Using Glycosyltransferase Mutants to Develop Effective C

Qasba, Pradman

Abstract

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: Here we show that the mutant Tyr289Leu-Gal-T1 enzyme] can transfer the C2-ketone derivative of galactose from its UDP derivative (synthesized by Dr. Linda Hsie-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 30C. 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. Development of a drug delivery System: The selective targeting of a drug to its planned site of action has therapeutic advantages because of the reduced toxicity and slighter dose levels needed for treatment. Because oligosaccharide moieties of glycoconjugates are involved in many biological processes, they have been used as a tool for the delivery of ligands to specific sites and tissues. The use of carbohydrates to create targeted drug delivery systems, termed """"""""glycotargetting"""""""", first demonstrated in 1971, has been used to target protein receptors at the site of location. The availability of mutant glycosyltransferases makes it possible to transfer sugar moieties with a variety of functional groups from their respective donor nucleotides to a specific residue of an oligosaccharide chain of a glycoprotein. The two glycoproteins, each carrying the sugar moiety with a reactive functional group, which can then be coupled by the heterobifunctional linkers, thus enabling two glycoproteins to be coupled at a specific site. One glycoprotein can be a monoclonal antibody, which carries a single N-linked oligosaccharide chain on the IgG heavy chain, while another can be a toxin. This coupling allows a controlled and specific linking of monoclonal antibodies to toxins. Furthermore, the monoclonal antibodies whose N-linked glycan moiety has been modified to have 2-keto-galactose can be linked to aminooxy linkers carrying chelating agents specific for gadolinium or other contrast agents for imaging. In the currently prevailing methods, generally two proteins are cross-linked, using a bi-functional cross-linker, at random sites to a protein residue, e.g., lysine, which is distributed at several places on the protein surface. This method of cross-linking often blocks the functional sites on the protein and thus reduces the bioefficacy of the protein.