Analysis of N- and O-Glycosylation of Plant Proteins

Fitchette-Lainé, Anne-Catherine; Denmat, Lise-Anne; Lerouge, Patrice; Faye, Loı̈c

doi:10.1007/978-1-60327-260-5_19

Cited by 13 publications

(6 citation statements)

References 34 publications

(12 reference statements)

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“…Immunodetections were performed using specific core ␤(1,2)-xylose and core ␣(1,3)-fucose antibodies as reported previously (32). Oxidation of the glycan moiety of glycoproteins was carried out on the blots using sodium periodate according to (33). Immunodetection with anti-V5 antibodies was performed following the instructions of the supplier for dilution of the antibody and revelation (ECL kit).…”

Section: Methodsmentioning

confidence: 99%

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

Baïet

Burel²,

Saint‐Jean³

et al. 2011

Journal of Biological Chemistry

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127

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N-Glycosylation, a major co-and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the Nglycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc 3 Man 9 GlcNAc 2 -PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the ␣-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

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Section: Methodsmentioning

confidence: 99%

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

Baïet

Burel²,

Saint‐Jean³

et al. 2011

Journal of Biological Chemistry

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127

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“…Affinodetection of high-mannose-type N-glycans was carried out using the concanavalin A/peroxidase method (Faye and Chrispeels, 1985). Affinodetection by RCA was carried out as reported in Fitchette-Lainé et al (1998).…”

Section: Sds-page and Immunoblotting Experimentsmentioning

confidence: 99%

N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants

et al. 1999

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Since plants are emerging as an important system for the expression of recombinant glycoproteins, especially those intended for therapeutic purposes, it is important to scrutinize to what extent glycans harbored by mammalian glycoproteins produced in transgenic plants differ from their natural counterpart. We report here the first detailed analysis of the glycosylation of a functional mammalian glycoprotein expressed in a transgenic plant. The structures of the N-linked glycans attached to the heavy chains of the monoclonal antibody Guy's 13 produced in transgenic tobacco plants (plantibody Guy's 13) were identified and compared to those found in the corresponding IgG1 of murine origin. Both N-glycosylation sites located on the heavy chain of the plantibody Guy's 13 are N-glycosylated as in mouse. However, the number of Guy's 13 glycoforms is higher in the plant than in the mammalian expression system. Despite the high structural diversity of the plantibody N-glycans, glycosylation appears to be sufficient for the production of a soluble and biologically active IgG in the plant system. In addition to high-mannose-type N-glycans, 60% of the oligosaccharides N-linked to the plantibody have beta(1, 2)-xylose and alpha(1, 3)-fucose residues linked to the core Man3GlcNAc2. These plant-specific oligosaccharide structures are not a limitation to the use of plantibody Guy's 13 for topical immunotherapy. However, their immunogenicity may raise concerns for systemic applications of plantibodies in human.

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“…The anti-M3GN2 antibody (clone 100-4g11-A) recognized fractions F11-0, F11-4, F11-6, and F13-6. In addition to the MAbs generated from infected mice, we also screened with a commercial anti-HRP antibody produced in rabbits (P7899), which is directed against core ␤-xylose and core ␣3-fucose determinants (43,44). The specificity of P7899 for core ␤-xylose and core ␣3-fucose determinants also was confirmed in our hands using defined glycan microarrays.…”

Section: Preparation Of N-glycans From Schistosoma Mansoni Eggsmentioning

confidence: 83%

“…An anti-LDN (IgG, clone Y1H5), an anti-LDNF (IgG, clone L6B8), an anti-Le x (IgM, clone 5F1), two anti-LDN-dF (IgM, clone 290-2D9-A; IgM, clone 290-4A8), and an anti-Man 3 GlcNAc 2 (M3GN2; IgM, clone 100-4G11-A) MAb, as well as an anti-FLNDF antibody (IgG, clone F2D2), were developed as MAbs by the production of hybridomas from spleens of mice that had been infected with S. mansoni by using methods described previously (16,(39)(40)(41)(42). The anti-horseradish peroxidase (HRP) antibody (anti-peroxidase, polyclonal rabbit IgG; P7899) was purchased from Sigma-Aldrich and used to detect core ␣3 fucose and core xylose (CF/CX) determinants (43,44). The secondary antibody conjugates goat anti-human, anti-mouse, anti-rabbit (Alexa-488 anti-human IgM, Alexa-555 anti-human IgG, Alexa-488 anti-mouse IgM, Alexa-555 anti-mouse IgG, Alexa-633 anti-mouse IgG, and Alexa-488 anti-rabbit IgG), and Cy5-streptavidin were from Invitrogen (Carlsbad, CA).…”

Section: Methodsmentioning

confidence: 99%

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

et al. 2016

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f Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fuc␣3GalNAc␤4(Fuc␣3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core ␤-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. Schistosomiasis is a major health problem in tropical and subtropical areas where it is endemic, with more than 200 million people actively infected and 800 million at risk of contracting the disease (1-3). Current treatment for disease is limited to the drug praziquantel (4), but cases of drug resistance have been reported (5). Decades of research on schistosomiasis vaccines have yielded only two candidates for clinical trials, and no encouraging results have been published yet (6-9). Thus, there is an urgent need to develop more sensitive diagnostic methods and to identify new vaccine candidates.Recent studies have shown that a major part of the host immune response to infection is directed against carbohydrate (glycan) antigens in glycoproteins and glycolipids (10-17). A wide variety of unusual antigenic determinants include glycans containing the LDN, fucosylated LDN sequences (LDNF, LDN-dF, FLDN, and FLDNF), Lewis X (Le x ), poly-Le x , core ␣3 fucose, and core ␤2 xylose structures (Fig. 1) (11, 14, 17), many of which are expressed by all developmental stages of schistosomes (18). Interestingly, monoclonal antibodies (MAbs) specific to these glycans recognize these antigens on the surface of 3-h-old schistosomula, and some anti-glycan antibodies can mediate killing in vitro in a complement-dependent fashion (18-21). Schistosoma mansoniinfected rhesus monkeys, which are known to self-cure after infection, have IgG to many glycan antigens, including Le x , LDN, LDNF, core fucose, and core xylose determinants, and their sera are effective in complement-mediated cytolysis of cells expressing Le x as well as schistosomulum larvae in ...

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Analysis of N- and O-Glycosylation of Plant Proteins

Cited by 13 publications

References 34 publications

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants

Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray

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