Combined N‑Glycome and N‑Glycoproteome Analysis of the <i>Lotus japonicus</i> Seed Globulin Fraction Shows Conservation of Protein Structure and Glycosylation in Legumes

Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. <i>Lotus japonicus</i> (<i>Lotus</i>) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the <i>Lotus</i> N-glycoproteome. The global and site-specific N-glycan structures of <i>Lotus</i> seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant <i>Lotus</i> seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, <i>Lotus</i> convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. <i>In silico</i> investigation confirmed that peanut Ara h 1 and <i>Lotus</i> LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that <i>Lotus</i> has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy