项目来源

美国卫生和人类服务部基金(HHS)

项目主持人

BOND, MICHELLE RUEFFER

项目受资助机构

UNIV OF MARYLAND, COLLEGE PARK

项目编号

2R01GM080374-10

立项年度

2018

立项时间

未公开

项目级别

国家级

研究期限

未知 / 未知

受资助金额

308016.00美元

学科

Biotechnology

学科代码

未公开

基金类别

Non-SBIR/STTR RPGs

关键词

未公开

参与者

WANG, LAI-XI

参与机构

NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

项目标书摘要：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.