An interaction between SLC35A1 and ST3Gal4 is differentially affected by CDG-causing mutations in the SLC35A1 gene
Wiertelak, W., Olczak, M. and Maszczak-Seneczko, D. (2022) An interaction between SLC35A1 and ST3Gal4 is differentially affected by CDG-causing mutations in the SLC35A1 gene. Biochem. Biophys. Res. Commun. 635, 46–51. DOI: 10.1016/j.bbrc.2022.10.019
ST3Gal4 is a mammalian sialyltransferase that uses the substrate CMP-sialic acid (CMP-Sia) to attach terminal sugars (sialic acids) to N-glycans via α2,3 linkage. The multitransmembrane transporter SLC35A1 delivers CMP-Sia from the nucleus to the Golgi lumen and has been shown to associate as homodimers. Mutations in the SLC35A1 gene are associated with a subtype of a congenital disorder of glycosylation (CDG). Earlier research showed that two CDG-causing mutations in the SLC35A1 gene (T156R and E196K) reduced or eliminated CMP-Sia transport.
The goal of this study was to determine if ST3Gal4 interacts with the wild-type SLC35A1. This interaction was tested using a two-subunit NanoBiT® luciferase fused in various orientations with ST3Gal4 and wild-type SLC35A1 then assessed in a luminescent protein interaction assay. The data showed that ST3Gal4 and wild-type SLC35A1 associated in HEK293T cells. To test if the CDG mutations in SLC35A1 could restore N-glycan sialylation, HPLC analysis of wild-type and SLC35A1 knockout HEK293T cells were compared with results from the knockout cells expressing T156R or E196K SLC35A1 mutations. Both mutations restored sialylation with the T156R mutant providing greater correction, nearly matching the wild type cells. To examine if the T156R and E196K SLC35A1 mutations also interacted with the ST3Gal4 sialyltransferase, the same two-subunit luciferase complementation assay used for the wild-type SLC35A1 was repeated with the CDG-causing variants. Results show that the T156R mutant interacted with STEGal4, generating luminescence similarly to wild-type SLC35A1. In contrast, the E196K mutation did not associate with STEGal4.The researchers noted that this study improved understanding about the interactions between nucleotide sugar transporters and sialyltransferases, and the role the association may play in CDG.
Keywords: sialylation, N-Glycans, SLC35A1, ST3Gal4, congenital disorder of glycosylation, split-luciferase complementation assay
