Glycosyltransferases Involved in<i>N</i>–and<i>O</i>–Glycan Biosynthesis

Brockhausen, Inka; Schachter, Harry

doi:10.1002/9783527614738.ch5

Cited by 55 publications

(66 citation statements)

References 296 publications

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“…22, 34 Our results are in agreement with the observation made for other proteins, where an N-glycan with a bisecting GlcNAc was not the substrate of other glycosyltransferases such as GnT-II, -IV, or -V. 21,35 Notably, molecular modeling exhibits that bisected GlcNAc residues may be accommodated without the need for conformational changes in the CxMab-Fc glycan structure (data not shown). Thus, the suppression of further processing and elongation of N-glycans due to bisecting GlcNAc modification may be attributed to a generally improper environment for glycosyltransferases created by this structure.…”

Section: Discussionsupporting

confidence: 88%

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

Castilho

Gruber

Thader

et al. 2015

mAbs

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(2015) Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation, mAbs, 7:5, 863-870, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Keywords: core fucosylation, sialylation, Nicotiana benthamiana, bisected glycans, glycan modelling, IgG, cetuximab Abbreviations: 3-FucT, Zea maize core a1,3-fucosyltransferase;3-FucT GnTIV, human a1,3-mannosyl-b1,4-N-acetyL-glucosaminyltransferase fused to the CTS region of the Arabidopsis thaliana core a1,3-fucosyltransferase (FUT11); 6-FucT, Mus musculus core a1,6-fucosyltransferase; CH2, constant domain of an IgG heavy chain; CTS, cytoplasmic tail, transmembrane domain and stem region; CxMab cetuximab (Erbitux Ò ); Fab, fragment, antigen-binding; Fc, Fragment crystallizable region of immunoglobulin G; GlcNAc, N-acetylglucosamine; IgG1, Immunoglobulin G subclass 1; LC-ESI-MS, Liquid chromatography-electrospray ionisationmass spectrometry; mAb, monoclonal antibody; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; ST GalT, b1,4-galactosyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; 6-SiaT, a2,6-sialyltransferase;ST GnT-III, b1,4-mannosyl-b1,4-N-acetylglucosaminyltransferase fused to the CTS region of the rat a2,6-sialyltransferase; DXT/FT, Nicotiana benthamiana glycosylation mutants lacking plant specific core b1,2-xylose and a1,3-fucose residuesWe investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (a1,3-and a1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF 3 , and GnGnF 6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core a1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies.

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Section: Discussionsupporting

confidence: 88%

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

Castilho

Gruber

Thader

et al. 2015

mAbs

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“…Their presence can definitely not be dismissed as a random event. As an initial measure to appraise the role of modifications it can give the synthetic route to mature Nglycans a clear direction (Brockhausen and Schachter, 1997). As an illuminating example for this role, the relative timing of adding a bisecting N-acetylglucosaminyl (GlcNAc) moiety by N-acetylglucosaminyltransferase-III (GnT-III) commits N-glycan synthesis either to hybrid or to complex-type N-glycans (Brockhausen and Schachter, 1997).…”

Section: Introductionmentioning

confidence: 99%

“…As an initial measure to appraise the role of modifications it can give the synthetic route to mature Nglycans a clear direction (Brockhausen and Schachter, 1997). As an illuminating example for this role, the relative timing of adding a bisecting N-acetylglucosaminyl (GlcNAc) moiety by N-acetylglucosaminyltransferase-III (GnT-III) commits N-glycan synthesis either to hybrid or to complex-type N-glycans (Brockhausen and Schachter, 1997). Also, its insertion precludes forming branches by GnT-IV/V and the a1,6-fucosylation of the core by fucosyltransferase-VIII (FucT-VIII), the latter substitution mode establishing a second common modification of the core Figure 1 Structure of a biantennary complex-type N-glycan (nonasaccharide: Bi9) with the two common core substitutions, i.e., introduction of core-fucose (F) and bisecting GlcNAc (B) moieties, to yield the two monosubstituted decasaccharides BiB10 and BiF10 and the disubstituted undecasaccharide BiBF11.…”

Section: Introductionmentioning

confidence: 99%

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

André

Kožár²,

Kojima³

et al. 2009

BCHM

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Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals (sugar coding). Here, we test the hypothesis that the common N-glycan modifications by core fucosylation and introduction of the bisecting Nacetylglucosamine moiety have long-range effects with functional consequences. Molecular dynamics simulations indicate a shift in conformational equilibria between linear extension or backfolding of the glycan antennae upon substitution. We also present a new fingerprint-like mode of presentation for this multi-parameter system. In order to delineate definite structure-function relationships, we strategically combined chemoenzymatic synthesis with bioassaying cell binding and the distribution of radioiodinated neoglycoproteins in vivo. Of clinical relevance, tailoring the core region affects serum clearance markedly, e.g., prolonging circulation time for the neoglycoprotein presenting the N-glycan with both substitutions. a2,3-Sialylation is another means toward this end, similarly seen for type II branching in triantennary N-glycans. This discovery signifies that rational glycoengineering along the given lines is an attractive perspective to optimize pharmacokinetic behavior of glycosylated pharmaproteins. Of general importance for the concept of the sugar code, the presented results teach the fundamental lesson that N-glycan core substitutions convey distinct characteristics to the concerned oligosaccharide relevant for cis and trans biorecognition processes. These modifications are thus molecular switches.

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“…Depending on the cell type and its activation and differentiation status, the core structures can be further elongated or terminated by various glycosyltrans-ferases, resulting in a large number of O-glycans, of which some are depicted in Scheme 12. Many of these glycosyltransferases have been isolated and their genes have been cloned [87,88]. There are glycosyltransferases which specifically act on O-glycans (mainly those responsible for assembly of the core structures) and there are others with an activity restricted to N-glycans.…”

Section: Structure Of O-glycansmentioning

confidence: 99%

Occurrence and Significance

Wittmann

2001

Glycoscience: Chemistry and Chemical Biology I–III

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Glycosyltransferases Involved inN–andO–Glycan Biosynthesis

Cited by 55 publications

References 296 publications

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation

From structural to functional glycomics: core substitutions as molecular switches for shape and lectin affinity of N-glycans

Occurrence and Significance

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