Characterizing Inhibition of Recombinant Sialyltransferases
Creators: Jerasi, Jeremy
Publisher: name: University of Alberta Library
Year: 2023

<p>Sialic acid is a monosaccharide found at the non-reducing end of cell surface glycans. Among the many important roles in mammals, sialic acid commonly serves as a ligand for glycan binding proteins, such as in host self-recognition (Siglecs), response to infection (Selectins), and in viral infection (Hemagglutinins). Sialic acid plays important roles in health and disease, as evidenced by upregulation of Neu5Ac in cancers, as well as in viral and bacterial attachment. The enzymes that catalyze the transfer of sialic acid to glycans are called sialyltransferases, and are localized in the trans-Golgi. In humans, twenty sialyltransferases are present and show expression across tissues. As such, the biological roles of sialic acid expression on the cell surface are difficult to probe, and vary between tissues. Chemical tools that modulate cell surface sialic acid have the potential to elucidate these roles, and offer therapeutic potential in pathologies characterized by dysregulated sialic acid expression. In the context of sialic acid, such a strategy involves the direct inhibition of sialyltransferases. While many small molecules have been studied for their ability to inhibit sialyltransferases in vitro, few have the ability to do so in cells.<br>
CMP-3Fax-Neu5Ac is reported as one of the only sialyltransferase inhibitors capable of decreasing cell surface sialic acid in cells. Two mechanisms-of-action have been proposed: direct inhibition of sialyltransferases and feedback inhibition of sialic acid biosynthesis. More studies are needed to identify which is the dominant mechanism. Studying CMP-3Fax-Neu5Ac inhibition of sialyltransferases in controlled enzymatic reactions, outside of the context of a living system, allows for testing of only the first mechanism-of-action. To do this, recombinant sialyltransferases are needed, as well as robust inhibition assays that can detect rates of reaction.<br>
Here, a recombinant cloning strategy was developed and implemented to generate soluble human sialyltransferases in a mammalian expression system. These constructs consist of the catalytic region of a given sialyltransferase fused to an IgG Fc domain, also containing two purification tags (His6 and Strep II Tag). The catalytic activities of purified constructs were<br>
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assessed using a quantitative enzymatic assay and found to exhibit similar properties to recombinant sialyltransferases generated previously in the literature. To investigate inhibition, a fluorescence polarization(FP)-based assay was developed. While optimizing the assay, reaction conditions which influenced the observed inhibitor potencies were characterized, finding that reaction buffer pH, buffer composition, and acceptor glycoprotein concentrations significantly altered the IC50 values generated in the FP-based assay. Using the optimized conditions, the inhibitory potency of CMP-3Fax-Neu5Ac and CMP-7F-Neu5Ac were determined towards human ST6Gal1 and ST3Gal1. The IC50 values measured for CMP-3Fax-Neu5Ac were approximately 15-30 μM towards both sialyltransferases, corroborating the literature evidence of the direct mode of inhibition. Then, IC50 values were measured for CMP-7F-Neu5Ac, with potencies of approximately 15-50 μM. This is the first demonstration of CMP-7F-Neu5Ac acting as a competitive sialyltransferase inhibitor, which will lay the groundwork for future studies of this compound in cells. Finally, the polysialyltransferase ST8Sia2 was assessed using the FP-based inhibition assay, finding that autopolysialylation activity could be detected and inhibited with nucleotide inhibitor CTP. The tools generated in this Thesis will pave the way for the identification of biologically relevant inhibitors which selectively inhibit sialyltransferases and may offer great therapeutic potential.</p>