Physical Properties of Intact Proteins May Predict Allergenicity or Lack Thereof

Citation: Singh S, Taneja B, Salvi SS, Agrawal A ( Physical Properties of Intact Proteins May Predict Allergenicity or Lack Thereof Suchita Singh 0 Bhupesh Taneja 0 Sundeep Santosh Salvi 0 Anurag Agrawal 0 Dominik Hartl, LMU University of Munich, Germany 0 1 Institute of Genomics & Integrative Biology , Delhi , India , 2 Chest Research Foundation, Pune , India Background: Predicting the allergenicity of proteins is challenging. We considered the possibility that the properties of the intact protein that may alter the likelihood of being taken up by antigen presenting cells, may be useful adjuncts in predicting allergens and non-allergens in silico. It has been shown that negatively charged acidic proteins are preferentially processed by dendritic cells. Methodology: Datasets (aeroallergen, food-allergen and non-allergen) for in-silico study were obtained from public databases. Isoelectric point (pI), net charge, and electrostatic potential (EP) were calculated from the protein sequence (for pI and net charge) or predicted structure (for EP). Result: Allergens and non allergens differed significantly in pI, net charge, and EP (p,0.0001). Cluster analysis based on these parameters resulted in well defined clusters. Non-allergens were characterized by neutral to basic pI (mean6SE, 7.660.16) and positive charge. In contrast allergens were acidic (5.760.15) and negatively charged. Surface electrostatic potentials calculated from predicted structures were mostly negative for allergens and mostly positive for non-allergens. The classification accuracy for non-allergens was superior to that for allergens. Thus neutral to basic pI, positive charge, and positive electrostatic potentials characterize non-allergens, and seem rare in allergens (p,0.0001). It may be possible to predict reduced likelihood of allergenicity in such proteins, but this needs to be prospectively validated. - Funding: This work was supported by OLP5502 grant from the Institute of Genomics & Integrative Biology, Council of Scientific & Industrial Research of India http://www.csir.res.in. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Allergy and other forms of hypersensitivity affect up to 1520% of the population in industrial nations [1,2]. Several forms of this disorder are described and a major one is designated IgE-mediated allergy [3]. In general, the differentiating feature between an allergens and non-allergens is the formers ability to induce a specific IgE response via a series of complex interactions with the immune system including uptake, processing and recognition. Since the specific IgE immunoglobulins are directed at precise epitopes within the protein, they have received much attention. However, in silico evaluation of allergenicity of proteins, in terms of peptide motifs, has been relatively unsuccessful, and in spite of many efforts to define structural motifs that distinguish allergens from non allergenic proteins, there has been little progress in formulating structure activity relationships of allergenicity [4]. A possibility is that the Bcell and even T-cell epitopes that have been the focus of such efforts are only second-hand actors, depending upon uptake and processing of the protein prior to recognition [5]. The protein is first seen by antigen presenting cells (APCs), which must internalize whole molecules before cutting them into pieces. Physico-chemical properties of the protein such as lipid binding, ionic charge and post-translational modifications may be important in this process [6]. Lack of these features would be anticipated to reduce allergenicity even in the presence of structural epitopes that may otherwise predict allergenicity. It could then be modeled that potent allergens would possess both such properties as well as relevant epitopes; while non-allergens would lack either. While past efforts have focused on epitope mapping, bioinformatic investigations for discovery of physical properties of the whole protein that may underlie allergenicity are lacking in published literature. Physical properties such as charge and pH have been shown to influence binding and uptake of ligands by antigen presenting cells (APC) [7]. Also, ions have important effects on respiratory health as well as particulate deposition and uptake patterns, and it is empirically known that electrical storms can precipitate asthma exacerbations [8]. From previous knowledge it has been found that electrostatic charge on inhaled particles can enhance and alter the level of deposition within the lung [9,10]. Negatively charged particles may also experience stronger binding to the lung surface once deposited facilitating an increased allergic reaction [11]. Therefore we hypothesized that pH and/or ionic charge on the surface of proteins may be determinants or predictors of allergenicity, and tested this in an in-silico study. Allergens are acidic compared to non-allergens Table 1 contains the summary data for 80 Aeroallergens, 50 Food-allergens and 80 Non-allergens whose complete data was Table 1. Calculated pI, net charge calculated from peptide sequence, net charge per amino acid, and a semiquantitative electrostatic potential score (EP) calculated from protein structure, are shown as mean6standard error mean (SEM) or median6interquartile range (IQR). Mean (SEM) Median (IQR) Mean (SEM) Median (IQR) Mean (SEM) Median (IQR) Mean (SEM) 23.76 (2.72) 23 (14.25) 20.01 (0.005) 20.01 (0.05) 20.04* (0.006) 20.025 (0.052) 20.02* (0.005) 20.021 (0.05) 0.005 (0.004) Asterix (*) denotes significant differences when compared to non-allergens. (1) denotes significant difference from random proteins. There were no significant differences between aeroallergens or dietary allergens. doi:10.1371/journal.pone.0006273.t001 available. (Full details are provided in supplement Data S1). It was found that aeroallergens were mostly acidic proteins (82% acidic; mean pI, 5.7), significantly different from non-allergens (20% acidic; mean pI, 7.6), but similar to food allergens (88% acidic; mean pI, 5.8). It followed that the calculated net charge for the entire protein in a neutral aqueous solution, was more negative for allergens compared to non-allergens. Allergens have more negative electrostatic potential compared to non-allergens The three dimensional electrostatic potential surface visualization of these proteins confirmed that there was an overall statistically significant tendency for allergens to have a negative electrostatic potential (83% of aeroallergens and 72% of food allergens versus 40% for non allergens, p,0.0001). Distinct clusters can be established for allergens and non-allergens Plots of the electrostatic potential (EP) scores and charge versus pI show distinct clusters for allergens and non-allergens with a classification accuracy of (...truncated)