and characterize the major antigenic components of sand swimming crab,and provide theoretical evidences for the research of standardization of allergenic vaccine.Methods:The crude extract of Linnaeus was prepared using classical method, and electrophoresis of SDS-PAGE was used to separate the proteins of each group. The allergen proteins of 26 crab were identified using Western blot. To distinguish between the primary allergen proteins and secondary allergen proteins, FPLC(Gel Chromatography and ion exchange chromatography) was used to filtrate and identify the allergen protein.Results:SDS-PAGE analysis revealed that sand swimming crab proteins were composed of at lease 9 discrete protein bands, which molecular weight ranging from 13 000 to 90 000. Major bands were of 20 900, 24 200, 27 100, 29 200, 33 700, 38 900, 48 700, 74 700, 89 100 respectively. Western blot assay indicated that the crude extract reacted with sera obtained from 26 crab allergenic subjects and contained 5 allergen bands altogether, and the bands of 74 400 and 48 700 were the major allergenic components. The positive rates of the two major allergen proteins were both 100%, indicating the allergenic components maintained the immunocompetence after yielding from chromatography.Conclusion:The 74 400 and 48 700 bands are the major allergens of sand swimming crab. The major allergen proteins can be purified by chromatography.