Molecular properties of food allergens
2005; Elsevier BV; Volume: 115; Issue: 1 Linguagem: Inglês
10.1016/j.jaci.2004.10.022
ISSN1097-6825
AutoresHeimo Breiteneder, E. N. Clare Mills,
Tópico(s)Occupational exposure and asthma
ResumoPlant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators. Plant food allergens belong to a rather limited number of protein families and are also characterized by a number of biochemical and physicochemical properties, many of which are also shared by food allergens of animal origin. These include thermal stability and resistance to proteolysis, which are enhanced by an ability to bind ligands, such as metal ions, lipids, or steroids. Other types of lipid interaction, including membranes or other lipid structures, represent another feature that might promote the allergenic properties of certain food proteins. A structural feature clearly related to stability is intramolecular disulfide bonds alongside posttranslational modifications, such as N-glycosylation. Some plant food allergens, such as the cereal seed storage prolamins, are rheomorphic proteins with polypeptide chains that adopt an ensemble of secondary structures resembling unfolded or partially folded proteins. Other plant food allergens are characterized by the presence of repetitive structures, the ability to form oligomers, and the tendency to aggregate. A summary of our current knowledge regarding the molecular properties of food allergens is presented. Although we cannot as yet predict the allergenicity of a given food protein, understanding of the molecular properties that might predispose them to becoming allergens is an important first step and will undoubtedly contribute to the integrative allergenic risk assessment process being adopted by regulators. Recent epidemiologic data suggest that almost 4% of the American population have food allergy.1Sicherer S.H. Munoz-Furlong A. Sampson H.A. Prevalence of seafood allergy in the United States determined by a random telephone survey.J Allergy Clin Immunol. 2004; 114: 159-165Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar This prevalence has notably risen from the previously reported 2%.2Sampson H.A. Food allergy. Part 1: immunopathogenesis and clinical disorders.J Allergy Clin Immunol. 1999; 103: 717-728Abstract Full Text Full Text PDF PubMed Google Scholar Food allergies are most prevalent during infancy, affecting up to 6% of young children.3Sampson H.A. 9. Food allergy.J Allergy Clin Immunol. 2003; 111: S540-S547Abstract Full Text Full Text PDF PubMed Google Scholar In a recent update on food allergy, Sampson4Sampson H.A. Update on food allergy.J Allergy Clin Immunol. 2004; 113: 805-819Abstract Full Text Full Text PDF PubMed Scopus (864) Google Scholar stated that although the diversity of the human diet is enormous, there are few foods that account for the majority of food allergies. In this context it is worth noting that plant food allergens also fall into rather few protein families.5Jenkins J.A. Griffiths-Jones Shewry P.R. Breiteneder H. Mills E.N.C. Structural relatedness of plant food allergens with specific reference to cross-reactive allergens—an in silico analysis.J Allergy Clin Immunol. 2005; 115: 163-170Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar The scientific community and regulatory authorities are both highly interested in assessing the allergenic potential of a given protein, an essential facet of the safety assessment of novel foods, including those derived from biotechnology. To this end, there has been considerable interest in using bioinformatic approaches to identifying potential allergens.6Brusic V. Petrovsky N. Gendel S.M. Millot M. Gigonzac O. Stelman S.J. Computational tools for the study of allergens.Allergy. 2003; 58: 1083-1092Crossref PubMed Scopus (36) Google Scholar A joint Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) expert consultation on foods derived from biotechnology held in 2001 on the evaluation of allergenicity of genetically modified foods (www.fao.org/es/ESN/food/pdf/allergygm.pdf) proposed that a query protein be regarded as potentially cross-reactive if it has an identity of at least 6 contiguous amino acids or more than 35% sequence similarity over a window of 80 amino acids when compared with known allergens. Subsequently, Hileman et al7Hileman R.E. Silvanovich A. Goodman R.E. Rice E.A. Holleschak G. Astwood J.D. et al.Bioinformatic methods for allergenicity assessment using a comprehensive allergen database.Int Arch Allergy Immunol. 