Abstract
N-glycosylation is a highly abundant protein modification present in all domains of life. Terminal sugar residues on complex-type N-glycans mediate various crucial biological processes in mammals such as cell-cell recognition or protein-ligand interactions. In plants, the Lewis A trisaccharide constitutes the only known outer-chain elongation of complex N-glycans. Lewis A containing complex N-glycans appear evolutionary conserved, having been identified in all plant species analyzed so far. Despite their ubiquitous occurrence, the biological function of this complex N-glycan modification is currently unknown. Here, we report the identification of Lewis A bearing glycoproteins from three different plant species: Arabidopsis thaliana, Nicotiana benthamiana, and Oryza sativa. Affinity purification via the JIM84 antibody, directed against Lewis A structures on complex plant N-glycans, was used to enrich Lewis A bearing glycoproteins, which were subsequently identified via nano-LC-MS. Selected identified proteins were recombinantly expressed and the presence of Lewis A confirmed via immunoblotting and site-specific N-glycan analysis. While the proteins identified in O. sativa are associated with diverse functions, proteins from A. thaliana and N. benthamiana are mainly involved in cell wall biosynthesis. However, a Lewis A-deficient mutant line of A. thaliana showed no change in abundance of cell wall constituents such as cellulose or lignin. Furthermore, we investigated the presence of Lewis A structures in selected accessions from the 1001 genome database containing amino acid variations in the enzymes required for Lewis A biosynthesis. Besides one relict line showing no detectable levels of Lewis A, the modification was present in all other tested accessions. The data provided here comprises the so far first attempt at identifying Lewis A bearing glycoproteins across different species and will help to shed more light on the role of Lewis A structures in plants.
Highlights
N-glycosylation is an abundant protein modification present in all domains of life
We report the isolation of 10 proteins from A. thaliana, seven proteins from O. sativa and nine proteins from N. benthamiana modified with Lewis A
In a recent study investigating Nglycan microheterogeneity in, among others, inflorescence stems of A. thaliana (Zeng et al, 2018), the authors report the detection of biantennary complex N-glycans with Lewis A structures on three glycoproteins, among them Laccase 4 (At2g38080.1), which was identified in our approach in both Arabidopsis stems and siliques
Summary
N-glycosylation is an abundant protein modification present in all domains of life. N-glycosylation is initiated by the transfer of a preassembled oligosaccharide to certain asparagine residues of nascent polypeptide chains either co-translationally as they enter the lumen of the endoplasmic reticulum (ER), or posttranslationally. In the model plant A. thaliana, Lewis A bearing complex N-glycans show organspecific expression, being absent in leaves but present in stems and siliques In these plant organs they constitute only a minor fraction of the total N-glycan content (Strasser et al, 2007) indicating that only few proteins are modified with this trisaccharide. Overall, this modification appears to be evolutionarily conserved, having been identified in mosses such as Physcomitrella patens (Koprivova et al, 2003), a wide variety of foodstuff (Wilson et al, 2001, Okada et al, 2017), and on diverse water plants (Maeda et al, 2016). We describe the identification of glycoproteins modified with Lewis A structures from three different plant species and show that Lewis A structures represent an evolutionarily conserved modification in natural accessions of A. thaliana
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