Abnormal N-glycosylation of a novel missense creatine transporter mutant, G561R, associated with cerebral creatine deficiency syndromes alters transporter activity and localization

Tatsuki Uemura, Shingo Ito, Yusuke Ohta, Masanori Tachikawa, Takahito Wada, Tetsuya Terasaki, Sumio Ohtsuki

Research output: Contribution to journalArticle

5 Citations (Scopus)

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 wildtype 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.

Original languageEnglish
Pages (from-to)49-55
Number of pages7
JournalBiological and Pharmaceutical Bulletin
Volume40
Issue number1
DOIs
Publication statusPublished - 2017 Jan 1

Keywords

  • Cerebral creatine deficiency syndromes
  • Creatine
  • Creatine transporter
  • N-linked glycosylation

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

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