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An automated platform for the enzyme-mediated assembly of complex oligosaccharides.

ABSTRACT: An automated platform that can synthesize a wide range of complex carbohydrates will greatly increase their accessibility and should facilitate progress in glycoscience. Here we report a fully automated process for enzyme-mediated oligosaccharide synthesis that can give easy access to different classes of complex glycans including poly-N-acetyllactosamine derivatives, human milk oligosaccharides, gangliosides and N-glycans. Our automated platform uses a catch and release approach in which glycosyltransferase-catalysed reactions are performed in solution and product purification is accomplished by solid phase extraction. We developed a sulfonate tag that can easily be installed and enables highly efficient solid phase extraction and product release using a single set of washing conditions, regardless of the complexity of the glycan. Using this custom-built synthesizer, as many as 15 reaction cycles can be performed in an automated fashion without a need for lyophilization or buffer exchange steps.

SUBMITTER: Li T

PROVIDER: S-EPMC6399472 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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An automated platform for the enzyme-mediated assembly of complex oligosaccharides.

Li Tiehai T Liu Lin L Wei Na N Yang Jeong-Yeh JY Chapla Digantkumar G DG Moremen Kelley W KW Boons Geert-Jan GJ

Nature chemistry 20190221 3

An automated platform that can synthesize a wide range of complex carbohydrates will greatly increase their accessibility and should facilitate progress in glycoscience. Here we report a fully automated process for enzyme-mediated oligosaccharide synthesis that can give easy access to different classes of complex glycans including poly-N-acetyllactosamine derivatives, human milk oligosaccharides, gangliosides and N-glycans. Our automated platform uses a catch and release approach in which glycos ...[more]

PMID: 30792508

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Project description: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??M/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 (ALG1?TM), 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.

Dataset Information

An automated platform for the enzyme-mediated assembly of complex oligosaccharides.

Publications

An automated platform for the enzyme-mediated assembly of complex oligosaccharides.

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