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