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.
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.
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