Molecular basis of <i>N</i>-acetylglucosaminyltransferase I deficiency in <i>Arabidopsis thaliana</i> plants lacking complex N-glycans

Strasser, Richard; Stadlmann, Johannes; Svoboda, Barbara; Altmann, Friedrich; Glössl, Josef; Mach, Lukas

doi:10.1042/bj20041686

Cited by 86 publications

(92 citation statements)

References 33 publications

(34 reference statements)

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“…SM6 heavy and light chains (19,20) were codon-optimized for N. benthamiana and cloned into Magnicon-based vectors pICHα31160 and pICHα26033, respectively (23). The codon-optimized joining chain (GenBank Accession NP_653247.1) lacking the 22-bp signal peptide was fused to the barley alfa amylase signal peptide and cloned into the binary vector pPT2 (34). HC-and LC-containing vectors were transformed into GV3101pMP90RK agrobacteria, the JC-containing vector into UIA143pMP90.…”

Section: Methodsmentioning

confidence: 99%

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Loos

Gruber

Altmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Significance IgM antibodies are increasingly gaining interest as therapeutics; however, knowledge about this antibody class is scarce. Specifically the impact of N-glycans on the functional mechanism of this heavily glycosylated molecule is entirely unknown. To address this issue we produced different IgM glycoforms in plants and characterized them. Moreover, we present a computer model that explains the characteristic N-glycosylation pattern of IgMs. With the successful in planta generation of recombinant IgMs largely resembling the plasma-derived orthologue, we offer an efficient alternative to mammalian cell-based expression systems. IgMs with targeted glycoengineered N-glycans now enable detailed structure–function studies and will lead to the production of IgMs with optimized in vivo activities.

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Section: Methodsmentioning

confidence: 99%

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Loos

Gruber

Altmann

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…For this, the catalytic domain of GalT was first amplified from the vector pCDNAI-GT (11) with the primers tataTCTAGAcccgggtaccgccatcgggcagtcct/tataGGATCCctagctcggtgtcccgatgtcc and ligated to pPT2M (12). The CTS region of the ST was amplified from the pGA482rST (13) with the primers tatATCTAGAatgattcataccaacttgaag/tataGGTACCggccactttctcctggctcttg and subsequently ligated to the vector containing GalT catalytic domain resulting in the vector ST-GalT.…”

Section: Methodsmentioning

confidence: 99%

Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous β1,4-Galactosylated N-Glycan Profile

Strasser

Castilho

Stadlmann

et al. 2009

Journal of Biological Chemistry

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157

174

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It is well established that proper N-glycosylation significantly influences the efficacy of monoclonal antibodies (mAbs). However, the specific immunological relevance of individual mAbassociated N-glycan structures is currently largely unknown, because of the heterogeneous N-glycan profiles of mAbs when produced in mammalian cells. Here we report on the generation of a plant-based expression platform allowing the efficient production of mAbs with a homogeneous ␤1,4-galactosylated N-glycosylation structure, the major N-glycan species present on serum IgG. This was achieved by the expression of a highly active modified version of the human ␤1,4-galactosyltransferase in glycoengineered plants lacking plant-specific glycosylation. Moreover, we demonstrate that two anti-human immunodeficiency virus mAbs with fully ␤1,4-galactosylated N-glycans display improved virus neutralization potency when compared with other glycoforms produced in plants and Chinese hamster ovary cells. These findings indicate that mAbs containing such homogeneous N-glycan structures should display improved in vivo activities. Our system, using expression of mAbs in tobacco plants engineered for post-translational protein processing, provides a new means of overcoming the two hurdles that limit the therapeutic use of anti-human immunodeficiency virus mAbs in global health initiatives, low biological potency and high production costs.

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“…High-Man N-glycans are processed in the endoplasmic reticulum (Lerouge et al, 1998). In plant cells, oligomannoside glycans account for only a small fraction of N-glycans and Man 3 GlcNAc 2 is rare (Strasser et al, 2004(Strasser et al, , 2005. The secretory pathway of sieve elements is limited (Van Bel, 2003), potentially limiting the formation of complex glycans and leading to an enrichment in glycoproteins with high-Man N-glycans.…”

Section: Pp2-a1 Binds To Different Classes Of Glycansmentioning

confidence: 99%

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

et al. 2010

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Phloem Protein2 (PP2) is a component of the phloem protein bodies found in sieve elements. We describe here the lectin properties of the Arabidopsis (Arabidopsis thaliana) PP2-A1. Using a recombinant protein produced in Escherichia coli, we demonstrated binding to N-acetylglucosamine oligomers. Glycan array screening showed that PP2-A1 also bound to highmannose N-glycans and 9-acyl-N-acetylneuraminic sialic acid. Fluorescence spectroscopy-based titration experiments revealed that PP2-A1 had two classes of binding site for N,N#,N$-triacetylchitotriose, a low-affinity site and a high-affinity site, promoting the formation of protein dimers. A search for structural similarities revealed that PP2-A1 aligned with the Cbm4 and Cbm22-2 carbohydrate-binding modules, leading to the prediction of a b-strand structure for its conserved domain. We investigated whether PP2-A1 interacted with phloem sap glycoproteins by first characterizing abundant Arabidopsis phloem sap proteins by liquid chromatography-tandem mass spectrometry. Then we demonstrated that PP2-A1 bound to several phloem sap proteins and that this binding was not completely abolished by glycosidase treatment. As many plant lectins have insecticidal activity, we also assessed the effect of PP2-A1 on weight gain and survival in aphids. Unlike other mannosebinding lectins, when added to an artificial diet, recombinant PP2-A1 had no insecticidal properties against Acyrthosiphon pisum and Myzus persicae. However, at mid-range concentrations, the protein affected weight gain in insect nymphs. These results indicate the presence in PP2-A1 of several carbohydrate-binding sites, with potentially different functions in the trafficking of endogenous proteins or in interactions with phloem-feeding insects.

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Molecular basis of N-acetylglucosaminyltransferase I deficiency in Arabidopsis thaliana plants lacking complex N-glycans

Cited by 86 publications

References 33 publications

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Expression and glycoengineering of functionally active heteromultimeric IgM in plants

Improved Virus Neutralization by Plant-produced Anti-HIV Antibodies with a Homogeneous β1,4-Galactosylated N-Glycan Profile

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

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