Engineering of an a2,6-Sialyltransferase from H. acinonychis

The project Enzyme Engineering of an a2,6-Sialyltransferase from Helicobacter acinonychis for Sialylation of Therapeutic Proteins aims at investigating and engineering of the sialyltransferase HAC1268 from the bacterium Helicobacter acinonychis. Sialyltransferases are enzymes that transfer sialic acids, which are acidic sugars, to a variety of acceptor molecules. These sugar acids play important roles in many physiological and pathological processes related to diseases in humans. Recently, the development of therapeutic proteins for the treatment of numerous diseases has gained enormous interest. There, correct glycosylation is particularly important, since structural instabilities pose a problem and may negatively influence therapeutic efficacies. The modification of therapeutic proteins by engineered, sialylated glycans offers a promising and elegant strategy to overcome this problem. Thus, the detailed characterization of sialyltransferases for the transfer of sialic acids as well engineering approaches to obtain improved enzymes is of high importance for the research community. The chosen a2,6-sialyltransferase HAC1268 is of particular interest, since it catalyzes the transfer of sialic acids to human glycoproteins making it suitable for engineering of therapeutic proteins with human-like glycosylation that would, thus, be tolerated by the human organism. HAC1268 is the so far only enzyme with that specificity within a specific group of sugar transferases. This novel enzyme shall be investigated in the laboratory of Prof. Warren W. Wakarchuk at the Ryerson University in Toronto, where relevant expertise is provided. We hypothesize that the detailed characterization of this enzyme by mutational analysis will significantly add to the understanding of the whole enzyme family. By directed evolution, stability, activity and substrate specificity of this enzyme shall be optimized and the improved mutant enzymes will be tested for their ability to modify therapeutic proteins with sialic acids serving to improve stability and half-life. This project will shed light on the mechanism of a specific group of sialyltransferases and open new possibilities for the generation of humanized therapeutic proteins with possible medical applications in the future.

The project Enzyme Engineering of an a2,6-Sialyltransferase from Helicobacter acinonychis for Sialylation of Therapeutic Proteins aimed at investigating and engineering of the sialyltransferase HAC1268 from the bacterium Helicobacter acinonychis. Sialyltransferases are enzymes that transfer sialic acids, which are acidic sugars, to a variety of acceptor molecules. These sugar acids play important roles in many physiological and pathological processes related to diseases in humans. The development of therapeutic proteins for the treatment of numerous diseases has gained enormous interest. There, correct glycosylation is particularly important, since structural instabilities pose a problem and may negatively influence therapeutic efficacies. The modification of therapeutic proteins by engineered, sialylated glycans offers a promising and elegant strategy to overcome this problem. Thus, the detailed characterization of sialyltransferases for the transfer of sialic acids as well engineering approaches to obtain improved enzymes is of high importance for the research community. A novel a2,6-Sialyltransferase from Helicobacter was investigated in the laboratory of Prof. Warren W. Wakarchuk at the Ryerson University in Toronto. Our initial plan to use directed evolution to generate mutants of HAC1268 and to screen the mutant library by a FACS-based ultra-high-throughput screening approach resulted in an accumulation of clones with higher activity that showed no mutations in the gene and increased activity was due to modifications in the vector backbone. We therefore had to modify the project and used a rational engineering approach of an uncharacterized but similar enzyme (79% sequence identity) from Helicobacter cetorum and compared this enzyme to a human ST6Gal1 and a Photobacterium sp. sialyltransferase using glycoprotein substrates in a 96-well microtiter-plate-based assay. By doing so, we were able to demonstrate that the recombinant a2,6-sialyltransferase from H. cetorum is an excellent catalyst for modification of N-linked glycans of different therapeutic proteins.