Development of streamlined chemoenzymatic glycan remodeling systems for antibodies and other important glycoproteins

Project abstract/summary The objectives of this proposal are to develop efficient and streamlined cheomoenzymatic systems for glycan remodeling of antibodies and other important glycoproteins, through enzyme immobilization, scalable glycan production, and kits development. Such immobilized enzymes, when combined with activated glycan library as kits, can help general academic and industrial users, particularly non-specialists, to prepare glycan-defined glycoproteins for structural and functional studies. The streamlined approach can also be applied in scale-up preparation of homogenous glycoproteins with therapeutic potential. Protein glycosylation is one of the most ubiquitous posttranslational modifications. It profoundly affects a protein’s properties such as folding, in vivo stability, immunogenicity, and pharmacokinetics, and also directly participate in many important biological processes, including cell adhesion, cancer progression, host-pathogen interactions, and immune responses. A major issue in glycoprotein studies is the structural heterogeneity and the difficulty in isolating homogeneous glycoforms for detailed structural and functional studies. Although significant progresses have been made for producing glycan-defined glycoprotein such as total chemical synthesis and biosynthetic pathway engineering in different host expression system, the pure glycoforms that can be achieved are still quite limited. To address this challenge, a novel chemoenzymatic method to produce homogeneous glycoprotein and glycopeptides has been developed recently. This convergent approach consists of two key steps: deglcosylation of glycoproteins with an endoglycosidase and subsequent attachment of a desired activated N-glycan to the protein-GlcNAc acceptor by a novel glycosynthase. This SBIR II application is built on the success of the Phase I study (1R43GM134816-01), where we have successfully finished the immobilization of endoglycosidase S2 (EndoS2) from S. pyogenes and its corresponding glycosynthase mutant EndoS2-D184M. The EndoS2-D184M glycosynthase was immobilized by a site-oriented approach which almost fully retains the activity of enzyme. We have demonstrated that the immobilized enzymes are highly efficient for glycan remodeling of therapeutic antibodies. In this Phase II research, we propose to pursue the following three specific aims. Aim 1 is to expand this immobilization strategy to other glycosidases/glycosynthases to establish a tool box of enzymes for glycan- remodeling. Aim 2 is optimize and scale up our production protocol for different oxazolines and significantly lower their production cost. Aim 3 is to develop glycan remodeling kits and establish scalable glycan remodeling protocols.