Emerging technologies for making glycan-defined glycoproteins

Abstract

Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity. Glycoproteins, the end products of such a modification, are typically produced as mixtures of glycoforms possessing the same polypeptide backbone but differing in the site of glycosylation and/or in the structures of pendant glycans, from which single glycoforms are difficult to isolate. The urgent need for glycan-defined glycoproteins in both detailed structure-function relationship studies and therapeutic applications has stimulated an extensive interest in developing various methods for manipulating protein glycosylation. This review highlights emerging technologies that hold great promise in making a variety of glycan-defined glycoproteins, with a particular emphasis in the following three areas: specific glycoengineering of host biosynthetic pathways, in vitro chemoenzymatic glycosylation remodeling, and chemoselective and site-specific glycosylation of proteins.

Figure 1

Structures of representative N- and O-linked glycans on glycoproteins. (a) high-mannose type N-glycan; (b) bi-antennary complex type N-glycan; (c) core 1 O-GalNAc glycan; (d) core 2 O-GalNAc glycan; (e) O-GlcNAc.

Figure 2

N-glycan biosynthetic pathways in eukaryotes and glycoengineering. (a) the shared early steps in the ER leading to the Man₈GlcNAc₂ (M8) glycoform, which is translocated to Golgi for further processing; (b) processing and branching in mammalian host leading to mature glycoproteins; (c) glycan processing in yeast leading to hypermannosylation and its engineering being directed to the mammalian glycosylation pathway; (d) glycan processing in plants leading to plant-specific glycoform and its engineering being directed to the mammalian glycosylation pathway.

Figure 3

Chemoenzymatic approaches to glycosylation remodeling of glycoproteins. (a) sugar chain extension by sequential glycosyltransferase-catalyzed reactions; (b) sugar chain extension by endoglycosidase-catalyzed transglycosylation.

Figure 4

Chemoenzymatic synthesis of different glycoforms by the ENGase-catalyzed transglycosylation. (a) examples of synthetic RNase glycoforms produced by using ENGase and related glycosynthase mutants; (b) glycoengineering of human IgG-Fc.

Figure 5

Chemo-selective and site-specific glycosylation of proteins. (a) a general chemo-selective and site-specific strategy; (b) a dual tagging approach to generating a functional PSGL-1 mimic.