Galactosylation and sialylation of terminal glycan residues of human immunoglobulin G using bacterial glycosyltransferases with in situ regeneration of sugar-nucleotides

Chung, Seung-Wook; Joo, Hwang-Soo; Jang, Kyoung‐Soon; Lee, Hwajin; Lee, Sun‐Gu; Kim, Byung‐Gee

doi:10.1016/j.enzmictec.2005.09.007

Cited by 12 publications

(6 citation statements)

References 19 publications

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“…Mammalian tissue culture cells such as CHO cells or murine cells are typically used in the biopharmaceutical industry to 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0 1 4 0 0 1 6 0 0 1 8 0 0 produce antibodies with appropriately processed carbohydrates. Recent researches have also demonstrated that antibodies with human like glycan structures can be produced in glycoengineered Escherichia coli (Chung et al, 2006) or yeast (Li et al, 2006) cell lines. Cumulative evidence indicates the correlation of the presence of glycan on an antibody to certain effector functions of the molecule (Jefferis, 2005).…”

Section: Glycan Structure and Profilingmentioning

confidence: 99%

Mass spectrometry for structural characterization of therapeutic antibodies

Zhang

Pan

Chen

2008

Mass Spectrometry Reviews

289

230

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Antibodies, also known as immunoglobulins, have emerged as one of the most promising classes of therapeutics in the biopharmaceutical industry. The need for complete characterization of the quality attributes of these molecules requires sophisticated techniques. Mass spectrometry (MS) has become an essential analytical tool for the structural characterization of therapeutic antibodies, due to its superior resolution over other analytical techniques. It has been widely used in virtually all phases of antibody development. Structural features determined by MS include amino acid sequence, disulfide linkages, carbohydrate structure and profile, and many different post-translational, in-process, and in-storage modifications. In this review, we will discuss various MS-based techniques for the structural characterization of monoclonal antibodies. These techniques are categorized as mass determination of intact antibodies, and as middle-up, bottom-up, top-down, and middle-down structural characterizations. Each of these techniques has its advantages and disadvantages in terms of structural resolution, sequence coverage, sample consumption, and effort required for analyses. The role of MS in glycan structural characterization and profiling will also be discussed.

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Section: Glycan Structure and Profilingmentioning

confidence: 99%

Mass spectrometry for structural characterization of therapeutic antibodies

Zhang

Pan

Chen

2008

Mass Spectrometry Reviews

289

230

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“…An alternative approach is the in vitro glycoengineering of proteins by modifying the glycostructure via enzymatic reactions with purified glycosyltransferases and nucleotide sugars (Thomann et al, ). Case studies have shown very promising results in terms of increasing the level of galactosylation and sialylation on IgG (Chung et al, ; Raju, Briggs, Chamow, Winkler, & Jones, ; Thomann et al, ). To satisfy the high demand of nucleotide sugars UDP‐galactose and CMP‐sialic acid for in vitro glycoengineering, Raju et al () have designed an in vitro enzymatic nucleotide sugar regeneration cascade for these two co‐substrates and demonstrated galactosylation and sialylation of tumor necrosis factor receptor IgGs.…”

Section: Introductionmentioning

confidence: 99%

“…To avoid product purification after one‐pot multi‐enzyme cascade synthesis of nucleotide sugars, the in vitro coupling of an enzyme cascade regenerating nucleotide sugars to the glycosyltransferase reactions is advantageous. For example, Chung et al () published 2006 the in vitro galactosylation and sialylation of human IgG in combination with the regenerating enzymes, all expressed in E. coli and used as raw extracts. Wang et al () have designed an in vitro enzyme cascade with pyruvate kinase, inorganic pyrophosphatase, and mannose‐1‐phosphate‐guanyltransferase to regenerate GDP‐man from mannose‐1‐phosphate.…”

