The proposed research focuses on the development of efficient and widely applicable chemoenzymatic methods for synthesizing N-glycopeptides and N-glycoproteins of biomedical significance. N-linked glycosylation is a ubiquitous posttranslational modification of proteins in eukaryotes. Glycoproteins play important roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. The covalently linked oligosaccharides can profoundly affect proteins'structure, function, and their serum half-life. However, homogeneous glycoproteins with structural defined oligosaccharides are difficult to obtain from natural or recombinant sources, since they are typically produced as a mixture of heterogeneous glycoforms. To obtain homogeneous materials for structural/biological studies and for biomedical applications, we propose to systematically explore the potential of endo-?-N-acetylglucosaminidases (ENGases), a special class of endoglycosidases, for constructing N-glycopeptides and N-glycoproteins. The biggest advantage is that some ENGases are able to transfer an intact oligosaccharide to a GlcNAc-containing peptide or protein in a single step without the need of any protecting groups, thus providing a highly convergent route to large glycopeptides and glycoproteins. But the method has hitherto suffered with low transglycosylation yield, the risk of product hydrolysis, and the limitation to the use of only natural N-glycans as donor substrates that themselves are difficult to prepare. Our preliminary studies have shown that the use of sugar oxazolines (the transition state mimics) as donor substrates not only expanded the substrate availability, but also resulted in a high-yield transglycosylation to form large glycopeptides and homogeneous glycoproteins. This proposal intends to systematically explore the scope and limitation of the novel methodology through pursuing five specific aims: 1) synthesis and examination of a range of oligosaccharide oxazolines as donor substrates for the enzymatic transglycosylation;2) evaluation of novel acceptor substrates for synthesizing complex fucose-containing N-glycopeptides and for detecting O-GlcNAc glycosylation;3) total synthesis of large HIV-1 gp120 fragments for structural and functional studies;4) exploitation of the methodology for synthesizing natural and tailor-made homogeneous glycoproteins;and 5) synthesis and functional studies of homogeneous glycoforms of human antibody IgG-Fc. The knowledge gained from the proposed research will eventually facilitate the development of glycoprotein-based therapeutics.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Synthetic and Biological Chemistry A Study Section (SBCA)
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Marino, Pamela
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University of Maryland Baltimore
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Yang, Qiang; An, Yanming; Zhu, Shilei et al. (2017) Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation. ACS Chem Biol 12:1665-1673
Li, Chao; Zhu, Shilei; Ma, Christopher et al. (2017) Designer ?1,6-Fucosidase Mutants Enable Direct Core Fucosylation of Intact N-Glycopeptides and N-Glycoproteins. J Am Chem Soc 139:15074-15087
Yang, Qiang; Wang, Lai-Xi (2017) Chemoenzymatic Glycan Remodeling of Natural and Recombinant Glycoproteins. Methods Enzymol 597:265-281
Tong, Xin; Li, Tiezheng; Orwenyo, Jared et al. (2017) One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes. Bioorg Med Chem :
Yang, Qiang; Zhang, Roushu; Cai, Hui et al. (2017) Revisiting the substrate specificity of mammalian ?1,6-fucosyltransferase reveals that it catalyzes core fucosylation of N-glycans lacking ?1,3-arm GlcNAc. J Biol Chem 292:14796-14803
Li, Tiezheng; Tong, Xin; Yang, Qiang et al. (2016) Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling. J Biol Chem 291:16508-18
Yamaguchi, Takahiro; Amin, Mohammed N; Toonstra, Christian et al. (2016) Chemoenzymatic Synthesis and Receptor Binding of Mannose-6-Phosphate (M6P)-Containing Glycoprotein Ligands Reveal Unusual Structural Requirements for M6P Receptor Recognition. J Am Chem Soc 138:12472-85
Toonstra, Christian; Amin, Mohammed N; Wang, Lai-Xi (2016) Site-Selective Chemoenzymatic Glycosylation of an HIV-1 Polypeptide Antigen with Two Distinct N-Glycans via an Orthogonal Protecting Group Strategy. J Org Chem 81:6176-85
Yang, Qiang; Wang, Lai-Xi (2016) Mammalian ?-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans. J Biol Chem 291:11064-71
Giddens, John P; Lomino, Joseph V; Amin, Mohammed N et al. (2016) Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins. J Biol Chem 291:9356-70

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