Conformational stability of digestion-resistant peptides of peanut conglutins reveals the molecular basis of their allergenicity

Conglutins represent the major peanut allergens and are renowned for their resistance to gastro-intestinal digestion. Our aim was to characterize the digestion-resistant peptides (DRPs) of conglutins by biochemical and biophysical methods followed by a molecular dynamics simulation in order to better understand the molecular basis of food protein allergenicity. We have mapped proteolysis sites at the N- and C-termini and at a limited internal segment, while other potential proteolysis sites remained unaffected. Molecular dynamics simulation showed that proteolysis only occurred in the vibrant regions of the proteins. DRPs appeared to be conformationally stable as intact conglutins. Also, the overall secondary structure and IgE-binding potency of DRPs was comparable to that of intact conglutins. The stability of conglutins toward gastro-intestinal digestion, combined with the conformational stability of the resulting DRPs provide conditions for optimal exposure to the intestinal immune system, providing an explanation for the extraordinary allergenicity of peanut conglutins.

[1]  C. Radauer,et al.  Cor a 14, the allergenic 2S albumin from hazelnut, is highly thermostable and resistant to gastrointestinal digestion , 2015, Molecular nutrition & food research.

[2]  J. Baumert,et al.  Anaphylaxis from passive transfer of peanut allergen in a blood product. , 2011, The New England journal of medicine.

[3]  J. V. van Bilsen,et al.  The protein structure determines the sensitizing capacity of Brazil nut 2S albumin (Ber e1) in a rat food allergy model , 2013, Clinical and Translational Allergy.

[4]  P. Argos,et al.  Knowledge‐based protein secondary structure assignment , 1995, Proteins.

[5]  E. Knol,et al.  The diagnostic accuracy of specific IgE to Ara h 6 in adults is as good as Ara h 2 , 2014, Allergy.

[6]  R. Helm,et al.  Identification and mutational analysis of the immunodominant IgE binding epitopes of the major peanut allergen Ara h 2. , 1997, Archives of biochemistry and biophysics.

[7]  M. Blanusa,et al.  Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases. , 2010, Journal of the science of food and agriculture.

[8]  Julian E. Fuchs,et al.  Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen , 2016, The Journal of allergy and clinical immunology.

[9]  Roy L. Fuchs,et al.  Stability of food allergens to digestion in vitro , 1996, Nature Biotechnology.

[10]  S. Dreskin,et al.  Effector activity of peanut allergens: a critical role for Ara h 2, Ara h 6, and their variants , 2009, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[11]  T. Velickovic,et al.  Reduction and alkylation of peanut allergen isoforms Ara h 2 and Ara h 6; characterization of intermediate- and end products. , 2013, Biochimica et biophysica acta.

[12]  M. Kulis,et al.  The 2S albumin allergens of Arachis hypogaea, Ara h 2 and Ara h 6, are the major elicitors of anaphylaxis and can effectively desensitize peanut‐allergic mice , 2012, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[13]  K Schulten,et al.  VMD: visual molecular dynamics. , 1996, Journal of molecular graphics.

[14]  A. Nowak‐Wegrzyn,et al.  Effect of chemical modifications on allergenic potency of peanut proteins. , 2015, Allergy and asthma proceedings.

[15]  Laxmikant V. Kalé,et al.  Scalable molecular dynamics with NAMD , 2005, J. Comput. Chem..

[16]  F Beaudoin,et al.  Plant protein families and their relationships to food allergy. , 2001, Biochemical Society transactions.

[17]  M. Blanca,et al.  Basophil response to peanut allergens in Mediterranean peanut-allergic patients. , 2014, Allergy.

[18]  E. Knol,et al.  Purification and immunoglobulin E‐binding properties of peanut allergen Ara h 6: evidence for cross‐reactivity with Ara h 2 , 2005, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[19]  Thomas Jacks,et al.  The major peanut allergen, Ara h 2, functions as a trypsin inhibitor, and roasting enhances this function. , 2003, The Journal of allergy and clinical immunology.

[20]  Klaus R Liedl,et al.  Stabilizing of a globular protein by a highly complex water network: a molecular dynamics simulation study on factor Xa. , 2010, The journal of physical chemistry. B.

[21]  L. Pedersen,et al.  The Molecular Basis of Peanut Allergy , 2014, Current Allergy and Asthma Reports.

[22]  J. Wal,et al.  Capacity of purified peanut allergens to induce degranulation in a functional in vitro assay: Ara h 2 and Ara h 6 are the most efficient elicitors , 2009, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[23]  John M. Walker,et al.  The Proteomics Protocols Handbook , 2005, Humana Press.

[24]  P. Shewry,et al.  Stability of sunflower 2S albumins and LTP to physiologically relevant in vitro gastrointestinal digestion. , 2013, Food chemistry.

