Chemoenzymatic Glycan Remodeling of Natural and Recombinant Glycoproteins

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 Man₉GlcNAc₂ glycoform, with the former showing significantly enhanced antibody-dependent cellular cytotoxicity. EPO was engineered to carry azide-tagged Man₃GlcNAc₂ glycans that could be further modified via click chemistry to introduce other functional groups.

Keywords: Antibody; Chemoenzymatic synthesis; Erythropoietin; Glycoprotein; Glycosynthase; Herceptin; Oxazoline.

Fig. 1

Reaction scheme for glycan remodeling of Herceptin.

Fig. 2

Reaction scheme for glycan remodeling of EPO.

Fig. 3

LC-MS analysis of intact Herceptin to monitor transfer of S2G2 glyan to Herceptin-GlcNAc acceptor. A) Herceptin. B) GlcNAc-Herceptin (Herceptin-Gn). C) Intermediate product during transfer of S2G2 to Herceptin-Gn at 30 min. SM, starting material. D) Final Herceptin-S2G2 transglycosylation product.

Fig. 4

LC-MS analysis under reducing conditions for the heavy and light chains of different glycoforms of Herceptin in the glycan remodeling with S2G2 glycan. A) Heavy chain of Herceptin. B) Heavy chain of Herceptin-Gn. C) Heavy chain of Herceptin-S2G2. D) Heavy chain of Herceptin-S2G2 treated with PNGase F. E) Light chain of Herceptin. F) Light chain of Herceptin-S2G2.

Fig. 5

LC-MS analysis under reducing conditions for the heavy and light chains of different glycoforms of Herceptin in the glycan remodeling with M9 glycan. A) Heavy chain of Fucα1,6GlcNAc-Herceptin (Herceptin-GnF). B) Heavy chain of Herceptin-M9F. C) Heavy chain of Herceptin-M9F treated with PNGase F. D) Light chain of Herceptin-M9F.

Fig. 6

Mass spectrometry analysis of glycan remodeling of EPO. A) MALDI-TOF analysis of glycan released from EPO with high-mannose type glycan. The glycan release and MALDI-TOF analysis was performed as described in (Yang & Wang, 2016). B) LC-MS analysis of GlcNAc-EPO (EPO-Gn). C) LC-MS analysis of EPO transferred with M3_N3 glycan.