Biomolecules & Therapeutics

The Korean Society of Applied Pharmacology (한국응용약물학회)
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Development of a Label-Free LC-MS/MS-Based Glucosylceramide Synthase Assay and Its Application to Inhibitors Screening for Ceramide-Related Diseases

  • Fu, Zhicheng (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Yun, So Yoon (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Won, Jong Hoon (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Back, Moon Jung (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Jang, Ji Min (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Ha, Hae Chan (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Lee, Hae Kyung (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Shin, In Chul (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Kim, Ju Yeun (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Kim, Hee Soo (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University) ;
  • Kim, Dae Kyong (Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University)
  • Received : 2018.07.04
  • Accepted : 2018.08.09
  • Published : 2019.03.01
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Abstract

Ceramide metabolism is known to be an essential etiology for various diseases, such as atopic dermatitis and Gaucher disease. Glucosylceramide synthase (GCS) is a key enzyme for the synthesis of glucosylceramide (GlcCer), which is a main ceramide metabolism pathway in mammalian cells. In this article, we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine GCS activity using synthetic non-natural sphingolipid C8-ceramide as a substrate. The reaction products, C8-GlcCer for GCS, could be separated on a C18 column by reverse-phase high-performance liquid chromatography (HPLC). Quantification was conducted using the multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z $588.6{\rightarrow}264.4$ for C8-GlcCer at positive ionization mode. The calibration curve was established over the range of 0.625-160 ng/mL, and the correlation coefficient was larger than 0.999. This method was successfully applied to detect GCS in the human hepatocellular carcinoma cell line (HepG2 cells) and mouse peripheral blood mononuclear cells. We also evaluated the inhibition degree of a known GCS inhibitor 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) on GCS enzymatic activity and proved that this method could be successfully applied to GCS inhibitor screening of preventive and therapeutic drugs for ceramide metabolism diseases, such as atopic dermatitis and Gaucher disease.

Keywords

References

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