title: Abnormal N-Glycosylation of a Novel Missense Creatine Transporter Mutant, G561R, Associated with Cerebral Creatine Deficiency Syndromes Alters Transporter Activity and Localization.
abstract: Cerebral creatine deficiency syndromes (CCDSs) are caused by loss-of-function mutations in creatine transporter (CRT, SLC6A8), which transports creatine at the blood-brain barrier and into neurons of the central nervous system (CNS). This results in low cerebral creatine levels, and patients exhibit mental retardation, poor language skills and epilepsy. We identified a novel human CRT gene missense mutation (c.1681 G>C, G561R) in Japanese CCDSs patients. The purpose of the present study was to evaluate the reduction of creatine transport in G561R-mutant CRT-expressing 293 cells, and to clarify the mechanism of its functional attenuation. G561R-mutant CRT exhibited greatly reduced creatine transport activity compared to wild-type CRT (WT-CRT) when expressed in 293 cells. Also, the mutant protein is localized mainly in intracellular membrane fraction, while WT-CRT is localized in plasma membrane. Western blot analysis revealed a 68 kDa band of WT-CRT protein in plasma membrane fraction, while G561R-mutant CRT protein predominantly showed bands at 55, 110 and 165 kDa in crude membrane fraction. The bands of both WT-CRT and G561R-mutant CRT were shifted to 50 kDa by N-glycosidase treatment. Our results suggest that the functional impairment of G561R-mutant CRT was probably caused by incomplete N-linked glycosylation due to misfolding during protein maturation, leading to oligomer formation and changes of cellular localization.
authors.affiliation: Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Uemura
authors.forename: Shingo
authors.initials: S
authors.lastname: Ito
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Ohta
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.forename: Takahito
authors.initials: T
authors.lastname: Wada
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
authors.forename: Sumio
authors.initials: S
authors.lastname: Ohtsuki
mesh_terms: D001692:Biological Transport; D001921:Brain / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003401:Creatine / Q000172:deficiency / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D020125:Mutation, Missense; D013577:Syndrome
chemical_list: D026901:Membrane Transport Proteins; C080679:creatine transporter; D003401:Creatine