Identification of immunoglobulin E (IgE)-binding epitopes and recombinant IgE reactivities of a latex cross-reacting Indian jujube Ziz m 1 allergen

Abstract

Ziz m 1 is a major Indian jujube (Zizyphus mauritiana) allergen involved in latex-fruit syndrome, and cDNA of the allergen has been cloned, sequenced and expressed in yeast by our laboratory previously. In this study, we performed an immunoglobulin E (IgE)-binding epitope analysis of Ziz m 1 using overlapping recombinant fragments. Eight overlapping recombinant fragments were generated from the recombinant Ziz m 1 allergen. The fragments were expressed in Escherichia coli and IgE-binding activities were evaluated by sera of latex-Indian jujube-allergic subjects and normal subjects using immunoblotting. Human allergic sera are not able to recognize fragments consisting of amino acid sequences 26-71, 119-280 and 119-291. However, residues at positions 26-199, 26-105, 26-86, 119-320 and 238-330 were found relevant in the IgE-binding. Our results indicate that (72)NISGHCSDCTFLGEE(86) and (292)VWNRYYDLKTNYSSSIILEYVNSGTKYLP(320) of Ziz m 1 are the sequences required for human IgE binding. Four corresponding peptides, (72)NISGHCSDCTE(86), (292)VWNRYYDLKT(301), (300)KTNYSSSIILEY(311) and (309)LEYVNSGTKYLP(320), were synthesized, and these peptides reacted with 70%, 100%, 70% and 70% of 10 allergic sera tested, as revealed by enzyme-linked immunosorbent assay. Sensitization to (292)VWNRYYDLKT(301) correlated significantly with the presence of allergic symptoms (P < 0.001). These findings will be useful in designing diagnostic and therapeutic approaches, thereby contributing to the development of specific immunotherapy for subjects with latex-fruit syndrome.

Fig. 1

Silver-stained sodium dodecyl sulphate-polyacrylamide gel electrophoresis (a) and immunoblotting (b) of purified proteins expressed from deleted Ziz m 1 cDNA. Mature Ziz m 1: 26–330 (lane 1), N1 (Ziz m 1: 26–199, lane 2), N2 (Ziz m 1: 26–105, lane 3), N3 (Ziz m 1: 26–86, lane 4), N4 (Ziz m 1: 26–71, lane 5), C1 (Ziz m 1: 119–280, lane 6), C2 (Ziz m 1: 119–291, lane 7), C3 (Ziz m 1: 119–320, lane 8) and C4 (Ziz m 1: 238–330, lane 9). Numbers on the left indicate sizes (kDa) of protein marker (lane M). The non-specific weak band in lanes 5 and 6 of (b) may be due to the high sensitivity of chemiluminescent exposure.

Fig. 2

B cell epitope mapping on Ziz m 1. Schematic map location of deletion mutants on the Ziz m 1. represents the region recognized by human IgE. Arrows (< or >) represent the direction of deletion construction.

Fig. 3

Binding profiles of immunoglobulin E antibodies to synthetic peptides by enzyme-linked immunosorbent assay. Sera from 10 latex–Indian jujube-allergic subjects (P1–P10) and five from non-allergic subjects (NA1–NA5) were analysed. The cut-off value was 0·2, as shown by the dotted line.