Identification and Purification of Novel Low-Molecular-Weight Lupine Allergens as Components for Personalized Diagnostics.

Nicola Wagner,Marisa Böttger,Franziska Ruëff,Susanne Abraham,Arabella Karstedt,Wolf-Meinhard Becker,Friedrich W. Riffelmann,Regina Treudler,Margitta Worm,Gerald Reese,Daniela Warneke,Sabine Dölle-Bierke,Uta Jappe,Saskia Hellmig,Lars Lange,André C. Knulst

doi:10.3390/nu13020409

Abstract

Lupine flour is a valuable food due to its favorable nutritional properties. In spite of its allergenic potential, its use is increasing. Three lupine species, Lupinus angustifolius, L. luteus, and L. albus are relevant for human nutrition. The aim of this study is to clarify whether the species differ with regard to their allergen composition and whether anaphylaxis marker allergens could be identified in lupine. Patients with the following characteristics were included: lupine allergy, suspected lupine allergy, lupine sensitization only, and peanut allergy. Lupine sensitization was detected via CAP-FEIA (ImmunoCAP) and skin prick test. Protein, DNA and expressed sequence tag (EST) databases were queried for lupine proteins homologous to already known legume allergens. Different extraction methods applied on seeds from all species were examined by SDS-PAGE and screened by immunoblotting for IgE-binding proteins. The extracts underwent different and successive chromatography methods. Low-molecular-weight components were purified and investigated for IgE-reactivity. Proteomics revealed a molecular diversity of the three species, which was confirmed when investigated for IgE-reactivity. Three new allergens, L. albus profilin, L. angustifolius and L. luteus lipid transfer protein (LTP), were identified. LTP as a potential marker allergen for severity is a valuable additional candidate for molecular allergy diagnostic tests.

Highlights

Flour from raw lupine seed is used increasingly as a protein source in Australia, NewZealand, The USA and European countries, where lupine serves as a replacement for animal proteins, i.e., milk, egg white, and potentially genetically modified soy products [1].Since lupine lacks gluten, lupine-containing products are recommended for patients with wheat protein allergy and coeliac disease
Investigations with sera from lupine-allergic patients revealed that seeds of different Lupinus species vary quantitatively in their allergen compositions
The patients reveal inter-individually different sensitization profiles and react differently to acidic and alkaline extractions of lupine flour. This is evident for the sera from P 3 and P 23 reacting to considerably more proteins in the alkaline L. angustifolius extract when compared to the acidic extraction, indicating the necessity to use more than one extraction method when searching for new single allergens

Summary

Introduction

Flour from raw lupine seed is used increasingly as a protein source in Australia, NewZealand, The USA and European countries, where lupine serves as a replacement for animal proteins, i.e., milk, egg white, and potentially genetically modified soy products [1].Since lupine lacks gluten, lupine-containing products are recommended for patients with wheat protein allergy and coeliac disease. The dietary value of lupine proteins is higher than that of beans or peas, which is mainly due to high concentrations of the essential amino acids lysine, leucine and threonine, which are higher only in soybeans. It has relatively high concentrations of protein and dietary fiber in contrast to digestible carbohydrates and lipids (summarized in [3]). Three lupine species (Lupinus albus, L. luteus, and L. angustifolius) are used as food as well as a food additive to fortify wheat flour, which may contain up to 10 or even 15% of lupine flour

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