Emerging Principles for the Therapeutic Exploitation of Glycosylation

Dalziel, Martin; Crispin, Max; Scanlan, Christopher N.; Zitzmann, Nicole; Dwek, Raymond A.

doi:10.1126/science.1235681

Cited by 406 publications

(313 citation statements)

References 98 publications

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“…2A), as expected for the removal of all the three highmannose N-glycans. Unexpectedly, MALDI-TOF MS analysis of the N-glycans released by PNGase F showed two major molecular ions: one with an m/z of 1257.3 that matches N-glycan Man 5 (Fig. 2B).…”

Section: Characterization Of Epo Produced From the Hek293smentioning

confidence: 99%

“…1). However, the discovery of core-fucosylated Man 5 GlcNAc 2 glycan from natural proteins of a GnT I knockout CHO cell lines by Lin et al (13) provided the first evidence suggesting the presence of a GnT I-independent pathway for core fucosylation of Man 5 GlcNAc 2 and paucimannose glycans in mammalian systems. Later on, Crispin et al (14) reported the detection of minor fractions (ϳ5%) of core-fucosylated, highmannose glycoforms in recombinant glycoproteins produced in GnT I knockout CHO and human HEK293S cell lines, confirming the presence of a GnT I-independent fucosylation pathway.…”

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confidence: 99%

“…For the recombinant production of therapeutic glycoproteins such as monoclonal antibodies, it is particularly important to control glycosylation to achieve the optimal therapeutic efficacy (3)(4)(5). The biosynthesis of asparagine-linked (i.e.…”

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confidence: 99%

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Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Yang

Wang

2016

Journal of Biological Chemistry

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Understanding the biosynthetic pathway of protein glycosylation in various expression cell lines is important for controlling and modulating the glycosylation profiles of recombinant glycoproteins. We found that expression of erythropoietin (EPO) in a HEK293S N-acetylglucosaminyltransferase I (GnT I) ؊/؊ cell line resulted in production of the Man 5 GlcNAc 2 glycoforms, in which more than 50% were core-fucosylated, implicating a clear GnT I-independent core fucosylation pathway. Expression of GM-CSF and the ectodomain of Fc␥IIIA receptor led to ϳ30% and 3% core fucosylation, suggesting that the level of core fucosylation also depends on the nature of the recombinant proteins.To elucidate the GnT I-independent core fucosylation pathway, we generated a stable HEK293S GnT I ؊/؊ cell line with either knockdown or overexpression of FUT8 by a highly efficient lentivirus-mediated gene transfer approach. We found that the EPO produced from the FUT8 knockdown cell line was the pure Man 5 GlcNAc 2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man 5 GlcNAc 2 Fuc). These results provide direct evidence that FUT8, the mammalian ␣1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man 5 GlcNAc 2 glycoform of EPO in the FUT8-overexpressed HEK293S GnT I ؊/؊ cell line represents the first example of production of fully core-fucosylated high-mannose glycoforms.

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Section: Characterization Of Epo Produced From the Hek293smentioning

confidence: 99%

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confidence: 99%

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Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Yang

Wang

2016

Journal of Biological Chemistry

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“…In addition to the complex type N-glycan, the specificity of Endo-S2 was further tested with high-mannose type (HM) Man 9 GlcNAc oxazoline (7) and sialo hybrid type (Hyb) Neu5AcGalGlcNAcMan 5 GlcNAc oxazoline (5) as donor substrate in the transglycosylation reactions. The reactions led to the formation of the corresponding homogeneous glycoforms, (8) and (6), respectively (Fig.…”

Section: Comparative Study On the Hydrolysis And Transglycosylationmentioning

confidence: 99%

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Tong

Yang

et al. 2016

Journal of Biological Chemistry

109

139

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Glycosylation can exert a profound impact on the structures and biological functions of antibodies. Glycosylation remodeling using the endoglycosidase-catalyzed deglycosylation and transglycosylation approach is emerging as a promising platform to produce homogeneous glycoforms of antibodies, but the broad application of this method will require the availability of highly efficient glycosynthase mutants. We describe in this paper a systematic site-directed mutagenesis of an endoglycosidase from Streptococcus pyogenes of serotype M49 (Endo-S2) and the evaluation of the resulting mutants for their hydrolysis and transglycosylation activities. We found that mutations at the Asp-184 residue gave mutants that demonstrated significantly different properties, some possessed potent transglycosylation activity with diminished hydrolysis activity but others did not, which would be otherwise difficult to predict without the comparative study. In contrast to the previously reported Endo-S mutants that are limited to action on complex type N-glycans, the Endo-S2 glycosynthases described here, including D184M and D184Q, were found to have remarkably relaxed substrate specificity and were capable of transferring three major types (complex, high-mannose, and hybrid type) of N-glycans for antibody glycosylation remodeling. In addition, the Endo-S2 glycosynthase mutants were found to be much more active in general than the Endo-S mutants for transglycosylation. The usefulness of these Endo-S2 glycosynthase mutants was exemplified by an efficient glycosylation remodeling of two therapeutic monoclonal antibodies, rituximab and trastuzumab (Herceptin).

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“…Recent advances in material chemistry, especially with introduction of various nanomaterials, together with launching of novel transducing protocols and biorecognition elements are behind a huge development in ultrasensitive and multiplexed analysis of a wide range of disease biomarkers [18][19][20][21][22][23]. It is well-known that presence of labels can negatively affect binding [24,25] and thus label-free methods of analysis are preferential option for diagnostic purposes.…”

Section: Introductionmentioning

confidence: 99%

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

Kluková

Bertók

Petrikova

et al. 2015

Analytica Chimica Acta

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Systemic sclerosis (SSc) is an autoimmune disease seriously affecting patient´s quality of life. The heterogeneity of the disease also means that identification and subsequent validation of biomarkers of the disease is quite challenging. A fully validated single biomarker for diagnosis, prognosis, disease activity and assessment of response to therapy is not yet available. The main aim of this study was to apply an alternative assay protocol to the immunoassay-based analysis of this disease by employment of sialic acid recognizing lectin Sambucus nigra agglutinin (SNA) to glycoprofile serum samples. To our best knowledge this is the first study describing direct lectin-based glycoprofiling of serum SSc samples. Three different analytical methods for glycoprofiling of serum samples relying on application of lectins are compared here from a bioanalytical point of view including traditional ELISA-like lectin-based method (ELLA), novel fluorescent lectin microarrays and ultrasensitive impedimetric lectin biosensors. Results obtained by all three bioanalytical methods consistently showed differences in the level of sialic acid present on glycoproteins, when serum from healthy people was compared to the one from patients having SSc. Thus, analysis of sialic acid content in human serum could be of a diagnostic value for future detection of SSc, but further work is needed to enhance selectivity of assays for example by glycoprofiling of a fraction of human serum enriched in antibodies for individual diagnostics.

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Emerging Principles for the Therapeutic Exploitation of Glycosylation

Cited by 406 publications

References 98 publications

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

Glycoprofiling as a novel tool in serological assays of systemic sclerosis: A comparative study with three bioanalytical methods

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