title: 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.
authors.affiliation: Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Joseph V
authors.initials: JV
authors.lastname: Lomino
authors.affiliation: Institute of Microbiology, Dept. of Biology, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Naegeli
authors.affiliation: Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Jared
authors.initials: J
authors.lastname: Orwenyo
authors.affiliation: Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
authors.forename: Mohammed N
authors.initials: MN
authors.lastname: Amin
authors.affiliation: Institute of Microbiology, Dept. of Biology, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.affiliation: Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, United States. Electronic address: LWang@som.umaryland.edu.
authors.forename: Lai-Xi
authors.initials: LX
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D006020:Glycopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D006031:Glycosylation; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006020:Glycopeptides; D010455:Peptides; D011134:Polysaccharides; D005964:Glucosyltransferases; C410788:N-glycosyltransferase, Nocardia aerocolonigenes