H: Health, Nutrition, & Food JFS H: Health, Nutrition, and Food Identification of a Specific IgE-Binding Protein from Narrow-Leafed Lupin ( L. Angustifolius ) Seeds PATRYCJA KLOS,EL˙ zBIETA POR  EBA,EWA SPRINGER,ELEONORA LAMPART -SZCZAPA, AND ANNA GO´ ZDZICKA J´ OZEFIAK ABSTRACT: Since lupin has been introduced as a food ingredient on the market there are more and more reports concerning its allergenic properties. However, only few narrow-leafed lupin proteins have yet been characterized as specific IgE-binding molecules and identified. The aim of the study has been to find and identify the main narrow- leafed lupin globulins that bind to specific IgEs from the sera of lupin-allergic people. Isolated lupin globulins were subjected to immunoblotting with the sera from people who suffered from lupin allergy. Incubation with α-methyl- D-galactopyranoside was performed to eliminate possible binding of unspecific human IgEs. The proteins binding specific IgEs from lupin-allergic patients’ sera were identified by means of mass spectrometry. Western blot analysis revealed 2 signals corresponding to lupin globulins that bound to specific IgEs from the sera of people allergic to lupin. The globulins were identified as conglutin-γ and its smaller subunit. The results suggested that individuals that displayed lupin allergy symptoms reacted to conglutin-γ . Practical Application: The results of the study can contribute to identification of yet undetected allergens of narrow- leafed lupin. This, in turn, can make lupin-fortified products safer for the consumers. Keywords: allergy, conglutin-γ , globulins, IgE, immunoblotting, mass spectrometry, narrow-leafed lupin Introduction L upins are a valuable component of human diet. Nutritional value of lupin seeds is mainly related to their high protein con- tent (Gueguen and Cerletti 1994; Sirtori and others 2004). Lupin seeds are also high in fiber (G´ orecka and others 2000), oligosac- charides (Zdu ´ nczyk and others 1998), and phenolic compounds (Lampart-Szczapa and others 2003a). Additionally, they are low in antinutritional components, such as lectins, trypsin inhibitors, or alkaloids (Dupont and others 1994; van Barneveld 1999). Due to their composition lupin seeds feature some beneficial properties. It has been shown that they have a cholesterol-lowering effect in pigs and in man (Martins and others 2005), as well as antioxidant and antimicrobial properties (Lampart-Szczapa and others 2003a, 2003b). In addition, it has been proved that lupin seed proteins have hypocholesterolemic effect (Sirtori and others 2004; Marchesi and others 2008) and attenuate the development of hypertension in rats (Pilvi and others 2006), whereas lupin conglutin γ increases LDL-uptake and degradation (Sirtori and others 2004), as well as reduces plasma glucose levels in hyperglycemic rats (Magni and others 2004). Since lupin seeds have become popular as a food ingredient there have been reports concerning allergic properties of their pro- tein. Most of the reports have dealt with the cases of hypersensitiv- ity reactions and cross-reactivity in people suffering from allergy to legumes, after consuming lupin-fortified products (Moneret- Vautrin and others 1999; Kanny and others 2000; Faeste and MS 20090580 Submitted 6/23/2009, Accepted 10/9/2009. Author Klos is with Dept. of General Pathology, Pomeranian Medical Univ., Al. Powsta´ nc´ ow Wielkopolskich 72, 70-111, Szczecin, Poland. Author Lampart-Szczapa is with Dept. of Food Biochemistry and Analysis, the Poznan Univ. of Life Sciences, ul. Mazowiecka 48, 60-623, Pozna´ n, Poland. Authors Por  eba and J´ ozefiak are with Dept. of Molecular Virology, the Adam Mickiewicz Univ., ul. Umultowska 89, 61-614, Pozna´ n, Poland. Author Springer is with NZOZ Alergologia Plus, Center of Diagnostics and Treatment of Allergy, ul. Drob- nika 49, 60-693, Pozna´ n, Poland. Direct inquiries to author Klos (E-mail: patrycja.klos@gmail.com). others 2004; Rojas-Hijazo and others 2006). Lupin flour also has been recognized as a cause of occupational airborne allergy (Cre- spo and others 2001). Some of lupin globulins that comprise the bulk of lupin proteins have been identified as IgE-binding molecules (Magni and others 2005; Dooper and others 2007; Holden and others 2008). However, not all of them have been char- acterized as proteins that bind to specific IgEs from the sera of lupin-allergic individuals. In our pilot study, we have investigated the chosen globulins of narrow-leafed lupin, by means of immunoblotting, to find which of them bind to specific IgEs from the sera of people who suffer from lupin allergy. As some of lupin seed proteins have the affinity for galactose, we have assumed that they can bind to Fc fragments of human IgEs, which are rich in this sugar (Robertson and Liu 1991). For this reason we have used α-methyl-D-galactopyranoside during the blotting procedures to eliminate possible unspecific binding of globulins to IgEs. Mass spectrometric identification of globulins that bind to specific IgEs of lupin-allergic people was also a major objective of this study. Materials and Methods Isolation and fractionation of lupin globulins Globulins were isolated (Freitas and others 2000) from defatted (n-hexane, room temperature, 4 h) narrow-leafed lupin flour (Lupi- nus angustifolius, var. Baron), supplied by the Plant Breeding and Acclimatization Inst. in Przeb  edowo, Poland, and fractionated by means of gel filtration chromatography (glass column packed with Sephadex G 200, Labart, Poland). The separation was run in 50 mM Tris-HCl, pH 7.5, at a flow rate of 0.76 mL/min, at room tempera- ture. The elution profile of the globulins is presented in Figure 1. Three globulin fractions with the protein concentration of 676, 177, and 144 μg/mL (Bradford 1976), respectively, were numbered from 1 to 3 and subjected to some further analysis. C  2009 Institute of Food Technologists R  Vol. 75, Nr. 1, 2010—JOURNAL OF FOOD SCIENCE H39 doi: 10.1111/j.1750-3841.2009.01440.x Further reproduction without permission is prohibited