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

We aimed to investigate whether 6-O-alpha-maltosyl-beta-cyclodextrin (Mal-beta CD) 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-beta CD in cells and lysosomes and extracellular release of lysosomal unesterified cholesterol were demonstrated by LC/MS/MS and LC/MS, respectively. Internalization of Mal-beta CD was greater in Npc1 KO cells than in JP17 cells. The majority of internalized Mal-beta CD in both cell types was metabolized by lysosomal alpha-glucosidase to 6-O-alpha-Dglucosyl- beta-cyclodextrin (Glc-beta CD). However, Mal-beta CD did not directly enter the lysosomes prepared from cell homogenates. Mal-beta CD-treated Npc1 KO cells and JP17 cells both released Mal-beta CD and Glc-beta CD, together with unesterified cholesterol, out of cells. The release of unesterified cholesterol by Mal-beta CD in Npc1 KO cells was much greater than that in JP17 cells. This study is the first to report the influx of Mal-beta CD into the lysosomes of Npc1 KO cells and JP17 cells, followed by generation of Glc-beta CD, and the efflux of Mal-beta CD/Glc-beta CD and unesterified cholesterol to the extracellular fluid, based on the quantitative LC/MS analysis. (C) 2017 Elsevier Ltd. All rights