A Biotin Capture Assay for Oligosaccharyltransferase

Srinivasan, Anjali; Coward, James K.

doi:10.1006/abio.2002.5699

Cited by 2 publications

(3 citation statements)

References 34 publications

(40 reference statements)

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“…345 Some early in vitro enzymatic studies from Coward, Imperiali, and several other laboratories have shown that OST is able to transfer truncated N-glycan core from the corresponding glycosyl-phosphate dolichol to synthetic peptides containing the consensus NXS/T sequence. 346–355 However, a practical application of the eukaryotic OST for in vitro glycoprotein synthesis has not been fulfilled. Major hurdles include the instability of the eukaryotic enzyme complex, the complexity of the membrane-bound multiple protein subunits that are required for catalytic activity of the STT3 subunit, and the difficulty to obtain the dolichol-associated glycolipids as the substrates.…”

Section: Direct Enzymatic Glycosylation Of Polypeptides and Proteinsmentioning

confidence: 99%

“…In the biosynthesis of eukaryotic N-glycoproteins, the oligosaccharyltransferase (OST) is the sole enzyme responsible for transferring a large N-glycan precursor (Glc 3 Man 9 GlcNAc 2 ) en bloc from a dolichol-phosphate glycolipid to a consensus sequon NXS/T on the nascent unfolded protein. − Eukaryotic OST is in fact a multisubunit protein complex consisting of at least 9 subunits, and all of them are transmembrane proteins . Some early in vitro enzymatic studies from Coward, Imperiali, and several other laboratories have shown that OST is able to transfer truncated N-glycan core from the corresponding glycosyl-phosphate dolichol to synthetic peptides containing the consensus NXS/T sequence. − However, a practical application of the eukaryotic OST for in vitro glycoprotein synthesis has not been fulfilled. Major hurdles include the instability of the eukaryotic enzyme complex, the complexity of the membrane-bound multiple protein subunits that are required for catalytic activity of the STT3 subunit, and the difficulty to obtain the dolichol-associated glycolipids as the substrates.…”

Section: Direct Enzymatic Glycosylation Of Polypeptides and Proteinsmentioning

confidence: 99%

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Chemoenzymatic Methods for the Synthesis of Glycoproteins

2018

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Glycosylation is one of the most prevalent posttranslational modifications that profoundly affects the structure and functions of proteins in a wide variety of biological recognition events. However, the structural complexity and heterogeneity of glycoproteins, usually resulting from the variations of glycan components and/or the sites of glycosylation, often complicates detailed structure-function relationship studies and hampers the therapeutic applications of glycoproteins. To address these challenges, various chemical and biological strategies have been developed for producing glycan-defined homogeneous glycoproteins. This review highlights recent advances in the development of chemoenzymatic methods for synthesizing homogeneous glycoproteins, including the generation of various glycosynthases for synthetic purposes, endoglycosidase-catalyzed glycoprotein synthesis and glycan remodeling, and direct enzymatic glycosylation of polypeptides and proteins. The scope, limitation, and future directions of each method are discussed.

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Section: Direct Enzymatic Glycosylation Of Polypeptides and Proteinsmentioning

confidence: 99%

Section: Direct Enzymatic Glycosylation Of Polypeptides and Proteinsmentioning

confidence: 99%

Chemoenzymatic Methods for the Synthesis of Glycoproteins

2018

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“…Early work from the Coward and Imperiali groups devised in vitro glycosylation assays to gain mechanistic understanding of the substrate specificity and activity of the yeast OST (Xu and Coward, 1997 ; Tai and Imperiali, 2001 ). Many of these studies were done using crude extract-containing detergent-solubilized OSTs from yeast microsomes to catalyze glycan transfer from dolichyl lipid-linked oligosaccharides (LLOs) onto peptide acceptors containing a glycosylation motif (Sharma et al, 1981 ; Srinivasan and Coward, 2002 ). Due to the inherent structural complexity, the preparation of membrane-bound OST complexes has proven difficult, often leading to inactive or unstable enzymes.…”

Section: Cell-based and Cell-free Biosynthesis Of Structurally-definementioning

confidence: 99%

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

Jaroentomeechai

Taw

et al. 2020

Front. Chem.

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Glycans and glycosylated biomolecules are directly involved in almost every biological process as well as the etiology of most major diseases. Hence, glycoscience knowledge is essential to efforts aimed at addressing fundamental challenges in understanding and improving human health, protecting the environment and enhancing energy security, and developing renewable and sustainable resources that can serve as the source of next-generation materials. While much progress has been made, there remains an urgent need for new tools that can overexpress structurally uniform glycans and glycoconjugates in the quantities needed for characterization and that can be used to mechanistically dissect the enzymatic reactions and multi-enzyme assembly lines that promote their construction. To address this technology gap, cell-free synthetic glycobiology has emerged as a simplified and highly modular framework to investigate, prototype, and engineer pathways for glycan biosynthesis and biomolecule glycosylation outside the confines of living cells. From nucleotide sugars to complex glycoproteins, we summarize here recent efforts that harness the power of cell-free approaches to design, build, test, and utilize glyco-enzyme reaction networks that produce desired glycomolecules in a predictable and controllable manner. We also highlight novel cell-free methods for shedding light on poorly understood aspects of diverse glycosylation processes and engineering these processes toward desired outcomes. Taken together, cell-free synthetic glycobiology represents a promising set of tools and techniques for accelerating basic glycoscience research (e.g., deciphering the “glycan code”) and its application (e.g., biomanufacturing high-value glycomolecules on demand).

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A Biotin Capture Assay for Oligosaccharyltransferase

Cited by 2 publications

References 34 publications

Chemoenzymatic Methods for the Synthesis of Glycoproteins

Chemoenzymatic Methods for the Synthesis of Glycoproteins

Cell-Free Synthetic Glycobiology: Designing and Engineering Glycomolecules Outside of Living Cells

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