Amino acid residues important for CMP-sialic acid recognition by the CMP-sialic acid transporter: analysis of the substrate specificity of UDP-galactose/CMP-sialic acid transporter chimeras
| Autor: | Nobuhiro Ishida, Kazuhisa Aoki, Masao Kawakita, Masayoshi Sakaguchi, Yasusato Sugahara, Taro Takeshima-Futagami, Eriko Uehara, Yutaka Sanai |
|---|---|
| Rok vydání: | 2012 |
| Předmět: |
Monosaccharide Transport Proteins
Recombinant Fusion Proteins Amino Acid Motifs Mutant Mutation Missense Context (language use) CHO Cells Biology Nucleotide sugar Biochemistry Uridine Diphosphate Substrate Specificity chemistry.chemical_compound Residue (chemistry) Chimera (genetics) Cricetulus Cricetinae Cytidine Monophosphate Animals Galactose Biological Transport Transporter N-Acetylneuraminic Acid Transmembrane protein Protein Structure Tertiary carbohydrates (lipids) Transmembrane domain chemistry Nucleotide Transport Proteins Tyrosine |
| Zdroj: | Glycobiology. 22:1731-1740 |
| ISSN: | 1460-2423 0959-6658 |
| DOI: | 10.1093/glycob/cws116 |
| Popis: | In our previous studies, we demonstrated that chimeric molecules of the CMP-sialic acid (CMP-Sia) transporter (CST) and the UDP-galactose (Gal) transporter (UGT) in which the seventh transmembrane helix-containing segment was derived from the CST could transport both CMP-Sia and UDP-Gal and that the CST-derived seventh transmembrane helix segment was sufficient for the chimera to recognize CMP-Sia in the otherwise UGT context. In this study, we continued to more precisely define the submolecular region that is necessary for CMP-Sia recognition, and we demonstrated that the N-terminal half of the seventh transmembrane helix of CST is essential for the CMP-Sia transport mediated by the chimeric transporters. We further showed that Tyr214Gly and Ser216Phe mutations of a chimeric transporter that was capable of transporting both CMP-Sia and UDP-Gal led to the selective loss of CMP-Sia transport activity without affecting UDP-Gal transport activity. Conversely, when a residue in a chimeric transporter that was active for UDP-Gal transport but not CMP-Sia transport was replaced by Tyr, so that Tyr occupied the same position as in the CMP-Sia transporter, the resulting mutant chimera acquired the ability to transport CMP-Sia. These results demonstrated that Tyr214 and Ser216, located in the seventh transmembrane helix of the human CST, are critically important for the recognition of CMP-Sia as a transport substrate. Identification of determinants critical for the discrimination between relevant and irrelevant substrates will advance our understanding of the mechanisms of substrate recognition by nucleotide sugar transporters. |
| Databáze: | OpenAIRE |
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