Lewis A Glycans Are Present on Proteins Involved in Cell Wall Biosynthesis and Appear Evolutionarily Conserved Among Natural Arabidopsis thaliana Accessions

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.

Keywords: N-glycans; carbohydrate epitope; glycoproteomics; glycosylation; posttranslational modification.

Figure 1

Lewis A bearing complex N-glycans are present on recombinantly produced plant proteins. KORRIGAN (A), g6145 (B), and CEBIP (C) were transiently expressed in leaves of wild-type and ΔXT/FT plants of N. benthamiana and purified via their fused tag. Expression of proteins was confirmed via immunoblotting using an antibody directed against the fused tag (GFP for KORRIGAN and mRFP for g6145 and CEBIP). Presence of Lewis A was shown using the JIM84 antibody.

Figure 2

Glycopeptides from recombinantly produced plant proteins display complex N-glycans with Lewis A structures. MS spectra of glycopeptides from KORRIGAN (A), g6145 (B), and CEBIP (C). Proteins were recombinantly expressed in leaves of wild-type N. benthamiana, purified via the fused tag and subjected to proteolytic digest using trypsin and chymotrypsin. Nomenclature of N-glycans is according to the ProGlycAn system (http://www.proglycan.com/). Only one N-glycan isoform is indicated. Lewis A bearing glycans are underlined.

Figure 3

Phenotypic comparison of wild-type and Lewis A-deficient (galt1 fut13) plants. rsw2-1 plants used as controls. The bottom and top of the boxes represent 25 and 75 percent quartiles, respectively, while the horizontal line inside the box indicates the median. The whiskers indicate 1,5x interquartile range (IQR). Asterisks indicate statistically significant differences to Col-0 plants (*p ≤ 0.05, **p ≤ 0.01). (A) Comparison of flowering time, length of the plants primary stem, and mass of dried primary stems. (B) Content of the primary stems. Cell wall residues (CWR) were determined gravimetrically after sequential removal of alcohol soluble residues. Cellulose and lignin content were determined photometrically as percentage of CWR.

Figure 4

Detection of Lewis A structures in Arabidopsis accessions carrying GALT1 polymorphisms. (A) Protein sequence of GALT1 from Arabidopsis Col-0. The transmembrane domain is shown in gray, the putative galactoside-binding lectin domain in green, and the galactosyltransferase domain in yellow. Additionally, the DXD-motif in the catalytic site is highlighted in light blue and the conserved GxxYxxS as well as the adjacent DxA motif are highlighted in magenta. Underlined amino acids indicate N-glycosylation sequons. Sites depicted in bold show positions of amino acids, for which we could find variations in the 1001 genome database. (B,C) Immunoblot analysis of the indicated Arabidopsis accessions. Total protein extracts from stems and siliques, respectively, were analyzed with antibodies directed against Lewis A bearing (JIM84) and plant N-glycans carrying β1,2-xylose and core α1,3-fucose (anti-HRP).