Abstract
The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities. Mutations in either induce congenital disorders of glycosylation. Despite the biomedical relevance, mechanisms of substrate specificity are unclear. To address this critical issue, we utilized a structure-guided mutagenesis strategy and assayed a series of SLC35A2 and SLC35A1 mutants using a rescue approach. Our results suggest that three pockets in the central cavity of each transporter provide substrate specificity. The pockets comprise (1) nucleobase (residues E52, K55, and Y214 of SLC35A1; E75, K78, N235, and G239 of SLC35A2); (2) middle (residues Q101, N102, and T260 of SLC35A1; Q125, N126, Q129, Y130, and Q278 of SLC35A2); and (3) sugar (residues K124, T128, S188, and K272 of SLC35A1; K148, T152, S213, and K297 of SLC35A2) pockets. Within these pockets, two components appear to be especially critical for substrate specificity. Y214 (for SLC35A1) and G239 (for SLC35A2) in the nucleobase pocket appear to discriminate cytosine from uracil. Furthermore, Q129 and Q278 of SLC35A2 in the middle pocket appear to interact specifically with the β-phosphate of UDP while the corresponding A105 and A253 residues in SLC35A1 do not interact with CMP, which lacks a β-phosphate. Overall, our findings contribute to a molecular understanding of substrate specificity and coordination in SLC35A1 and SLC35A2 and have important implications for the understanding and treatment of diseases associated with mutations or dysregulations of these two transporters.
Highlights
The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities
All were substituted with alanine, except G239, which was replaced with tyrosine because the corresponding residue in SLC35A1 is tyrosine that may contribute to substrate specificity
We proposed a three-pocket model for substrate coordination by nucleotide sugar transporters SLC35A1 and SLC35A2, and utilized this model to identify potential elements that determine the substrate specificity of the two transporters
Summary
The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities. SLC35 members are divided into seven subfamilies, from SLC35A to SLC35G [3] They transport a wide range of nucleotide sugars, including CMP-sialic acid, UDP-glucose, UDP-galactose, etc [1, 3]. Two of the best-characterized NSTs are CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 [3, 4]. The crystal structures of three NSTs, yeast GDP-mannose transporter Vrg4 [20, 21], maize CMP-sialic acid transporter [22], and murine SLC35A1 [23] have been reported. In maize CMP-sialic acid transporter, residues E42, K45, Y82, N86, K108, Y199, and K262 Though crystal structures of the two CMP-sialic acid transporters provide insights into the mechanisms of CMP-sialic acid coordination by CMP-sialic acid transporters, no crystal structure is available for UDP-galactose transporters, and little is known about how the two most distantly related transporters differentiate between different substrates
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