2002; 128: 280-291Crossref PubMed Google Scholar analyzed a total of 59 proteins using the FASTA algorithm8Pearson W.R. Flexible sequence similarity searching with the FASTA3 program package.Methods Mol Biol. 2000; 132: 185-219PubMed Google Scholar and suggested that an 8-amino-acid window was more appropriate because it reduced the frequency of spurious matches. A new strategy to predict allergenic proteins on the basis of 52 sequence motifs identified from a comprehensive allergen database combined with a pairwise sequence alignment was proposed by Stadler and Stadler.9Stadler M.B. Stadler B.M. Allergenicity prediction by protein sequence.FASEB J. 2003; 17: 1141-1143PubMed Google Scholar This method identified potential allergens with greater sensitivity and improved precision compared with other methods. Recently, Soeria-Atmadja et al10Soeria-Atmadja D. Zorzet A. Gustafsson M.G. Hammerling U. Statistical evaluation of local alignment features predicting allergenicity using supervised classification algorithms.Int Arch Allergy Immunol. 2004; 133: 101-112Crossref PubMed Scopus (35) Google Scholar introduced new bioinformatic procedures to be used as part of an integrated assessment scheme for potential protein allergenicity. These in silico methods generally provide a quantitative measure of the global similarity of a given protein sequence with that of known allergens. Two other types of approach have been described that are aimed at identifying potential IgE epitopes, especially relevant to identifying potential cross-reactive allergens. One combines homology searching to identify short contiguous sequences between allergens and query proteins, followed by an analysis of their potential antigenicity.11Kleter G.A. Peijnenberg A.C.M. Screening of transgenic proteins expressed in genetically modified food crops for the presence of six amino acid sequences identical to potential, IgE-binding linear epitopes of allergens.BMC Struct Biol. 2002; 2: 8Crossref PubMed Google Scholar Another combines sequence and structural information to identify whether query sequences match known IgE epitopes. This has been used to identify potential cross-reactive IgE epitopes for the thaumatin-like pathogenesis-related protein (PR) 5 protein allergen of cedar pollen (Jun a 3).12Ivanciuc O. Mathura V. Midoro-Horiuti T. Braun W. Goldblum R.M. Schein C.H. Detecting potential IgE-reactive sites on food proteins using a sequence and structure database, SDAP-Food.J Agric Food Chem. 2003; 51: 4830-4837Crossref PubMed Scopus (28) Google Scholar However, as is emerging for inhalant allergens,13Pomes A. Intrinsic properties of allergens and environmental exposure as determinants of allergenicity.Allergy. 2002; 57: 673-679Crossref PubMed Scopus (24) Google Scholar the structure and function of a protein and the habits of food consumption coupled with any genetic predisposition of a given individual to become atopic, rather than sequence per se, are probably more important determinants of food protein allergenicity. This is reflected in the more recent deliberations of the third session of the FAO/WHO Codex Alimentarius Commission Ad Hoc Intergovernmental Task Force on Foods Derived from Biotechnology (FAO/WHO Report of the third session of the Codex Ad Hoc Intergovernmental Task Force on Foods derived from Biotechnology, ALINORM 03/34, March 4-8, 2002, ftp://ftp.fao.org/codex/alinorm03/Al03_34e.pdf), during which it was decided not to elaborate the decision tree approach. Because no single criterion is sufficiently predictive of allergenicity, they recommended that the risk assessment process should adopt an integrated stepwise case-by-case approach that takes account of information of several types. Thus, Aalberse14Aalberse R.C. Structural biology of allergens.J Allergy Clin Immunol. 2000; 106: 228-238Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar simply considered structural aspects of allergens using a proximity plot as a 2-dimensional representation of protein structure. He came to the conclusion that allergens have no characteristic structural features that set them aside from all other proteins. The author, however, did not address molecular properties that might be shared by food allergens. When studying the question of what establishes a protein as an allergen, Bredehorst and David15Bredehorst R. David K. What establishes a protein as anallergen?.J Chromatogr B Biomed Sci Appl. 2001; 756: 33-40Crossref PubMed Scopus (67) Google Scholar concluded that functional aspects of some allergens might play a role in sensitization and the allergic response beside the structural aspects of allergens. In a recent review, Bannon16Bannon G.A. What makes a