Project Summary/Abstract The objectives of the proposed research are to develop facile chemoenzymatic methods for synthesizing homogeneous N-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 chemoenzymatic method that exploits the transglycosylation 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 have discovered novel endoglycosidase-based mutants, the glycosynthases, that are capable of using highly active glycan oxazolines for transglycosylation but lack the product hydrolysis activity. The glycosynthase-catalyzed native ligation permits independent manipulations of the sugar and protein portions and provides a highly convergent approach to glycoprotein assembly. This method has been successfully applied for the synthesis of a series of complex glycopeptides such as the HIV-1 glycopeptide antigens and CD52 glycoproteins. It has also been explored for glycan remodeling of recombinant glycoproteins including human erythropoietin (EPO) and therapeutic antibodies. In this application, we aim at improving the chemoenzymatic method, expanding its scope, and speeding up its application as a general method for the synthesis of homogeneous glycoforms of glycoproteins. Four specific aims will be pursued to achieve the goals. The first two aims are focused on generating new endoglycosynthases from bacterial endoglycosidases and novel -fucoligases from an array of -fucosidases with distinct (1,6, 1,3/1,4, and 1,2)-fucosidic linkages; the third aim is to develop an E. coli co-expression system to produce GlcNAc- and Glc-containing proteins that will serve as the key precursor for a combined synthesis of glycosylated therapeutic proteins with a goal of enhancing the serum half-life of therapeutic proteins; and the fouth aim is to establish a method for glycosylation remodeling of lysosomal enzymes such as the recombinant human -glucosidase with mannose-6-phosphate (M6P) oligosaccharides for improving their cellular uptake in enzymatic replacement therapy. A successful completion of the proposed research will significantly expand the scope of the chemoenzymatic method; provide new enabling technologies to the community of glycobiology and biotechnology; and speed up the applications of the chemoenzymatic method for improving the efficacy of therapeutic proteins.