Bioinformatic Methods for Allergenicity Assessment Using a Comprehensive Allergen Database

Abstract

Background: A principal aim of the safety assessment of genetically modified crops is to prevent the introduction of known or clinically cross-reactive allergens. Current bioinformatic tools and a database of allergens and gliadins were tested for the ability to identify potential allergens by analyzing 6 Bacillus thuringiensis insecticidal proteins, 3 common non-allergenic food proteins and 50 randomly selected corn (Zea mays) proteins. Methods: Protein sequences were compared to allergens using the FASTA algorithm and by searching for matches of 6, 7 or 8 contiguous identical amino acids. Results: No significant sequence similarities or matches of 8 contiguous amino acids were found with the B. thuringiensis or food proteins. Surprisingly, 41 of 50 corn proteins matched at least one allergen with 6 contiguous identical amino acids. Only 7 of 50 corn proteins matched an allergen with 8 contiguous identical amino acids. When assessed for overall structural similarity to allergens, these 7 plus 2 additional corn proteins shared ≧35% identity in an overlap of ≧80 amino acids, but only 6 of the 7 were similar across the length of the protein, or shared >50% identity to an allergen. Conclusions: An evaluation of a protein by the FASTA algorithm is the most predictive of a clinically relevant cross-reactive allergen. An additional search for matches of 8 amino acids may provide an added margin of safety when assessing the potential allergenicity of a protein, but a search with a 6-amino-acid window produces many random, irrelevant matches.

A Pepsin-Resistant 20 kDa Protein Found in Red Kidney Bean (Phaseolus vulgarisL.) Identified as Basic Subunit of Legumin

“…11,12) Further analysis of the sequence and structure of bean legumin should reveal its allergenic properties by the bioinformatics method of allergenicity assessment. 13) The 20-kDa polypeptide appeared to have rigid intramolecular disulfide bonds, which may contribute to tolerance to proteolysis. De Val et al suggested that controlling specific disulfide bonds in protein can lower the allergenicity of milk.…”

Recent advances in the development of transgenic papaya technology

“…Using the minimum length of six amino acids recommended by an FAO/WHO Expert Consultation [126] in 2001, the PRSV-CP sequence (EKQKEK), which is present in transgenic SunUp and Rainbow, is identical to a putative allergen determinant (ABA-1) of roundworms [127]. However, Hileman et al [128] concluded that a threshold of six amino acids will not distinguish allergenic from non-allergenic proteins and will result in a large number of false positives. He instead recommended a minimum threshold of eight amino acids which is consistent with the International Life Science Institute/ Institute of Food Biotechnology Council (ILSI/IFBC) recommendations in 1996 in their decision tree approach which has been adopted internationally by GM food evaluators [129].…”

Practical and predictive bioinformatics methods for the identification of potentially cross‐reactive protein matches

“…The practice of searching for identical short amino acid matches between the introduced GE protein and sequences from known allergens had not been evaluated in terms of efficiency until recently [9,10]. However, short peptide sequence matches have been used to evaluate most, if not all, of the proteins introduced into commercially available GE crops that have been reviewed by US, Japan and EU regulators.…”

“…Such a match was thought to indicate that the GE protein might cause cross-reactions in those allergic to the matched allergen as proteins having an overall alignment which suggests overall structural similarity and evolutionary divergence, which also has significant short sequence matches, may have a higher probability of sharing IgE epitopes. However, in practice, these searches have been performed using simple algorithms that were developed to find identical strings, or words, much like the search function of a word processing program [9]. Each individual possible contiguous amino acid sequence segment of the protein was used to search a selected allergen database for an exact match.…”

“…The FAO/WHO panel report stimulated a number of efforts as reported in three studies that demonstrated that more appropriate criteria are needed. Hileman et al [9] found that roughly 80% of randomly chosen protein sequences of maize match an allergen if a six amino acid match is used, while comparisons for eight amino acid matches or those with >35% identity over 80 amino acids were more accurate in identifying proteins with overall FASTA alignments that would indicate an increased potential for shared sequential and conformational epitopes. Kleter and Peijnenburg [8] found also that six amino acid matches also identify matches unlikely to cause cross-reactions, and they investigated using a subsequent evaluation for antigenicity prediction for any matched proteins.…”