food protein an allergen?.Curr Allergy Asthma Rep. 2004; 4: 43-46Crossref PubMed Google Scholar discussed some biochemical characteristics associated with food allergens, such as the presence of multiple, linear IgE-binding epitopes and the resistance of the protein to digestion and processing. Recent years have seen an extensive effort in the allergy community to identify and characterize many food allergens. Do they show common properties and functions, and how might they influence allergenic potential? It is the aim of this review to summarize, with examples, a number of potentially important functional and physicochemical characteristics shared by many food allergens that might promote allergenicity. A number of food allergens are able to bind ligands, ranging from metal ions to lipids. Certain ligands, such as metal ions, become integrated into the 3-dimensional structure of a protein, often buried deep within the molecule. The loss of a metal ion frequently disrupts protein folding, with an increase in polypeptide mobility and a transition in some instances into a partially folded form. Some proteins form a cavity into which a ligand fits: this might be a metal ion, steroids, or a variety of lipid molecules. Other proteins possess a tunnel into which ligands fit, whereas yet others bind ligands through superficial surface interactions. Ligand binding can have the overall effect of reducing mobility on the polypeptide backbone, increasing both thermal stability and resistance to proteolysis, with many proteases requiring flexibility in substrate proteins. Proteins such as the lipocalins and nonspecific lipid-transfer proteins (nsLTPs), which possess a lipid-binding pocket, show increased stability when the pocket is occupied. Thus the thermostability of β-lactoglobulin (βLg) increases on lipid binding,17Creamer L.K. Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine beta-lactoglobulin.Biochemistry. 1995; 34: 7170-7176Crossref PubMed Google Scholar as does that of the nsLTP of wheat.18Douliez J.P. Jegou S. Pato C. Molle D. Tran V. Marion D. Binding of two mono-acylated lipid monomers by the barley lipid-transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.Eur J Biochem. 2001; 268: 384-388Crossref PubMed Google Scholar These properties are described in more detail below for several notable allergens, including the major fish allergen parvalbumin, the cow's milk allergens casein and βLg, and the nsLTPs and Bet v 1 homologues found in a variety of plant foods. A widely found calcium-binding domain is known as the EF-hand,19Lewit-Bentley A. Rety S. EF-hand calcium-binding proteins.Curr Opin Struct Biol. 2000; 10: 637-643Crossref PubMed Scopus (253) Google Scholar a motif that consists of a 12-residue loop flanked on both sides by a 12-residue α-helical domain. Parvalbumins comprise 3 such EF-hand motifs,20Ikura M. Calcium binding and conformational response in EF-hand proteins.Trends Biochem Sci. 1996; 21: 14-17Abstract Full Text PDF PubMed Scopus (463) Google Scholar 2 capable of binding calcium and the remaining silent domain forming a cap covering the hydrophobic surface of the 2 functional domains (Fig 1, A).21Declercq J.P. Tinant B. Parello J. Rambaud J. Ionic interactions with parvalbumins. Crystal structure determination of pike 4.10 parvalbumin in four different ionic environments.J Mol Biol. 1991; 220: 1017-1039Crossref PubMed Scopus (95) Google Scholar Abundant (up to 5 mg/g fresh weight) proteins in the white muscle of many fish species, parvalbumins, are thought to be important for the relaxation of muscle fibers through the binding of free intracellular calcium.22Pauls T.L. Cox J.A. Berchtold M.W. The Ca2+(-)binding proteins parvalbumin and oncomodulin and their genes: new structural and functional findings.Biochim Biophys Acta. 1996; 1306: 39-54Crossref PubMed Google Scholar Loss of the protein-bound calcium triggers a large change in conformation and an associated loss of conformation-dependent IgE epitopes.23Bugajska-Schretter A. Elfman L. Fuchs T. Kapiotis S. Rumpold H. Valenta R. et al.Parvalbumin, a cross-reactive fish allergen, contains IgE-binding epitopes sensitive to periodate treatment and Ca2+ depletion.J Allergy Clin Immunol. 1998; 101: 67-74Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 24Hilger C. Thill L. Grigioni F. Lehners C. Falagiani P. Ferrara A. et al.IgE antibodies of fish allergic patients cross-react with frog parvalbumin.Allergy. 2004; 59: 653-660Crossref PubMed Scopus (44) Google Scholar, 25Bugajska-Schretter A. Grote M. Vangelista L. Valent P. Sperr W.R. Rumpold H. et al.Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin.Gut. 2000; 46: 661-669Crossref PubMed Scopus (92) Google Scholar Parvalbumins show a remarkable resistance to heat, denaturing chemicals, and proteolytic enzymes.26Elsayed S. Aas K. Characterization of a major allergen (cod). Observations on effect of denaturation on the allergenic activity.J Allergy. 1971; 47: 283-291PubMed Scopus (57) Google Scholar However, although circular dichroism analysis of carp parvalbumin revealed a remarkable stability and refolding capacity of the calcium containing form after heating,25Bugajska-Schretter A. Grote M. Vangelista L. Valent P. Sperr W.R. Rumpold H. et al.Purification, biochemical, and immunological characterisation of a major food allergen: different immunoglobulin E recognition of the apo- and calcium-bound forms of carp parvalbumin.Gut. 2000; 46: 661-669Crossref PubMed Scopus (92) Google Scholar the IgE-binding activity of canned fish has been estimated to be 100 to 200 times lower than in boiled fish.27Bernhisel-Broadbent J. Strause D. Sampson H.A. Fish hypersensitivity. II: Clinical relevance of altered fish allergenicity caused by various preparation methods.J Allergy Clin Immunol. 1992; 90: 622-629Abstract Full Text PDF PubMed Scopus (98) Google Scholar Nevertheless, sufficient IgE-reactive epitopes remain after cooking to trigger allergic reactions in individuals, as demonstrated by the double-blind, placebo-controlled food challenge performed by Bernhisel-Broadbent et al.28Bernhisel-Broadbent J. Scanlon S.M. Sampson H.A. Fish hypersensitivity. I. In vitro and oral challenge results in fish-allergic patients.J Allergy Clin Immunol. 1992; 89: 730-737Abstract Full Text PDF PubMed Scopus (168) Google Scholar Major cow's milk allergens, caseins, also bind calcium but in a very different way than parvalbumins. Clusters of phosphoserine residues, phosphothreonine residues, or both in αs1-, αs2-, and β-caseins chelate metal ions, including calcium. These form microstructures called nanoclusters29Holt C. Timmins P.A. Errington N. Leaver J. A core-shell model of calcium phosphate nanoclusters stabilized by beta-casein phosphopeptides, derived from sedimentation equilibrium and small-angle X-ray and neutron-scattering measurements.Eur J Biochem. 1998; 252: 73-78Crossref PubMed Google Scholar in which amorphous calcium phosphate is sequestered within a shell formed from casein polypeptides. These then assemble into much larger structures comprising around 1000 nanoclusters that correspond to the casein micelles found in milk.30Tuinier R. de Kruif C.G. Stability of casein micelles in milk.J Chem Phys. 2002; 117: 1290-1295Crossref Scopus (134) Google Scholar Such structures allow calcium levels in milk to exceed the solubility limit of calcium phosphate. The significance of nanoclusters for determining the allergenic potential of caseins remains to be shown, although dephosphorylation reduces IgE binding to caseins.31Bernard H. Meisel H. Creminon C. Wal J.M. Post-translational phosphorylation affects the IgE binding capacity of caseins.FEBS Lett. 2000; 467: 239-244Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar βLg belongs to the lipocalin superfamiliy of extracellular lipid-binding proteins and is one of the best characterized lipid-binding proteins known (Fig 1, B). It binds a diverse range of molecules, including retinol and its analogues, β-carotene, saturated and unsaturated fatty acids ranging in chain length from C12-C18, and aliphatic hydrocarbons.32O'Neill T.E. Kinsella J.E. Binding of alkanone flavours to beta-lactoglobulin: Effects of conformational and chemical modification.J Agric Food Chem. 1987; 35: 770-774Crossref Google Scholar The affinities for these compounds range from 4.8 × 105 M−1 for heptane to 4.8 × 107 M−1 for retinol.32O'Neill T.E. Kinsella J.E. Binding of alkanone flavours to beta-lactoglobulin: Effects of conformational and chemical modification.J Agric Food Chem. 1987; 35: 770-774Crossref Google Scholar These ligands bind in the central calyx of the characteristic lipocalin β-barrel,33Kontopidis G. Holt C. Sawyer L. The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?.J Mol Biol. 2002; 318: 1043-1055Crossref PubMed Scopus (147) Google Scholar and it is has been proposed that ligand binding is determined by a pH-dependent opening-closing of the EF loop, akin to a lid opening on the ligand-binding site.34Ragona L. Fogolari F. Catalano M. Ugolini R. Zetta L. Molinari H. EF loop conformational change triggers ligand binding in beta-lactoglobulins.J Biol Chem. 2003; 278: 38840-38846Crossref PubMed Scopus (46) Google Scholar Unlike βLg, nsLTPs are generally able to bind lipids in a tunnel lined with hydrophobic residues running through the protein (Fig 1, C) that varies in size for the different nsLTPs structurally characterized thus far. Wheat nsLTP binds a range of fatty acids and phospholipids varying in chain length with high affinity, with dissociation constants in the order of 0.2 to 0.3 μM, depending on the lipid.18Douliez J.P. Jegou S. Pato C. Molle D. Tran V. Marion D. Binding of two mono-acylated lipid monomers by the barley lipid-transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.Eur J Biochem. 