Analysis of avocado allergen (Prs a 1) IgE-binding peptides generated by simulated gastric fluid digestion.
BACKGROUND
Resistance to pepsin digestion has been claimed to be a characteristic of food allergens that can induce severe adverse reactions. Moreover, pepsin treatment is included in protocols to evaluate the potential allergenicity of transgenic foods. Allergenic plant class I chitinases, such as avocado Prs a 1, are the panallergens involved in the latex-fruit syndrome. Previous reports indicated their susceptibility to simulated gastric fluid (SGF) digestion.


OBJECTIVE
We sought to evaluate the IgE-binding capacity and the in vivo reactivity of the SGF products of the avocado allergen Prs a 1.


METHODS
Patients with a clinical history of latex-fruit allergy syndrome, a positive skin prick test (SPT) response to Prs a 1, and specific IgE to avocado were selected. Untreated and SGF-digested Prs a 1 samples were analyzed by means of IgE and IgG immunoblotting, IgE immunoblotting and ELISA-inhibition assays, and SPTs. Peptides from SGF-digested samples were fractionated by means of HPLC, characterized by N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization analysis, and tested for in vivo reactivity with SPTs.


RESULTS
Neither protein staining nor IgE immunoblotting with a pool of sera from allergic patients resulted in the detection of any band after SDS-PAGE separation of an SGF-digested sample of Prs a 1. However, this sample showed a similar inhibitory potency to that of untreated Prs a 1 in both immunoblot- and ELISA-inhibition assays (up to 70% inhibition of the IgE binding to crude avocado extract) and induced positive SPT responses in 5 of 8 allergic patients. Peptides from SGF-digested Prs a 1 were separated by means of HPLC, and 4 of them reached more than 50% inhibition values when using avocado extract as the solid phase in ELISA-inhibition assays. Reactive peptides were located both in the N-terminal hevein-like domain and in the catalytic domain of Prs a 1. Those corresponding to the hevein-like domain (approximately 5100 d) produced positive SPT responses in 5 of 8 allergic patients, whereas 2 peptides located in the catalytic domain (approximately 1400 and 2500 d) were reactive in 2 or 3 of the 8 patients.


CONCLUSIONS
Prs a 1 was extensively degradated when subjected to SGF digestion. However, the resulting peptides, particularly those corresponding to the hevein-like domain, were clearly reactive both in vitro and in vivo.
Díaz-Perales
Araceli
A
Departamento de Biotecnología, E.T.S. Ingenieros Agrónomos, UPM, Madrid, Spain.
Blanco
Carlos
C
Sánchez-Monge
Rosa
R
Varela
Javier
J
Carrillo
Teresa
T
Salcedo
Gabriel
G
D000485:Allergens / Q000276:immunology* / Q000378:metabolism*; D052179:Antigens, Plant; D002688:Chitinases / Q000276:immunology* / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004063:Digestion / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005750:Gastric Juice / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology*; D010446:Peptide Fragments / Q000096:biosynthesis / Q000276:immunology; D027421:Persea / Q000276:immunology; D010940:Plant Proteins; D013194:Staining and Labeling
D000485:Allergens; D052179:Antigens, Plant; D010446:Peptide Fragments; D010940:Plant Proteins; D007073:Immunoglobulin E; D002688:Chitinases; C115191:Prs a 1 allergen