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.

A common glycan structure on immunoglobulin G for enhancement of effector functions

“…During this study, it was discovered that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimal structure that is able to enhance the activities of antibodies against cancer, influenza, and inflammatory diseases. (22) and later applied to antibody glycoengineering (25)(26)(27). The strategy starts with the use of exoglycosidases or endoglycosidases to cleave most of the N-glycans to form a homogeneous glycoform containing a well-defined glycan, followed by extension of the glycan using glycosyltransfer enzymes.…”

“…Many methods have been developed for the preparation of homogeneous glycoproteins with well-defined glycans, including native chemical ligation (NCL) (15,16), expressed protein ligation (EPL) (17), Staudinger ligation (18)(19)(20), sugar-assisted ligation (21), and glycoprotein remodeling in vitro using endoglycosidases and glycosyltransfer enzymes (22). Similarly, glycosylation pathway engineering has been developed to improve the biological function and reduce the heterogenecity of therapeutic antibodies (23,24).…”

Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation

“…Common glycosyltransferases can extend sugar chains by adding monosaccharides one at a time. 32 In contrast, the endoglycosidase-catalyzed transglycosylation reaction can attach a large intact oligosaccharide to a GlcpNAc polypeptide in a single-step, thus providing a highly convergent approach. 33, 34 A major disadvantage of this endoglycosidase-based chemoenzymatic approach is the relatively low transglycosylation efficiency and the issue of product hydrolysis, which is the focus of discussion of this review.…”

The yeast expression system for recombinant glycosyltransferases

“…Witte and coworkers [123] reported the synthesis of an unnatural glycoform of ribonuclease. Ribonuclease B contains a single N-linked glycosylation site and exists as a series of high-mannose glycoforms.…”