2001; 268: 384-388Crossref PubMed Google Scholar, 35Douliez J.P. Michon T. Marion D. Steady-state tyrosine fluorescence to study the lipid-binding properties of a wheat non-specific lipid-transfer protein (nsLTP1).Biochim Biophys Acta. 2000; 1467: 65-72Crossref Scopus (47) Google Scholar The cavity is highly plastic and can bind a wide range of lipophilic molecules, including sphingolipids, prostaglandins, amphoteracin B, and other hydrophobic drugs.36Tassin-Moindrot S. Caille A. Douliez J.P. Marion D. Vovelle F. The wide binding properties of a wheat nonspecific lipid transfer protein. Solution structure of a complex with prostaglandin B2.Eur J Biochem. 2000; 267: 1117-1124Crossref PubMed Scopus (47) Google Scholar, 37Pato C. Le Borgne M. Le Baut G. Le Pape P. Marion D. Douliez J.P. Potential application of plant lipid transfer proteins for drug delivery.Biochem Pharmacol. 2001; 62: 555-560Crossref PubMed Scopus (33) Google Scholar It can actually accommodate 2 lipid molecules, which, on the basis of molecular modeling, appear to lay side by side in the tunnel.18Douliez J.P. Jegou S. Pato C. Molle D. Tran V. Marion D. Binding of two mono-acylated lipid monomers by the barley lipid-transfer protein, LTP1, as viewed by fluorescence, isothermal titration calorimetry and molecular modelling.Eur J Biochem. 2001; 268: 384-388Crossref PubMed Google Scholar These properties seem to be a general feature of nsLTPs and might be related to their putative role in plants in transporting lipids involved in the synthesis of waxy cutin and suberin layers in outer plant tissues in seeds and pollen. The 3-dimensional scaffold found in nsLTPs is characteristic of the prolamin superfamily, and it is becoming evident that in some forms of the protein (eg, AceAMP1 from onion roots), the tunnel has been lost, along with the ability to bind lipids, the cavity being filled up with tryptophan and phenylalanine side chains.38Tassin S. Broekaert W.F. Marion D. Acland D.P. Ptak M. Vovelle F. et al.Solution structure of Ace-AMP1, a potent antimicrobial protein extracted from onion seeds. Structural analogies with plant nonspecific lipid transfer proteins.Biochemistry. 1998; 37: 3623-3637Crossref PubMed Scopus (71) Google Scholar The first indication that Bet v 1–homologous proteins might be involved in the transport of steroid ligands came from the discovery of a high structural similarity between Bet v 1 and the steroid-binding domain of the human MLN64 protein, which possibly plays a role in promoting steroidogenesis in the placenta and brain.39Tsujishita Y. Hurley J.H. Structure and lipid transport mechanism of a StAR-related domain.Nat Struct Biol. 2000; 7: 408-414Crossref PubMed Scopus (328) Google Scholar This structural homology led Neudecker et al40Neudecker P. Schweimer K. Nerkamp J. Scheurer S. Vieths S. Sticht H. et al.Allergic cross-reactivity made visible: solution structure of the major cherry allergen Pru av 1.J Biol Chem. 2001; 276: 22756-22763Crossref PubMed Scopus (112) Google Scholar to use NMR spectroscopy to indicate that the Bet v 1 homologue from sweet cherry, Pru av 1, interacted with phytosteroids. Molecular modeling showed that the hydrophobic cavity of Pru av 1 is large enough to hold 2 such molecules.40Neudecker P. Schweimer K. Nerkamp J. Scheurer S. Vieths S. Sticht H. et al.Allergic cross-reactivity made visible: solution structure of the major cherry allergen Pru av 1.J Biol Chem. 2001; 276: 22756-22763Crossref PubMed Scopus (112) Google Scholar Subsequently, Markovic-Housley et al41Markovic-Housley Z. Degano M. Lamba D. von Roepenack-Lahaye E. Clemens S. Susani M. et al.Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier.J Mol Biol. 2003; 325: 123-133Crossref PubMed Scopus (193) Google Scholar provided evidence that suggested a plant steroid carrier function for Bet v 1 and other PR-10 proteins by showing the interaction of the isoform Bet v 1l with 2 molecules of the plant steroid brassinolide (Fig 1, D).41Markovic-Housley Z. Degano M. Lamba D. von Roepenack-Lahaye E. Clemens S. Susani M. et al.Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier.J Mol Biol. 2003; 325: 123-133Crossref PubMed Scopus (193) Google Scholar It is generally accepted that oral allergy syndrome symptoms are associated with labile allergens, whereas anaphylactic reactions are attributed to stable food allergens. Generally, pollen-related allergens tend to be labile during heating and digestion.42Vieths S. Scheurer S. Ballmer-Weber B. Current understanding of cross-reactivity of food allergens and pollen.Ann N Y Acad Sci. 2002; 964: 47-68Crossref PubMed Google Scholar Surprisingly, the Bet v 1–homologous allergen Gly m 4 of soybean was found to be able to induce an oral allergy syndrome of extraordinary severity and severe systemic reactions in individuals with pollen allergy.43Kleine-Tebbe J. Vogel L. Crowell D.N. Haustein U.F. Vieths S. Severe oral allergy syndrome and anaphylactic reactions caused by a Bet v 1-related PR-10 protein in soybean, SAM22.J Allergy Clin Immunol. 2002; 110: 797-804Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar It is tempting to speculate that the difference in stability between Gly m 4 and other PR-10 allergens might be contributed to by a high-affinity binding of Gly m 4 of its ligands, a theory that will have to be tested in the future. Many plant food allergens are also able to associate with cell membranes and other types of lipid structures formed in foods. One commonly observed mode of action whereby proteins can protect plants against microbial pathogens is through destabilization of bacterial or fungal membranes resulting in leakage. Proteins acting in this way include thionins, thaumatin-like proteins (TLPs), 2 types of prolamin superfamily members (2S albumins and nsLTPs), and some defensins.44Selitrennikoff C.P. Antifungal proteins.Appl Environ Microbiol. 2001; 67: 2883-2894Crossref PubMed Scopus (341) Google Scholar Many plant species that are used for human consumption produce proteins that share sequence and structure similarities with thaumatin, an intensely sweet-tasting protein from the fruit of the West African ketemfe (Thaumatococcus daniellii) plant. These proteins are called TLPs. TLPs, also known as family 5 of the PR proteins, have been isolated from a wide range of plant species.44Selitrennikoff C.P. Antifungal proteins.Appl Environ Microbiol. 2001; 67: 2883-2894Crossref PubMed Scopus (341) Google Scholar Several allergenic TLPs from fruit and pollen have been described.45Breiteneder H. Thaumatin-like proteins—a new family of pollen and fruit allergens.Allergy. 2004; 59: 479-481Crossref PubMed Scopus (94) Google Scholar Many TLPs have been reported to inhibit the growth of fungi, including the apple allergen Mal d 2.46Krebitz M. Wagner B. Ferreira F. Peterbauer C. Campillo N. Witty M. et al.Plant-based heterologous expression of Mal d 2, a thaumatin-like protein and allergen of apple (Malus domestica), and its characterization as an antifungal protein.J Mol Biol. 2003; 329: 721-730Crossref PubMed Scopus (83) Google Scholar Although the precise mechanism of such action is not clear, it has been proposed to result from permeabilization of fungal membranes.47Vigers A.J. Roberts W.K. Selitrennikoff C.P. A new family of plant antifungal proteins.Mol Plant Microbe Interact. 1991; 4: 315-323Crossref PubMed Google Scholar TLPs might also interact directly with the lipid bilayer of the fungal plasma membrane, which is composed of sphingolipids, phospholipids, and sterols, components that occur in membranes of all living organisms. The membrane-permeabilizing activity of linusitin, an antifungal TLP from flax seeds, was shown to depend on membrane composition and was amplified by an increased content of negatively charged phospholipids and the presence of sterols.48Anzlovar S. Dalla Serra M. Dermastia M. Menestrina G. Membrane permeabilizing activity of pathogenesis-related protein linusitin.Mol Plant Microbe Interact. 1998; 11: 610-617Crossref Google Scholar Although a major function of the 2S albumins is undoubtedly as seed storage proteins, other functions have been ascribed to them, including antifungal activity resulting from membrane permeabilization in radish (Raphanus sativus) and rape (Brassica napus).49Terras F.R. Schoofs H.M. De Bolle M.F. Van Leuven F. Rees S.B. Vanderleyden J. et al.Analysis of two novel classes of plant antifungal proteins from radish (Raphanus sativus L.) seeds.J Biol Chem. 1992; 267: 15301-15309Abstract Full Text PDF PubMed Google Scholar, 50Terras F. Schoofs H. Thevissen K. 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