Molecular characterization and allergenic activity of Lyc e 2 (β‐fructofuranosidase), a glycosylated allergen of tomato

Abstract

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two‐step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE‐binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Manα1–6(Xylβ1–2)Manβ1–4GlcNAcβ1–4(Fucα1–3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N‐glycosylation sites. By application of MALDI‐TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Manα1–6(Manα1–3)(Xylβ1–2)Manβ1–4GlcNAcβ1–4(Fucα1–3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

The Role of Protein Glycosylation in Allergy

“…Such IgE binding has been observed with insect venoms, grass and tree pollens and foods [2, 11, 35, 36, 38, 39, 44, 76,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104]. Possibly the first work measuring anti-CCD IgE in individual sera was on a panel of patients allergic to bee venom [24].…”

“…This structure occurs so frequently on plant proteins that trying to list all known carriers would be futile. Examples among non-allergens are HRP [18] and many lectins [33, 34], and among allergens, Phl p 1, Phl p 13 [35], Lyc e 2 [36] Hev b 4 [37], Lol p 11 [38], and Api g 5 [39]. Tree pollens [30] and tree pollen allergens [39] tend to bear terminal non-reducing GlcNAc residues, a feature which may reduce IgE binding (see paragraph on specificity of IgG).…”

A proteomic study of the major allergens from yellow jacket venoms

“…CCDs are a frequent cause of cross-reactions of patients' sera with allergens from insects and other sources [3,14,15] but appear to be of low clinical significance [13,16,17]. The relative contributions of the protein and the carbohydrate moiety of insect venom Hyases to in vitro IgE binding and to eliciting allergic symptoms in vivo has not yet been defined.…”

Reduction of Allergenic Proteins by the Effect of theripening inhibitor(rin) Mutant Gene in an F1Hybrid of therinMutant Tomato

“…4,28) Indeed, -fructofuranosidase and PG-2A that were focused on in this study are known as N-glycosidic proteins. 29,30) The total amount of Nglycosidic proteins obviously increases in tomato fruit during ripening, 13) suggesting that the accumulation of N-glycosidic proteins in fruit would also be affected by LeMADS-RIN. Thus, the N-glycosidic proteins are candidates for the undetermined IgE-reactive bands in Fig.…”