Molecular characterisation of Lac s 1, the major allergen from lettuce (Lactuca sativa).

BACKGROUND

IgE sensitisation to non-specific lipid transfer proteins (nsLTP), e.g., Pru p 3 the major allergen from peach and most important allergenic LTP, is strongly associated with severe symptoms in food allergic patients. Lac s 1, a member of the nsLTP protein family, was recently identified as major allergen in lettuce (Lactuca sativa), but has not yet been investigated on the molecular basis.

OBJECTIVE

Molecular characterisation and immunological comparison of Lac s 1 to peach allergen Pru p 3.

METHODS

Lac s 1 cDNA was cloned by RT-PCR and natural (n) Lac s 1 was purified by a two-step chromatography. Protein structure was verified by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy. Immunoblotting, ImmunoCAP, and competitive IgE binding experiments were performed to study the IgE sensitisation pattern and cross-reactivity with Pru p 3. Allergenic potency was analysed by histamine release assay.

RESULTS

Twenty-nine lettuce allergic patients, with or without concomitant peach allergy, and 19 peach allergic patients without lettuce allergy were included in this study. IgE reactivity to lettuce was due to mono-sensitisation to Lac s 1 or cross-reactive glycan structures. Two Lac s 1 isoforms were identified which showed amino acid identity (aa-id) of 62% to each other, up to 66% to Pru p 3, and 72% to the N-terminal peptide of plane pollen LTP Pla a 3. The prevalence of IgE binding to nLac s 1 was 90% using lettuce extract in immunoblotting experiments. Enhanced sensitivity was observed in ImmunoCAP using purified nLac s 1 in comparison to extracts (93% versus 76%). Although IgE sensitisation to Lac s 1 and Pru p 3 was strongly associated, the two LTPs showed different IgE binding properties. Sensitisation to LTPs does not necessarily reflect the clinical disease, but Lac s 1 was capable of triggering histamine release as shown by positive skin test results in Lac s 1 mono-sensitised patients and by in vitro mediator release assays.

CONCLUSION

Purified nLac s 1 will enhance the sensitivity in component resolved diagnosis of lettuce allergy. Similar to other cross-reactive food allergies, exclusive testing of IgE reactivities to LTP cannot be used as biomarker for clinical relevance. Our data provide indirect evidence that Pru p 3 might act as the primary sensitising agent in patients allergic to both lettuce and peach.