Utilization of glycosyltransferase mutants for linking glycoconjugates via glycan moieties: The mutant enzymes that have been generated in our laboratory can transfer a sugar residue with a chemically reactive unique functional group to a sugar moiety of a glycoprotein or glycolipids (glycoconjugates). The presence of a unique modified sugar moiety on a glycoconjugate makes it possible to link bioactive molecules via the modified residue on the glycan chain. We have tested this strategy, using a few model systems described here and demonstrate the feasibility of this approach. Transfer of galactose derivative to GlcNAc residues on the glycan chains of Ovalbumin and IgG with the mutant enzyme Tyr289Leu and coupling of the aminooxy-biotin to the modified galactose residue: We showed that the mutant Tyr289Leu-Gal-T1 enzyme can transfer the C2-ketone derivative of galactose from its UDP derivative (originally synthesized by Dr. Linda Hsieh-Wilson at CalTech) to the GlcNAc residue on the N-linked glycan chain on Ovalbumin or to an IgG molecule which does not have a fully matured N-glycan chain. The transfer of the modified sugar by the mutant enzyme is carried out either at room temperature or at 30 C. The transfer has been followed by coupling to the ketone group at the C2 position of galactose with the biotinylated aminooxy ligand, which was then detected by chemiluminescence after treating with the streptavidin-HRP system. The transfer is strictly dependent on both the presence of the mutant enzyme and the ketone derivative of galactose. The wild-type enzyme can not utilize the ketone derivative of galactose. That the biotinylated aminooxy ligand is linked only to the N-glycan chain of Ovalbumin has been confirmed by treatment of the proteins after the transfer of ketone derivatives with PNGase F, which removes N-glycan chains from the protein. We have followed the transfer of the modified sugars, like 2-keto-galactose or 2-azido-galactose, by MS analysis of the sugar chain, before and after the transfer reaction with IgGs, e.g Avastin, Remicade, Rituxan and Herceptin, and established the conditions where the transfer of modified sugar is nearly 100%. MALDI-TOF methodology was used to monitor the conditions for the complete de-galactosylation of the IgGs (100%) to G0 glycoform and the re-galactosylation to G2 glycoform. Using mutant enzyme b4Gal-T1-Y289L, modified sugars were transferred from the respective UDP-derivatives to the de-galactosylated MAb. The MAb carrying modified sugar could be completely linked to biotinylated derivatives carrying an orthogonal reactive group as monitored by MS analysis or chemiluminescence methods. Use of ELISA methodology shows that antigenantibody interactions have not been disturbed by the transfer of modified sugars to the N-linked glycans of the IgGs. Thus, the Fc N-glycans of therapeutic MAb can be specifically modified in vitro by the addition of C2-modified galactose having a chemical handle, such as ketone or azide, from its UDP-derivative using the mutant enzyme b4Gal-T1-Y289L, and that this modification permits the coupling of the modified galactose to a bio-molecule that carries an orthogonal reactive group. Re-galactosylation or linking of the IgGs does not affect the specificity of the Fab domain of the antibodies measured by indirect ELISA techniques. Thus, the possibility of linking cargo molecules to therapeutic monoclonal IgGs via glycans could prove to be an invaluable tool for (1) detection of GlcNAc residues on glycoconjugates, (2) potential drug targeting by immunotherapeutic methods, and (3) for developing contrast agents for MRI. Method to use polypeptide-alpha-N-acetylgalactosaminyltransferase (ppGalNAc-T2) for the glycoconjugation of non-glycoproteins: Here we describe a new method for bioconjugation of a non-glycoprotein with bio-molecules. Using ppGalNAc-T2, we transfer a C2-modified galactose that has a chemical handle, such as ketone or azide, from its respective UDP-sugars to the Ser/Thr residue(s) of an acceptor polypeptide fused to the non-glycoprotein. The protein with the modified galactose is then coupled to a bio-molecule that carries an orthogonal reactive group. As a model system for the non-glycoprotein, we engineered glutathione-S-transferase (GST) protein with a 17-amino-acid-long fusion peptide at the C-terminal end that was expressed as a soluble protein in E. coli. The ppGalNAc-T2 protein, the catalytic domain with the C-terminal lectin domain, was expressed as inclusion bodies in E. coli, and an in vitro folding method was developed to produce milligram quantities of the active enzyme from a liter of bacterial culture. This ppGalNAc-T2 enzyme transfers from the UDP-sugars, not only GalNAc, but also C2-modified galactose that has a chemical handle, to the Ser/Thr residue(s) in the fusion peptide. The chemical handle at the C2 of galactose is used for conjugation and assembly of bionanoparticles and preparation of immuno-liposomes for a targeted drug delivery system. This novel method enables one to glycosylate with ppGalNAc-T2 the important biological non-glycoproteins, such as single-chain antibodies, growth factors, or bacterial toxins, with an engineered 17-residue peptide sequence at the C-terminus of the molecule, for conjugation and coupling. Patent has been filled on this glyco bioconjugation method of non-glycoproteins. The paper is in print in the Bioconjugate Chemistry Journal of Am. Chem. Soc. Development of an in vitro folding method for ppGalNAc-Ts, a1,2Fuc-Ts: Glycosyltransferases expressed in E. coli often appear as inclusion bodies. A refolding method is required to generate soluble and active protein. In order to glycosylate non-glycoproteins using ppGalNAc-Ts for bioconjugation using glycan moiety, we have expressed the ppGalNAc-T2 in E. coli and developed an in vitro refolding method from inclusion bodies based on the one used for beta-1,4-galactosyltransferase to generate milligram quantities of soluble and active ppGalNAc-T2. This enzyme has bee used to glycosylate non-glycoproteins for bioconjugation (see above). Using the same refolding method we find that alpha-1,4-fucosyltransferase-I can also be refolded in vitro to generate soluble and active protein. We have tested its activity against an oligosaccharide having terminal galactose moiety. The active enzyme transfers fucose from GDP-fucose to the terminal galactose as judged by the MALDI-TOF spectroscopy. A Patent has been filed on the ppGalNAc-T refolding method. Synthesis of Modified Sugar Nucleotides: Linking of various glycoproteins and non-glycoproteins via glycan chains with glycosyltransferases requires modified sugars that carry orthogonal reactive groups. The design of the modified sugars is determined based on the structure of the catalytic cavity of the glycosyltransferase and their mutants that are being generated in our laboratory. We are developing convenient synthetic routes to functionalize carbohydrate nucleotides with C-2 modifications. Glycosylamine-1-phosphates are used as starting materials which can be acylated with a number of functionalized compounds or probes. This preparation attaches a chemical reactive group, a dye moiety, a conjugated substance, [summary truncated at 7800 characters]
| Mercer, Natalia; Ramakrishnan, Boopathy; Boeggeman, Elizabeth et al. (2013) Use of novel mutant galactosyltransferase for the bioconjugation of terminal N-acetylglucosamine (GlcNAc) residues on live cell surface. Bioconjug Chem 24:144-52 |
| Qasba, Pradman K; Boeggeman, Elizabeth; Ramakrishnan, Boopathy (2008) Site-specific linking of biomolecules via glycan residues using glycosyltransferases. Biotechnol Prog 24:520-6 |
