title: Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes.
abstract: Arabidopsis N-glycosylation mutants with enhanced salt sensitivity show reduced immunoreactivity of complex N-glycans. Among them, hybrid glycosylation 1 (hgl1) alleles lacking Golgi α-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry β1,2-xylose and α1,3-fucose epitopes. To dissect the contribution of xylose and core fucose residues to plant stress responses and immunogenic potential, we prepared Arabidopsis hgl1 xylT double and hgl1 fucTa fucTb triple mutants by crossing previously established T-DNA insertion lines and verified them by mass spectrometry analyses. Root growth assays revealed that hgl1 fucTa fucTb but not hgl1 xylT plants are more salt-sensitive than hgl1, hinting at the importance of core fucose modification and masking of xylose residues. Detailed immunoblot analyses with anti-β1,2-xylose and anti-α1,3-fucose rabbit immunoglobulin G antibodies as well as cross-reactive carbohydrate determinant-specific human immunoglobulin E antibodies (present in sera of allergy patients) showed that xylose-specific reactivity of hgl1 N-glycans is indeed reduced. Based on three-dimensional modeling of plant N-glycans, we propose that xylose residues are tilted by 30° because of untrimmed mannoses in hgl1 mutants. Glycosidase treatments of protein extracts restored immunoreactivity of hgl1 N-glycans supporting these models. Furthermore, among allergy patient sera, untrimmed mannoses persisting on the α1,6-arm of hgl1 N-glycans were inhibitory to immunoreaction with core fucoses to various degrees. In summary, incompletely trimmed glycoprotein N-glycans conformationally prevent xylose and, to lesser extent, core fucose accessibility. Thus, in addition to N-acetylglucosaminyltransferase I, Golgi α-mannosidase II emerges as a so far unrecognized target for lowering the immunogenic potential of plant-derived glycoproteins.
authors.affiliation: Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, 48149 Münster, Germany.
authors.forename: Heidi
authors.initials: H
authors.lastname: Kaulfürst-Soboll
authors.forename: Stephan
authors.initials: S
authors.lastname: Rips
authors.forename: Hisashi
authors.initials: H
authors.lastname: Koiwa
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kajiura
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Fujiyama
authors.forename: Antje
authors.initials: A
authors.lastname: von Schaewen
mesh_terms: D000483:Alleles; D000818:Animals; D017360:Arabidopsis / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D005643:Fucose / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011134:Polysaccharides / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D014994:Xylose / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D043323:alpha-Mannosidase / Q000276:immunology / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D000939:Epitopes; D007074:Immunoglobulin G; D011134:Polysaccharides; D005643:Fucose; D014994:Xylose; D043323:alpha-Mannosidase