Abstract
The molecular characterization of bioactive food components is necessary for understanding the mechanisms of their beneficial or detrimental effects on human health. This study focused on γ-conglutin, a well-known lupin seed N-glycoprotein with health-promoting properties and controversial allergenic potential. Given the importance of N-glycosylation for the functional and structural characteristics of proteins, we studied the purified protein by a mass spectrometry-based glycoproteomic approach able to identify the structure, micro-heterogeneity and attachment site of the bound N-glycan(s), and to provide extensive coverage of the protein sequence. The peptide/N-glycopeptide mixtures generated by enzymatic digestion (with or without N-deglycosylation) were analyzed by high-resolution accurate mass liquid chromatography–multi-stage mass spectrometry. The four main micro-heterogeneous variants of the single N-glycan bound to γ-conglutin were identified as Man2(Xyl) (Fuc) GlcNAc2, Man3(Xyl) (Fuc) GlcNAc2, GlcNAcMan3(Xyl) (Fuc) GlcNAc2 and GlcNAc 2Man3(Xyl) (Fuc) GlcNAc2. These carry both core β1,2-xylose and core α1-3-fucose (well known Cross-Reactive Carbohydrate Determinants), but corresponding fucose-free variants were also identified as minor components. The N-glycan was proven to reside on Asn131, one of the two potential N-glycosylation sites. The extensive coverage of the γ-conglutin amino acid sequence suggested three alternative N-termini of the small subunit, that were later confirmed by direct-infusion Orbitrap mass spectrometry analysis of the intact subunit.
Highlights
IntroductionWidely-consumed legume seeds (e.g. soybean, beans, peanut and lupin) have been studied with the specific aim of identifying and characterizing the proteins accounting for their health-promoting properties [1,2] and/or allergenic effects [3,4]
The molecular characterization of bioactive food components is essential for understanding the mechanisms of their beneficial or detrimental effects on human health.Widely-consumed legume seeds have been studied with the specific aim of identifying and characterizing the proteins accounting for their health-promoting properties [1,2] and/or allergenic effects [3,4]
The allergenic properties of the lupin seed have been ascribed to the abundant components β- and α-conglutin [14], while for γ-conglutin the allergenic potential remains controversial, ranging from strong to weak in different in vitro and/or in vivo settings [14,15,16,17,18,19,20]
Summary
Widely-consumed legume seeds (e.g. soybean, beans, peanut and lupin) have been studied with the specific aim of identifying and characterizing the proteins accounting for their health-promoting properties [1,2] and/or allergenic effects [3,4]. The large γconglutin subunit reportedly carries one N-linked oligosaccharide chain [22], but the structure, the possible micro-heterogeneity, and the actual attachment site of the Nglycan have not been investigated. This subunit has two potential N-glycosylation sites, Asn131 within the canonical eukaryote N-glycosylation consensus sequence Asn–Xaa– Ser/Thr (where Xaa is not Pro), and Asn132 within the less common sequon (Asn–Xaa–Cys), recently described in plant cells [23]
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