Utilization of wild-type and mutant glycosyltransferase 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). Also wild-type polypeptide-alpha-N-acetylgalactosaminyltransferase (ppGalNAc-T2) that has been expressed in E. coli and in vitro folded from inclusion bodies transfers GalNAc moiety with a chemically reactive unique functional group to a polypeptide chain. The b4Gal-T1 enzyme transfers Gal from UDP-Gal to GlcNAc present at the non-reducing end of an acceptor substrate, and its double mutant R228K-Y289L-b4Gal-T1 exhibits better GlcNAc-transferase activity where it transfers GlcNAc from UDP-GlcNAc to the same acceptor substrate. In the present study we find that this double-mutant enzyme can transfer C2-keto-Glc from UDP-C2-keto-Glc;however, only to GlcNAc, not to its analogue, C2-keto-Glc. Furthermore, we also show that the two wild-type N-acetylglucosaminyltransferases, human b3GN-T2 that synthesizes the poly-N-acetyl-lactosamine, and the human MFng that is involved in the synthesis of glycan of epidermal growth factor (EGF) repeats of the extracellular domains of the Notch receptor, which accommodate the N-acetyl group of the donor sugar GlcNAc, can also transfer the C2-modified Glc, C2-keto-Glc, to their corresponding acceptors, LacNAc on the N-glycans of Asialofetuin (ASF) and O-fucosylated EGF repeat from Factor VII, respectively. Thus our results suggest that the N-acetyl groups of the donor sugars GlcNAc and GalNAc of the N-acetylglucosaminyl- and N-acetylgalactosaminyl-transferases are generally embedded in a cavity or a hydrophobic pocket which can also accommodate a ketone group or an azido group in the N-acetyl-binding pocket. The transfer of a modified sugar residue that has a chemical handle by the mutant or wild type glycosyltransferases to a specific sugar residue on a glycoconjugate or to a specific site in a polypeptide engineered in the non-glycoprotein makes it possible to link bioactive molecules via the modified sugar residue. We have tested this strategy, using a few model systems described here and demonstrate the feasibility of this approach. The presence of a unique modified sugar moiety with a chemical handle at a specific site on a glycoconjugate or a non-glycoprotein makes it possible to transfer galactose derivative to GlcNAc residues on the glycan chains of Ovalbumin and IgG with the mutant enzyme Tyr289Leu and coupling of the aminooxy-biotin or aminooxy- fluoroprobes 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 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 is 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 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 or fluoroprobe probes carrying an orthogonal reactive group as monitored by MS analysis or chemiluminescence or florescence methods. Use of ELISA methodology shows that antigen-antibody 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 or fluorescence activated cell sorting (FACS) methods. 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. 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 chemoenzymatic methods of synthesis of functionalize carbohydrate nucleotides with C-2 modifications.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010742-08
Application #
8763195
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2013
Total Cost
$171,181
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
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State
Country
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