Role of 6-O-α-maltosyl-β-cyclodextrin in lysosomal cholesterol deprivation in Npc1-deficient Chinese hamster ovary cells

• Published in: Carbohydrate research 2018-01, Vol.455, p.54-61
• Main Authors: Okada, Yasuyo, Ueda, Erika, Kondo, Yuki, Ishitsuka, Yoichi, Irie, Tetsumi, Higashi, Taishi, Motoyama, Keiichi, Arima, Hidetoshi, Matuso, Muneaki, Higaki, Katsumi, Ohno, Kousaku, Nishikawa, Junichi, Ichikawa, Atsushi
• Format: Article
• Language: English
• Subjects: 6-O-α-maltosyl-β-cyclodextrin (Mal-βCD); Animals; beta-Cyclodextrins - chemistry; CHO Cells; Cholesterol - chemistry; Cholesterol/efflux; Chromatography, Liquid; Cricetinae; Cricetulus; Endocytosis; Endocytosis - physiology; Liquid chromatography-mass spectrometry; Lysosomal unesterified cholesterol; Lysosomes - chemistry; Mass Spectrometry; Npc1-deficient cells
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/j.carres.2017.11.003

We aimed to investigate whether 6-O-α-maltosyl-β-cyclodextrin (Mal-βCD) is incorporated into cells and lysosomes during the release of unesterified cholesterol in Npc1-deficient Chinese hamster ovary (CHO) cells (Npc1 KO cells) and CHO-JP17 cells (JP17 cells). Internalization of Mal-βCD in cells and lysosomes and extracellular release of lysosomal unesterified cholesterol were demonstrated by LC/MS/MS and LC/MS, respectively. Internalization of Mal-βCD was greater in Npc1 KO cells than in JP17 cells. The majority of internalized Mal-βCD in both cell types was metabolized by lysosomal α-glucosidase to 6-O-α-D-glucosyl-β-cyclodextrin (Glc-βCD). However, Mal-βCD did not directly enter the lysosomes prepared from cell homogenates. Mal-βCD-treated Npc1 KO cells and JP17 cells both released Mal-βCD and Glc-βCD, together with unesterified cholesterol, out of cells. The release of unesterified cholesterol by Mal-βCD in Npc1 KO cells was much greater than that in JP17 cells. This study is the first to report the influx of Mal-βCD into the lysosomes of Npc1 KO cells and JP17 cells, followed by generation of Glc-βCD, and the efflux of Mal-βCD/Glc-βCD and unesterified cholesterol to the extracellular fluid, based on the quantitative LC/MS analysis. [Display omitted] •6-O-α-Maltosyl(Mal)-βCD reduces lysosomal cholesterol storage of Npc1-mutant cells.•Mal-βCD is endocytosed, and accumulated in the lysosome of Npc1-mutant cells.•Mal-βCD accumulated in the lysosome is partly metabolized to Glc-βCD.•Mal/Glc-βCD and cholesterol are released from the lysosome and Npc1-mutant cells.•Intracellular behavior of Mal-βCD and cholesterol is quantitated by LC/MS.