A two-step enzymatic glycosylation of polypeptides with complex N -glycans

Abstract

A chemoenyzmatic method for direct glycosylation of polypeptides is described. The method consists of two site-specific enzymatic glycosylation steps: introduction of a glucose moiety at the consensus N-glycosylation sequence (NXS/T) in a polypeptide by an N-glycosyltransferase (NGT) and attachment of a complex N-glycan to the glucose primer by an endoglycosidase (ENGase)-catalyzed transglycosylation. Our experiments demonstrated that a relatively small excess of the UDP-Glc (the donor substrate) was sufficient for an effective glucosylation of polypeptides by the NGT, and different high-mannose and complex type N-glycans could be readily transferred to the glucose moiety by ENGases to provide full-size glycopeptides. The usefulness of the chemoenzymatic method was exemplified by an efficient synthesis of a complex glycoform of polypeptide C34, a potent HIV inhibitor derived from HIV-1 gp41. A comparative study indicated that the Glc-peptide was equally efficient as the natural GlcNAc-peptide to serve as an acceptor in the transglycosylation with sugar oxazoline as the donor substrate. Interestingly, the Glc–Asn linked glycopeptide was completely resistant to PNGase F digestion, in contrast to the GlcNAc–Asn linked natural glycopeptide that is an excellent substrate for hydrolysis. In addition, the Glc–Asn linked glycopeptide showed at least 10-fold lower hydrolytic activity toward Endo-M than the natural GlcNAc–Asn linked glycopeptide. The chemoenzymatic glycosylation method described here provides an efficient way to introducing complex N-glycans into polypeptides, for gain of novel properties that could be valuable for drug discovery.

Molecular Analysis of an Alternative N-Glycosylation Machinery by Functional Transfer from Actinobacillus pleuropneumoniae to Escherichia coli

“…Unlike many other glycosylation systems that often yield many different glycoforms, such glycoproteins are homogeneously modified with glucose with no detectable amounts of other glycoforms. Such N-linked hexoses that are the product of the ApNGT reaction can be used as starting material to produce defined glycoproteins by transglycosylation (40), for glycoPEGylation (41), or for coupling of desired ligands using hydrazide chemistry (42).…”

Bacterial Glycosidases in Pathogenesis and Glycoengineering

“…In this study, an initial glucose residue was attached to the peptide backbone by the glycosyl transferase from Actinobacillus pleuropneumoniae and the transfer of a complex glycan to the specific site by either EndoA from Arthrobacter protophormiae or EndoM from Mucor hiemalis was carried out (Figure 2B) [69]. The resulting glycan attached to the peptide was resistant to PNGaseF hydrolysis, also demonstrating that the approach is potentially useful for glycosylating peptides to gain novel properties.…”

Glycosylation in Cell Culture

“…A mutated Endo S with glycosynthase activity allowed efficient addition of the glycan to the Fc site of the Mab. Introduction of a glucose residue to N-glycosylation site (NXS/T) using an N-glycosyltransferase allowed further addition of large glycan to the polypeptide by enzymatic transglycosylation (Lomino et al 2013), and may be useful for creating unique glycosylation sites within a peptide to modify its characteristics.…”