Establishment of a novel, cell-based autotaxin assay

Anal Biochem. 2021 Oct 1:630:114322. doi: 10.1016/j.ab.2021.114322. Epub 2021 Jul 31.

Abstract

Autotaxin (ATX) plays an important role in (patho-)physiological lysophosphatidic acid (LPA) signaling. Here we describe the establishment of novel cell-based ATX assay formats. ATX-mediated LPA generation is detected by using a stable LPA receptor reporter cell line. In a first assay variant, ATX-mediated LPA generation is started in the absence of cells and the reaction mix is transferred to the reporter cells after stopping the reaction (two-tube assay). In a second assay variant, ATX is added to the reporter cells expressing the known autotaxin binding partners integrin β1, integrin β3 and the LPA receptor 1. LPA generation is started in the presence of cells and is detected in real-time (one-tube assay). Structurally diverse ATX inhibitors with different binding modes were characterized in both cell-based assay variants and were also tested in the well-established biochemical choline release assay. ATX inhibitors displayed similar potencies, regardless if the assay was performed in the absence or presence of cells, and comparable results were obtained in all three assay formats. In summary, our novel cell-based ATX assay formats are well-suited for sensitive detection of enzyme activity as well as for the characterization of ATX inhibitors in the presence and absence of cells.

Keywords: ATX; Cell-based assay; LPA receptor; Lipid metabolism; Method.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cells, Cultured
  • Humans
  • Lysophospholipids / chemistry
  • Lysophospholipids / metabolism
  • Models, Molecular
  • Phosphoric Diester Hydrolases / analysis*
  • Phosphoric Diester Hydrolases / metabolism

Substances

  • Lysophospholipids
  • Phosphoric Diester Hydrolases
  • alkylglycerophosphoethanolamine phosphodiesterase
  • lysophosphatidic acid