Combined N-Glycome and N-Glycoproteome Analysis of the Lotus japonicus Seed Globulin Fraction Shows Conservation of Protein Structure and Glycosylation in Legumes

Abstract

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. Lotus japonicus (Lotus) 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 Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus 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 Lotus 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, Lotus 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. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

“…Mass spectrometry (MS)‐based N ‐glycome profiling of Lotus japonicus seeds identified a total of 19 N ‐glycan structures spanning the full spectrum of high mannose, complex and paucimannosidic type N ‐glycans (Dam et al ., ). This observation indicated that Lotus japonicus possesses a typical plant N ‐glycan maturation pathway (Figure S1).…”

“…To verify the altered N ‐glycosylation of the generated Lotus japonicus mutants, N ‐glycans released from their carrier proteins were characterised from seed extracts using PGC‐LC‐MS/MS. As expected, the N ‐glycan profile of wild type seed proteins, which included 24 N ‐glycan structures spanning 18 monosaccharide compositions, strongly resembled our previously reported profile of Lotus japonicus (Dam et al ., ) (see Table S2 for details of the identified N ‐glycans). The N ‐glycan type distribution of the wild type proteins differed in general from the distribution of the mutant proteins (Figure a).…”

Human Neutrophils Secrete Bioactive Paucimannosidic Proteins from Azurophilic Granules into Pathogen-Infected Sputum

“…Outside the vertebrate subphylum, invertebrates such as Caenorhabditis elegans (16,18,(22)(23)(24) and Drosophila melanogaster (15,17,25,26), plants (27), and other "lower" organisms (28) abundantly produce protein paucimannosylation. Paucimannose synthesis in these species is facilitated by high ␤-Nacetylhexosaminidase activity, allowing partial suppression of the complex N-glycan biosynthetic route.…”

Mapping the N-linked glycosites of rice (Oryza sativa L.) germinating embryos

“…N- glycosylation is also involved in food allergy, as several identified, N- glycosylated albumin proteins in wheat shared high sequence similarities with known food allergens [22]. Glycoproteomic characterization has also been attempted in cotton and various bio-fuel grasses and trees [17,19,21,23,28]. To the best of our knowledge, the identification of 242 glycosites on 191 proteins in the current study represents the largest scale profiling of glycoproteome in crops so far (S3 Table).…”