Analysis of the N-glycosylation of recombinant glycoproteins produced in transgenic plants

Bardor, Muriel; Faye, Loı̈c; Lerouge, Patrice

doi:10.1016/s1360-1385(99)01461-2

Cited by 52 publications

(34 citation statements)

References 36 publications

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“…Whether the lectin-like domain glycans are involved in protein sorting requires further experimentation. Studies, mainly on animal cells and yeast (Helenius and Aebi, 2001) but also on plant-expressed proteins (Bardor et al, 1999), suggest that N-linked glycans may play a variety of roles including protein folding, oligomerization, quality control, sorting, and transport.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

et al. 2003

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To study the role of LecRK (lectin-like receptor kinase) genes in the legume-rhizobia symbiosis, we have characterized the four Medicago truncatula Gaernt. LecRK genes that are most highly expressed in roots. Three of these genes, MtLecRK7;1, MtLecRK7;2, and MtLecRK7;3, encode proteins most closely related to the Class A LecRKs of Arabidopsis, whereas the protein encoded by the fourth gene, MtLecRK1;1, is most similar to a Class B Arabidopsis LecRK. All four genes show a strongly enhanced root expression, and detailed studies on MtLecRK1;1 and MtLecRK7;2 revealed that the levels of their mRNAs are increased by nitrogen starvation and transiently repressed after either rhizobial inoculation or addition of lipochitooligosaccharidic Nod factors. Studies of the MtLecRK1;1 and MtLecRK7;2 proteins, using green fluorescent protein fusions in transgenic M. truncatula roots, revealed that they are located in the plasma membrane and that their central transmembrane-spanning helix is required for correct sorting. Moreover, their lectin-like domains appear to be highly glycosylated. Of the four proteins, only MtLecRK1;1 shows a high conservation of key residues implicated in monosaccharide binding, and molecular modeling revealed that this protein may be capable of interacting with Nod factors. However, no increase in Nod factor binding was found in roots overexpressing a fusion in which the kinase domain of this protein had been replaced with green fluorescent protein. Roots expressing this fusion protein however showed an increase in nodule number, suggesting that expression of MtLecRK1;1 influences nodulation. The potential role of LecRKs in the legumerhizobia symbiosis is discussed.The lectin-like receptor kinases (LecRKs) are a class of proteins originally described from Arabidopsis (Hervé et al., 1996). They have a structure similar to other plant receptor-like kinases (RLKs; Shiu and Bleecker, 2001;Cock et al., 2002) with an N-terminal targeting signal, a presumably extracellular domain, a single transmembrane (TM)-spanning helix, and a cytosolic kinase domain. The Arabidopsis genome contains over 610 RLKs that have been shown to be monophylogenetic with respect to the kinase domain and most closely related to the fruitfly (Drosophila melanogaster) Pelle and related animal cytoplasmic kinases (Shiu and Bleecker, 2001). In cases in which they have been tested, these kinases have been shown to have specificity for phosphorylation on Ser/Thr residues (Shiu and Bleecker, 2001).The plant RLKs can be grouped into more than 21 structural classes based on their extracellular domains (Shiu and Bleecker, 2001). The LecRKs represent one such class in which the extracellular domain is related to legume lectins. The Arabidopsis genome contains at least 42 LecRK sequences that have been grouped into three classes (A-C) plus an additional nine sequences of related soluble lectins (Barre et al., 2002). Searches in plant databases reveal that LecRK genes are widespread in higher plants, but apart from Arabidopsis (Hervé et al., 1996,...

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Section: Discussionmentioning

confidence: 99%

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

et al. 2003

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“…As eukaryotes, plants offer the advantage of all forms of posttranslational modifications, including glycosylation, which, however, differs in details from that in mammals (Bardor et al, 1999;Kusnadi et al, 1997). These differences can contribute to the small differences in kinetic parameters between the plant-derived enzyme and its mammalian counterparts.…”

Section: Discussionmentioning

confidence: 99%

Expression of recombinant human acetylcholinesterase in transgenic tomato plants

Mor

Sternfeld

Soreq

et al. 2001

Biotech & Bioengineering

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Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).

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“…Within the plant Golgi, an α(1 → 2)-mannosidase (α-Man I) can trim the core glycan down to structures as small as Man 5 GlcNAc 2 prior to construction of complex-and paucimannosidic-type glycans. Key features of these glycans include α(1 → 3)-fucosylation of the Asn-linked core GlcNAc, core xylosidation, and decoration of the glycan antennae with galactose, N-acetylglucosamine and fucose [57,60].…”

Section: Discussionmentioning

confidence: 99%

“…Based upon the known plant core glycan structure and the mode of action of α(1 → 2)-mannosidase in the Golgi [57], a likely structure for the cauliflower XET glycan is Man(α1-6)[Man(α1-3)]Man(α1-6)[Man(α1-3)]Man(β1-4)GlcNAc(β1-4)GlcNAc (IUPAC condensed notation) bearing an additional α(1 → 2)-linked Man on one of the three terminal Man residues [60]. Core fucosylation of the glycan was not indicated due to the sensitivity of the glycoprotein to PNGase F, which does not cleave complex-type glycopeptides bearing Fuc on the first core GlcNAc.…”

Section: Discussionmentioning

confidence: 99%

N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A

HENRIKSSON¹,

DENMAN²,

CAMPUZANO³

et al. 2004

Biochemical Journal

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The gene encoding a XET (xyloglucan endotransglycosylase) from cauliflower (Brassica oleracea var. botrytis) florets has been cloned and sequenced. Sequence analysis indicated a high degree of similarity to other XET enzymes belonging to glycosyl hydrolase family 16 (GH16). In addition to the conserved GH16 catalytic sequence motif EIDFE, there exists one potential Nlinked glycosylation site, which is also highly conserved in XET enzymes from this family. Purification of the corresponding protein from extracts of cauliflower florets allowed the fractionation of a single, pure glycoform, which was analysed by MS techniques. Accurate protein mass determination following the enzymic deglycosylation of this glycoform indicated the presence of a high-mannose-type glycan of the general structure GlcNAc 2 Man 6 . LC/MS and MS/MS (tandem MS) analysis provided supporting evidence for this structure and confirmed that the glycosylation site (underlined) was situated close to the predicted catalytic residues in the conserved sequence YLSSTNNEHD-EIDFEFLGNRTGQPVILQTNVFTGGK. Heterologous expression in Pichia pastoris produced a range of protein glycoforms, which were, on average, more highly mannosylated than the purified native enzyme. This difference in glycosylation did not influence the apparent enzymic activity of the enzyme significantly. However, the removal of high-mannose glycosylation in recombinant cauliflower XET by endoglycosidase H, quantified by electrospray-ionization MS, caused a 40 % decrease in the transglycosylation activity of the enzyme. No hydrolytic activity was detected in native or heterologously expressed BobXET16A, even when almost completely deglycosylated.

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Analysis of the N-glycosylation of recombinant glycoproteins produced in transgenic plants

Cited by 52 publications

References 36 publications

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

Characterization of Four Lectin-Like Receptor Kinases Expressed in Roots of Medicago truncatula. Structure, Location, Regulation of Expression, and Potential Role in the Symbiosis with Sinorhizobium meliloti

Expression of recombinant human acetylcholinesterase in transgenic tomato plants

N-linked glycosylation of native and recombinant cauliflower xyloglucan endotransglycosylase 16A

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