The goal of the proposed research is to develop a general and highly efficient chemo enzymatic method for making diverse homogeneous glycopeptides and glycoproteins of biomedical significance. A major problem in functional glycomics studies and glycoprotein therapeutic applications is the lack of efficient methods to produce glycan-defined glycoproteins. We have recently developed a chemo enzymatic method that exploits the trans glycosylation activity of a class of endoglycosidases (ENGases) that enables the """"""""native ligation"""""""" between free glycan and GlcNAc- tagged protein to form homogeneous glycoproteins with native glycosidic linkage. We found that synthetic glycan oxazoline and ENGase-based glycosynthase that we created is an excellent pair for an efficient transglycosylation. This method permits independent manipulations of the sugar and protein portions, providing a highly convergent and potentially general approach to glycoprotein assembly. In this renewal application, we aim at expanding the scope of the chemo enzymatic method and speeding up its application by pursuit of the following four specific aims.
Aim 1 is to expand the synthetic repertoire by evaluating new enzymes, mutants, and distinct donor and acceptor substrates for glycoprotein synthesis.
Aim 2 is to determine the crystal structures of EndoS and EndoF3, and their complexes with substrates/substrate analogs, which will provide a molecular level understanding of how these ENGases differentially recognize the substrates in hydrolysis and transglycosylation.
Aim 3 is to explore a two-step enzymatic strategy for direct glycosylation of polypeptides and proteins, including the study of N-glycosyltransferase (NGT) for directly introducing a monosaccharide primer into polypeptide and the subsequent extension of the sugar chains via ENGase-catalyzed transglycosylation.
Aim 4 is to remodel lysosomal enzymes with synthetic mannose 6-phosphate glycans aiming to enhance the therapeutic efficiency in enzyme replacement therapy (ERT). The proposed study is expected to provide important new tools in chemo enzymatic synthesis and the knowledge gained will speed up glycoprotein therapeutic applications.

Public Health Relevance

The proposed research aims at developing combined chemical and enzymatic methods for producing biologically important glycoproteins for functional studies. This study will provide new information on how glycosylation affects the functions of proteins and will speed up glycoprotein therapeutic applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM080374-06
Application #
8534511
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Marino, Pamela
Project Start
2007-06-01
Project End
2016-12-31
Budget Start
2013-03-01
Budget End
2013-12-31
Support Year
6
Fiscal Year
2013
Total Cost
$299,325
Indirect Cost
$104,325
Name
University of Maryland Baltimore
Department
Biochemistry
Type
Schools of Medicine
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Li, Chao; Li, Tiezheng; Wang, Lai-Xi (2018) Chemoenzymatic Defucosylation of Therapeutic Antibodies for Enhanced Effector Functions Using Bacterial ?-Fucosidases. Methods Mol Biol 1827:367-380
Tong, Xin; Li, Tiezheng; Li, Chao et al. (2018) Generation and Comparative Kinetic Analysis of New Glycosynthase Mutants from Streptococcus pyogenes Endoglycosidases for Antibody Glycoengineering. Biochemistry 57:5239-5246
Thomas, Julie A; Orwenyo, Jared; Wang, Lai-Xi et al. (2018) The Odd ""RB"" Phage-Identification of Arabinosylation as a New Epigenetic Modification of DNA in T4-Like Phage RB69. Viruses 10:
Trastoy, Beatriz; Klontz, Erik; Orwenyo, Jared et al. (2018) Structural basis for the recognition of complex-type N-glycans by Endoglycosidase S. Nat Commun 9:1874
Li, Tiezheng; Li, Chao; Quan, David N et al. (2018) Site-specific immobilization of endoglycosidases for streamlined chemoenzymatic glycan remodeling of antibodies. Carbohydr Res 458-459:77-84
Giddens, John P; Lomino, Joseph V; DiLillo, David J et al. (2018) Site-selective chemoenzymatic glycoengineering of Fab and Fc glycans of a therapeutic antibody. Proc Natl Acad Sci U S A 115:12023-12027
Li, Chao; Wang, Lai-Xi (2018) Chemoenzymatic Methods for the Synthesis of Glycoproteins. Chem Rev 118:8359-8413
Bennett, Lindsay D; Yang, Qiang; Berquist, Brian R et al. (2018) Implementation of Glycan Remodeling to Plant-Made Therapeutic Antibodies. Int J Mol Sci 19:
Tong, Xin; Li, Tiezheng; Orwenyo, Jared et al. (2018) One-pot enzymatic glycan remodeling of a therapeutic monoclonal antibody by endoglycosidase S (Endo-S) from Streptococcus pyogenes. Bioorg Med Chem 26:1347-1355
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

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