Abstract
N-glycosylation plays important roles in modulating the biological functions of glycoproteins, such as protein folding, stability, and immunogenicity. However, acquiring homogeneous glycoforms of glycoproteins has been a challenging task for functional studies and therapeutic applications. In this chapter, we describe an efficient chemoenzymatic glycan remodeling protocol for making homogeneous glycoproteins that involves enzymatic deglycosylation and subsequent reglycosylation procedures. Two therapeutic glycoproteins, Herceptin (trastuzumab, a therapeutic monoclonal antibody) and erythropoietin (EPO, a glycoprotein hormone), were chosen as the model systems. The detailed protocol includes the deglycosylation of the Herceptin or EPO with a wild-type endo-β-N-acetylglucosaminidase, to remove the heterogeneous N-glycans, leading to the GlcNAc-protein or Fucα1,6GlcNAc-protein intermediate. Then desired homogeneous N-glycans are attached to the acceptor by using an activated sugar oxazoline as the donor substrate and a specific glycosynthase (mutant of endoglycosidase) as the catalyst to reconstitute a homogeneous glycoform. Using this approach, Herceptin was remodeled to an afucosylated complex glycoform and a Man(9)GlcNAc(2) glycoform, with the former showing significantly enhanced antibody-dependent cellular cytotoxicity. EPO was engineered to carry azide-tagged Man(3)GlcNAc(2) glycans that could be further modified via click chemistry to introduce other functional groups.