Enzymatic Glycoprotein Synthesis: Preparation of Ribonuclease Glycoforms via Enzymatic Glycopeptide Condensation and Glycosylation

Abstract

In order to study the effects carbohydrates have on glycoprotein structure and function, it is imperative to be able to synthesize the appropriate natural and non-natural glycoprotein variants in a single form. Because the available in vivo techniques provide only heterogeneous mixtures of different glycoforms, enzymatic in vitro methodologies have been pursued. Using the N-glycoprotein RNase B as a model system, the oligosaccharide was removed leaving only the N-acetylglucosamine as a “tag” to the site of glycosylation. Glycosyltransferases were then used to build a unique carbohydrate moiety. A new RNase glycoform containing the branched oligosaccharide, sialyl Lewis X or the Hg derivative, was synthesized enzymatically to demonstrate the feasibility of the method. In addition, the monoglycosylated protein was digested into several smaller pieces by subtilisin BPN‘. These fragments were religated by subtilisin 8397 to the full length RNase by addition of glycerol; this method points to a new chemical-enzymatic process for the synthesis of glycoproteins using synthetic peptides and glycopeptides as substrates for enzymatic ligation followed by further enzymatic glycosylations.