title: 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.
abstract: 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.
authors: Department of Applied Chemistry, Faculty of Engineering,  Kogakuin University, 2,665-1 Nakano-cho, Hachioji, Tokyo 192-0015,  Japan.
authors: Taro
authors: T
authors: Takeshima-Futagami
authors: Masayoshi
authors: M
authors: Sakaguchi
authors: Eriko
authors: E
authors: Uehara
authors: Kazuhisa
authors: K
authors: Aoki
authors: Nobuhiro
authors: N
authors: Ishida
authors: Yutaka
authors: Y
authors: Sanai
authors: Yasusato
authors: Y
authors: Sugahara
authors: Masao
authors: M
authors: Kawakita
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003568:Cytidine Monophosphate / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D033704:Nucleotide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000235:genetics; D014530:Uridine Diphosphate / Q000378:metabolism; D000099237:UDP-Galactose Translocators
chemical_list: D003568:Cytidine Monophosphate; D005690:Galactose; D009004:Monosaccharide Transport Proteins; D019158:N-Acetylneuraminic Acid; D033704:Nucleotide Transport Proteins; D011993:Recombinant Fusion Proteins; D014443:Tyrosine; D014530:Uridine Diphosphate; D000099237:UDP-Galactose Translocators