Molecular Cloning, Expression, and Purification of the Human Sialyltransferase ST8Sia6

Abstract

Glycans linked to cell surface proteins are one of the most significant and complex biological molecules that play a role in human health and disease. Sialic acid (Sia) is found at the outermost position of glycan chains, facilitating a key role in cell-to-cell signaling and ligand-receptor interactions. PolySia is a long homopolymer consisting of 8 to 400+ residues of α2,8-linked Sias, which differs from widely expressed α2,3- and α2,6-linked monoSia. There is considerable knowledge surrounding the critical roles of polySia in cell migration, and more recently, the relationship between sialylation and immune attenuation. However, oligoSia, which consists of 2 to 8 Sia residues, is often overlooked. Due to the lack of tools, such as commercial antibodies, there is very limited data on this glycan. This project aims to shed light on the mystery that is oligoSia by investigating the biosynthesis of oligosialylated proteins. The sialyltransferase enzymes ST8Sia3 and ST8Sia6 are likely responsible for synthesizing oligoSia. ST8Sia3 is mostly limited to the nervous system, whereas ST8Sia6 mRNA is more broadly expressed and increases in many cancers, including breast cancer and melanoma, suggesting a potential role in cancer progression. However, the endogenous protein targets of ST8Sia6 remain unknown. Here, ST8Sia6 truncations were cloned and expressed using both a bacterial and eukaryotic system. The ST8Sia6 constructs were tested for activity on various Core 1 substrates to evaluate substrate specificity. The protein expression from the bacterial system was strong, however, little to no activity on the test proteins was observed. Expression in the eukaryotic system yielded insight into the potential post-translational modifications of the enzyme and the folding efficiency of the truncated variants. Together, elucidating the substrate specificity of ST8Sia6 will advance our understanding of the role of oligoSia in both health and disease.