[25]  S. Koppelman,et al.  Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: a comparative in vitro study and partial characterization of digestion-resistant peptides. , 2010, Molecular nutrition & food research.

[26]  M. Gaspari,et al.  Reversible denaturation of Brazil nut 2S albumin (Ber e1) and implication of structural destabilization on digestion by pepsin. , 2005, Journal of agricultural and food chemistry.

[27]  A. Knulst,et al.  Ara h 2 is the best predictor for peanut allergy in adults. , 2013, The journal of allergy and clinical immunology. In practice.

[28]  S. Vieths,et al.  Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions. , 2006, The Biochemical journal.

[29]  C. Grimm,et al.  In vitro digestion of soluble cashew proteins and characterization of surviving IgE-reactive peptides. , 2014, Molecular nutrition & food research.

[30]  E. Mills,et al.  Thermostability and in vitro digestibility of a purified major allergen 2S albumin (Ses i 1) from white sesame seeds (Sesamum indicum L.). , 2005, Biochimica et biophysica acta.

[31]  T. Yeates,et al.  Verification of protein structures: Patterns of nonbonded atomic interactions , 1993, Protein science : a publication of the Protein Society.

[32]  W. Thomas,et al.  Isoforms of the Major Peanut Allergen Ara h 2: IgE Binding in Children with Peanut Allergy , 2004, International Archives of Allergy and Immunology.

[33]  E. Knol,et al.  IgE binding to peanut components by four different techniques: Ara h 2 is the most relevant in peanut allergic children and adults , 2013, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[34]  V. Verhasselt,et al.  Peanut allergens are rapidly transferred in human breast milk and can prevent sensitization in mice. , 2014, Allergy.

[35]  P. Vadas,et al.  Detection of peanut allergens in breast milk of lactating women. , 2001, JAMA.

[36]  Amelie Stein,et al.  Improvements to Robotics-Inspired Conformational Sampling in Rosetta , 2013, PloS one.

[37]  J. Wal,et al.  Trypsin resistance of the major peanut allergen Ara h 6 and allergenicity of the digestion products are abolished after selective disruption of disulfide bonds. , 2012, Molecular nutrition & food research.

[38]  Adnan Custovic,et al.  Quantification of specific IgE to whole peanut extract and peanut components in prediction of peanut allergy. , 2011, The Journal of allergy and clinical immunology.

[39]  A. Burks,et al.  Protein Structure Plays a Critical Role in Peanut Allergen Stability and May Determine Immunodominant IgE-Binding Epitopes , 2002, The Journal of Immunology.

[40]  J. Nordlee,et al.  Distribution of Intact Peanut Protein and Digestion-Resistant Ara h 2 Peptide in Human Serum and Saliva , 2009 .

[41]  W. Becker,et al.  Isolation and characterization of natural Ara h 6: evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat. , 2004, Molecular nutrition & food research.

[42]  Alexander D. MacKerell,et al.  Extending the treatment of backbone energetics in protein force fields: Limitations of gas‐phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations , 2004, J. Comput. Chem..

[43]  Ramu Anandakrishnan,et al.  H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations , 2012, Nucleic Acids Res..

[44]  R. Van Ree,et al.  Ara h 6 complements Ara h 2 as an important marker for IgE reactivity to peanut. , 2014, Journal of agricultural and food chemistry.

[45]  D S Moss,et al.  Main-chain bond lengths and bond angles in protein structures. , 1993, Journal of molecular biology.

[46]  E. Knol,et al.  Does skin prick test reactivity to purified allergens correlate with clinical severity of peanut allergy? , 2007, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[47]  E. Coutsias,et al.  Sub-angstrom accuracy in protein loop reconstruction by robotics-inspired conformational sampling , 2009, Nature Methods.

[48]  A. Knulst,et al.  Relevance of Ara h1, Ara h2 and Ara h3 in peanut‐allergic patients, as determined by immunoglobulin E Western blotting, basophil–histamine release and intracutaneous testing: Ara h2 is the most important peanut allergen , 2004, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[49]  Yonghua Zhuang,et al.  Redefining the major peanut allergens , 2013, Immunologic research.

[50]  J. Chatel,et al.  Isolation and Characterization of Two Complete Ara h 2 Isoforms cDNA , 2003, International Archives of Allergy and Immunology.

[51]  C. Fenselau,et al.  Primary sequence and site‐selective hydroxylation of prolines in isoforms of a major peanut allergen protein Ara h 2 , 2009, Protein science : a publication of the Protein Society.

[52]  G. Ya. Wiederschain,et al.  The proteomics protocols handbook , 2006, Biochemistry (Moscow).

[53]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..