Section: Introductionmentioning

confidence: 99%

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Rexer

Schildbach

Klapproth

et al. 2017

Biotech & Bioengineering

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Glycosylation of proteins is a key function of the biosynthetic‐secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell‐cell adhesion, blood‐group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein‐based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose‐1‐phosphate‐guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1‐domain polyphosphate kinase 2 (1D‐Ppk2) expressed in E. coli for the cell‐free production and regeneration of GDP‐mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP‐mannose is produced at various conditions, that is pH 7–8, temperature 25–35°C and co‐factor concentrations of 5–20 mM MgCl2. The maximum reaction rate of GDP‐mannose achieved was 2.7 μM/min at 30°C and 10 mM MgCl2 producing 566 nmol GDP‐mannose after a reaction time of 240 min. With respect to the initial GDP concentration (0.8 mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane‐deleted Alg1 (ALG1ΔTM), the first mannosyltransferase in the ER‐associated lipid‐linked oligosaccharide (LLO) assembly. Thereby, in a one‐pot reaction, phytanyl‐PP‐(GlcNAc)2‐Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl‐PP‐(GlcNAc)2‐Man1 can serve as a substrate for the synthesis of LLO for the cell‐free in vitro glycosylation of proteins. A high‐performance anion exchange chromatography method with UV and conductivity detection (HPAEC‐UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP‐mannose regenerating cascade and can further be used to study coupling of the GDP‐mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell‐free production of LLOs as precursors for in vitro glycoengineering of proteins.

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“…For the regeneration of UDP-Gal in the GT reactions, extracts of recombinant E. coli expressing all enzymes were used. We previously constructed the UDPGal regeneration system that included regeneration of UMP kinase, acetate kinase, galactokinase, UDP-Glc pyrophosphorylase, and Gal-1-P uridyltransferase [30]. These five genes were inserted into one plasmid such that all proteins were expressed in situ (unpublished data).…”

Section: Overexpression Of Galgts In Bk=åçäá and Production Of α-Gal mentioning

confidence: 99%

“…To overcome these problems, several groups have developed efficient sugar-nucleotide synthetic systems or regeneration systems [18,19,[21][22][23][24] and screened new GTs [25][26][27][28][29]. In our previous study, we developed an in situ sugar-nucleotide regeneration system and employed it to produce oligosaccharides and to re-construct the glycans of human IgG in vitro [30][31][32].…”

Section: Introductionmentioning

confidence: 98%

Selective removal of anti-α-Gal antibodies from human serum by using synthetic α-Gal epitope on a core-shell type resin

Jang

Chung

Kim

et al. 2008

Biotechnol Bioproc E

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A novel α-Gal resin was chemo-enzymatically synthesized for the efficient adsorption of anti-α-Gal antibodies in human serum for xenotransplantation. To covalently conjugate a hexanoate linker with lactose and k-acetylglucosamine, both acceptor sugars were acetylated and brominated. Then, α-and β-galactoses were sequentially added to the linker-containing saccharides at their non-reducing ends by using recombinant α-(1,3)-and β-(1,4)-galactosyltransferases from bK=Åçäá. Finally, the synthesized α-Gal derivatives were immobilized on eáCore, a core-shell type resin, that was functionalized with amino groups on the shell region, as a packing material on-column. Using this method we were able to demonstrate that the α-Gal eáCore resin had a reduced level of non-specific protein adsorption compared with the commercially available polystyrene supports, TentaGel, and agarose-based supports, when Lectin BS-I was used as the model binding protein. Furthermore, the α-Gal eáCore resin was more efficient at eliminating anti-α-Gal IgGs from the total human IgGs through immunoadsorption than the other two α-Gal resins, α-Gal TentaGel and α-Gal agarose. The α-Gal eáCore resin developed in this study can be utilized in a wide range of applications including Éñ=îáîç immunoadsorption and as a quantitative assay of anti-Gal antibody in human sera. © KSBB hÉóïçêÇëW=αJd~ä=ÉéáíçéÉI=ÅçêÉJëÜÉää=íóéÉ=êÉëáåI=Hi`çêÉI=~åíáJαJd~ä=~åíáÄçÇóI=ÜóéÉê~ÅìíÉ=êÉàÉÅíáçåI=ñÉåçíê~åëéä~åí~íáçåI= = áããìåç~Çëçêéíáçå= = = = =

show abstract

Galactosylation and sialylation of terminal glycan residues of human immunoglobulin G using bacterial glycosyltransferases with in situ regeneration of sugar-nucleotides

Cited by 12 publications

References 19 publications

Mass spectrometry for structural characterization of therapeutic antibodies

Mass spectrometry for structural characterization of therapeutic antibodies

One pot synthesis of GDP‐mannose by a multi‐enzyme cascade for enzymatic assembly of lipid‐linked oligosaccharides

Selective removal of anti-α-Gal antibodies from human serum by using synthetic α-Gal epitope on a core-shell type resin

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