Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody clone #86

Abstract

A human-mouse hybridoma clone #86 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was newly established. To detect an antibody-binding sequence (epitope) on Ara h1, the monoclonal antibody #86 was reacted with multi-pin apparatus with a series of peptides synthesized from the amino acid sequence of Ara h1. The antibody #86 was found to bind to a peptide with amino acid sequence of 481EEEEDEDEEEEGSNREVRRY500. Further analysis with shorter pin-peptides with ten amino acid-long showed that the peptides reacted with the antibody #86 contained a sequence of 485DEDEEEE491. This might be an essential linear sequence of this epitope. When the 485DED487 part of the peptide was replaced by alanine, decreased binding of antibody #86 was observed.

Combined N-Glycome and N-Glycoproteome Analysis of the Lotus japonicus Seed Globulin Fraction Shows Conservation of Protein Structure and Glycosylation in Legumes

“…In the vast majority of people suffering from a peanut allergy, the immune system triggers a response against one or several protein epitopes from Ara h 1 . Identified epitopes are located throughout the protein sequence with no preference for a specific area of the protein. − The three-dimensional protein structure of Ara h 1 indicates that several epitopes are located at the protein surface which is in good agreement with the fact that Ara h 1 is resistant to both proteolysis and heat. , …”

“…Food allergy, where some of the major allergens are legume seed globulins, is a universal problem with 1–5% of the total human population displaying clinical symptoms. , Interestingly, the legume seed globulins seem to be highly conserved in protein sequences between species judging from the high similarity between the Lotus LCP2 and the known globulin allergens from other legumes, that is, mung bean, lupin, lentil, and peanut (Figure ). All five proteins have a high protein sequence similarity around the single conserved N-glycosylation site (where several of the Ara h 1 allergen epitopes are located) and, together with the similar high mannosylated glycoprofiles, reinforce the potential for LCP2 to be an allergen. − To further support this, in-silico digestion of LCP2 and Ara h 1, based on cleavage sites of proteases present in the gastrointestinal tract (i.e., pepsin, trypsin, and chymotrypsin) produces similar peptides (data not shown). LCP2 and Ara h 1 protein sequences are predominantly degraded into single or few amino acid residues by the in-silico digestion using the combination of the three proteases.…”

“…Few peptides of more than 8 amino acid residues were found for both proteins with complete digestion, which is well above the minimum peptide epitope size required for recognition by the immune system. However, these potentially undigested regions were not overlapping with the identified immunogenic epitopes for Ara h 1 − and, thus, cannot explain why Ara h 1 is an allergen.…”