Core fucosylation of N-glycoproteins plays a crucial role in modulating the biological functions of glycoproteins. Yet, the synthesis of structurally well-defined, core-fucosylated glycoproteins remains a challenging task due to the complexity in multistep chemical synthesis or the inability of the biosynthetic α1,6-fucosyltransferase (FUT8) to directly fucosylate full-size mature N-glycans in a chemoenzymatic approach. We report in this paper the design and generation of potential α1,6-fucosynthase and fucoligase for direct core fucosylation of intact N-glycoproteins. We found that mutation at the nucleophilic residue (D200) did not provide a typical glycosynthase from this bacterial enzyme, but several mutants with mutation at the general acid/base residue E274 of the Lactobacillus casei α1,6-fucosidase, including E274A, E274S, and E274G, acted as efficient glycoligases that could fucosylate a wide variety of complex N-glycopeptides and intact glycoproteins by using α-fucosyl fluoride as a simple donor substrate. Studies on the substrate specificity revealed that the α1,6-fucosidase mutants could introduce an α1,6-fucose moiety specifically at the Asn-linked GlcNAc moiety not only to GlcNAc-peptide but also to high-mannose and complex-type N-glycans in the context of N-glycopeptides, N-glycoproteins, and intact antibodies. This discovery opens a new avenue to a wide variety of homogeneous, core-fucosylated N-glycopeptides and N-glycoproteins that are hitherto difficult to obtain for structural and functional studies.

The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics. We have previously reported a chemoenzymatic glycan remodeling method for producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man5GlcNAc2Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed transglycosylation. Interestingly, a remarkable site-selectivity was observed in the transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans.

IgG antibodies contain a conserved N-glycosylation site on the Fc domain to which a complex, biantennary glycan is attached. The fine structures of this glycan modulate antibody effector functions by affecting the binding affinity of the Fc to diverse Fc receptor family members. For example, core fucosylation significantly decreases antibody-dependent cellular cytotoxicity (ADCC), whereas terminal α2,6-sialylation plays a critical role in the anti-inflammatory activity of human i.v. immunoglobulin therapy. The effect of specific combinations of sugars in the glycan on ADCC remains to be further addressed, however. Therefore, we synthesized structurally well-defined homogeneous glycoforms of antibodies with different combinations of fucosylation and sialylation and performed side-by-side in vitro FcγR-binding analyses, cell-based ADCC assays, and in vivo IgG-mediated cellular depletion studies. We found that core fucosylation exerted a significant adverse effect on FcγRIIIA binding, in vitro ADCC, and in vivo IgG-mediated cellular depletion, regardless of sialylation status. In contrast, the effect of sialylation on ADCC was dependent on the status of core fucosylation. Sialylation in the context of core fucosylation significantly decreased ADCC in a cell-based assay and suppressed antibody-mediated cell killing in vivo. In contrast, in the absence of fucosylation, sialylation did not adversely impact ADCC.

We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.

Mannose-6-phosphate (M6P)-terminated oligosaccharides are important signals for M6P-receptor-mediated targeting of newly synthesized hydrolases from Golgi to lysosomes, but the precise structural requirement for the M6P ligand–receptor recognition has not been fully understood due to the difficulties in obtaining homogeneous M6P-containing glycoproteins. We describe here a chemoenzymatic synthesis of homogeneous phosphoglycoproteins carrying natural M6P-containing N-glycans. The method includes the chemical synthesis of glycan oxazolines with varied number and location of the M6P moieties and their transfer to the GlcNAc-protein by an endoglycosynthase to provide homogeneous M6P-containing glycoproteins. Simultaneous attachment of two M6P-oligosaccahrides to a cyclic polypeptide was also accomplished to yield bivalent M6P-glycopeptides. Surface plasmon resonance binding studies reveal that a single M6P moiety located at the low α-1,3-branch of the oligomannose context is sufficient for a high-affinity binding to receptor CI-MPR, while the presence of a M6P moiety at the α-1,6-branch is dispensable. In addition, a binding study with the bivalent cyclic and linear polypeptides reveals that a close proximity of two M6P-oligosaccharide ligands is critical to achieve high affinity for the CI-MPR receptor. Taken together, the present study indicates that the location and valency of the M6P moieties and the right oligosaccharide context are all critical for high-affinity binding with the major M6P receptor. The chemoenzymatic method described here provides a new avenue for glycosylation remodeling of recombinant enzymes to enhance the uptake and delivery of enzymes to lysosomes in enzyme replacement therapy for the treatment of lysosomal storage diseases.

A convergent chemoenzymatic approach for sequential installation of different N-glycans in a polypeptide is described. The method includes introduction of distinguishably protected GlcNAc-Asn building blocks during automated solid phase peptide synthesis (SPPS), followed by orthogonal deprotection of the GlcNAc primers and site-selective sequential extension of the sugar chains through glycosynthase-catalyzed transglycosylation reactions. It was observed that the protecting groups on one neighboring GlcNAc moiety have an impact on the substrate activity of another GlcNAc acceptor toward some endoglycosynthases in transglycosylation. The usefulness of this synthetic strategy was exemplified by an efficient synthesis of the glycopeptide neutralizing epitope of broadly HIV-neutralizing antibody PG9. The method should be generally applicable for the synthesis of complex glycopeptides carrying multiple different N-glycans.

We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.