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 ␣-mannosidase II are unique, because their glycoprotein N-glycans are hardly labeled by anti-complex glycan antibodies, even though they carry ␤1,2-xylose and ␣1,3-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-␤1,2-xylose and anti-␣1,3-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°b ecause 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 ␣1,6-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.

Multiple N-Glycans Cooperate in the Subcellular Targeting and Functioning of Arabidopsis KORRIGAN1

“…Stronger effects caused by cgl1 or hgl1 could be attributed to persisting mannoses on the a1,6-branch, but in the case of hgl1, also alter the availability/ accessibility of core fucoses. We previously demonstrated that the a1,3-fucose epitope present in hgl1 mutants was recognized by complex glycan-and fucose-specific antibodies only after enzymatic digestion of the bulky mannoses on the a1,6-arm (Kaulfürst-Soboll et al, 2011). These immunologic and modeling results support our hypothesis that complex N-glycans could function as an important interface between glycoproteins and their interacting partners.…”

“…These results reveal that core a1,3-fucose attachment is an important host N-glycan modification for in vivo functionality of KOR1. However, this is not more important than mannose trimming ( Figure 9B) because persisting mannoses on the a1,6-branch produce more severely impaired phenotypes in rsw2-1 hgl1-1 whose N-glycans still receive core a1,3-fucose (Strasser et al, 2006;Kaulfürst-Soboll et al, 2011). Neither hgl1-1 nor fucTa/b double mutants without rsw2-1 showed growth defects in the absence of osmotic stress (Kang et al, 2008).…”

“…Antibodies used for immunoblotting were anticomplex glycan (PHA-L antigen) (Kaulfürst-Soboll et al, 2011), anti-GFP (Genscript), and anti-RGS-His (Qiagen).…”

Toward Stable Genetic Engineering of Human O-Glycosylation in Plants

“…Problems with degradation and plant-specific PTMs were identified, highlighting the need for additional engineering for plants to become versatile production platforms of therapeutics. Although in its infancy, engineering O-glycosylation capacities in plants may eventually be combined with engineered human N-glycosylation (Strasser et al, 2008;Kaulfürst-Soboll et al, 2011;Nagels et al, 2011;Schoberer and Strasser, 2011) to generate more general platforms for the expression of glycoprotein pharmaceuticals in plants.…”

“…However, stoichiometry and subcellular localization of the introduced glycoenzymes and subcellular substrate pools are increasingly difficult to control as the engineering effort increases in complexity. Stable engineering of especially the glycosylation machineries is thus generally needed at an early point, as seen in the engineering efforts to humanize plants with respect to N-linked protein glycosylation (Strasser et al, 2008;Castilho et al, 2010;Kaulfürst-Soboll et al, 2011;Nagels et al, 2011;Schoberer and Strasser, 2011;Yin et al, 2011).…”