β-N-Acetylhexosaminidases HEXO1 and HEXO3 Are Responsible for the Formation of Paucimannosidic N-Glycans in Arabidopsis thaliana

Liebminger, Eva; Veit, Christiane; Pabst, Martin; Batoux, Martine; Zipfel, Cyril; Altmann, Friedrich; Mach, Lukas; Strasser, Richard

doi:10.1074/jbc.m110.178020

Cited by 78 publications

(95 citation statements)

References 42 publications

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“…Reduced reactivity of Arabidopsis hgl1 N-glycans probably applies to most kinds of CCD-specific human IgE antibodies but seems to be best in the hgl1 fucTab combination. Another approach, likely based on conformational principles reported in this study, would be the down-regulation of ␤-N-acetylhexosaminidase activities recently suggested by Liebminger et al (46). How in these respects modified crop plants perform in the field remains to be investigated, especially concerning the enhanced salt sensitivity observed for Arabidopsis hgl1 fucTab null mutants described in this study.…”

Section: Discussionmentioning

confidence: 99%

“…3B) with proposed shielding of core fucose by untrimmed mannoses persisting on the ␣1,6-arm (blocking symbol). dases residing in vacuoles or in the cell wall (45,46). Alternatively, N-glycans with untrimmed ␣1,6-mannoses might be poor substrates for the hexosaminidases or selectively affected by protein degradation.…”

Section: Discussionmentioning

confidence: 99%

“…The dotted lines indicate terminal residues removed by mannosidase(s) (49) or GlcNac-specific hexosaminidases in vacuoles or the cell wall (45). The latter seems to occur less efficiently in the apoplast of leaf tissue analyzed here (46). The fully processed, complex N-glycan of wild type is circled by a dashed line.…”

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confidence: 99%

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Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

Kaulfürst-Soboll

Rips

Koiwa

et al. 2011

Journal of Biological Chemistry

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

Kaulfürst-Soboll

Rips

Koiwa

et al. 2011

Journal of Biological Chemistry

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“…The first committed step in the formation of complex N-glycans in the GA is catalyzed by b1,2-N-acetylglucosaminyltransferase I (GlcNac transferase I [GnTI]) (von Schaewen et al, 1993) and is followed by steps catalyzed by a-mannosidase II (Strasser et al, 2006), b1,2-N-acetylglucosaminyltransferase II (GnTII) (Strasser et al, 1999), b1,2-xylosyltransferase (Strasser et al, 2000), and a1,3-fucosyltransferase (Strasser et al, 2004). The two terminal GlcNac residues of some N-glycans are further modified by b1,3-galactosyltransferase (Strasser et al, 2007b) and a1,4-fucosyltransferase (Léonard et al, 2002) to produce Lewis a epitopes (Fitchette-Lainé et al, 1997), whereas the terminal GlcNac residues of most N-glycans are trimmed by hexosaminidases in the apoplast and vacuoles, resulting in paucimannosidic Man 3 XylFucGlcNac 2 structures (Strasser et al, 2007a;Liebminger et al, 2011). In Arabidopsis thaliana, about three quarters (73.9%) of all N-linked glycans are converted to complex-types.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-b1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Dall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.

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“…36 In addition, paucimannosidic, that derives from the removal of terminal GlcNAc residues from complex N-glycans, are present in vacuolar and secreted proteins. [36][37][38] Humans IgG1 and IgG4 were expressed, in transgenic suspension-cultured of tobacco BY2, sorted to different compartments, resulting in secretory versions producing higher yields than ER and vacuolar versions. 30,31 In addition, a mouse IgG fused to the sporamin Nt ssVSS (NPIRL) was produced also in transgenic BY2 cells, in intact form at levels of 8.5-80 ng/g and paucimannose Man3FucXylGlcNAc2 as main N-glycan structure.…”

Section: Introductionmentioning

confidence: 99%

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

2016

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Delivery of recombinant proteins to vegetative tissue vacuoles was considered inconvenient since this compartment was expected to be hydrolytic; nevertheless there is growing evidence that certain foreign proteins accumulate at high yields in vacuoles. For example avidin, cellulolytic enzymes, endolysin, and transglutaminases were produced at high yields when were sorted to leaf central vacuole avoiding the detrimental effect of these proteins on plant growth. Also, several secretory mammalian proteins such as collagen, a1-proteinase inhibitor, complement-5a, interleukin-6 and immunoglobulins accumulated at higher yields in leaf vacuoles than in the apoplast or cytosol. To reach this final destination, fusions to sequence specific vacuolar sorting signals (ssVSS) typical of proteases or proteinase inhibitors and/or Ct-VSS representative of storage proteins or plant lectins were used and both types of motifs were capable to increase accumulation. Importantly, the type of VSSs or position, either the N or C-terminus, did not alter protein stability, levels or pos-translational modifications. Vacuolar sorted glycoproteins had different type of oligosaccharides indicating that foreign proteins reached the vacuole by 2 different pathways: direct transport from the ER, bypassing the Golgi (high mannose oligosaccharides decorated proteins) or trafficking through the Golgi (Complex oligosaccharide containing proteins). In addition, some glycoproteins lacked of paucimannosidic oligosaccharides suggesting that vacuolar trimming of glycans did not occur. Enhanced accumulation of foreign proteins fused to VSS occurred in several plant species such as tobacco, Nicotiana benthamiana, sugarcane, tomato and in carrot and the obtained results were influenced by plant physiological state. Ten different foreign proteins fused to vacuolar sorting accumulated at higher levels than their apoplastic or cytosolic counterparts. For proteins with cytotoxic effects vacuolar sorted forms yields were superior than ER retained variants, but for other proteins the results were the opposite an there were also examples of similar levels for ER and vacuolar variants. In conclusion vacuolar sorting in vegetative tissues is a satisfactory strategy to enhance protein yields that can be used in several plant species.

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β-N-Acetylhexosaminidases HEXO1 and HEXO3 Are Responsible for the Formation of Paucimannosidic N-Glycans in Arabidopsis thaliana

Cited by 78 publications

References 42 publications

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

Reduced Immunogenicity of Arabidopsis hgl1 Mutant N-Glycans Caused by Altered Accessibility of Xylose and core Fucose Epitopes

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

Vacuolar deposition of recombinant proteins in plant vegetative organs as a strategy to increase yields

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