Sphingosine 1-phosphate lyase enzyme assay using a BODIPY-labeled substrate

Sphingosine 1-phosphate (S1P) lyase (SPL) is a highly conserved enzyme responsible for irreversible catabolism of phosphorylated sphingoid bases generated through the degradation and recycling of membrane sphingolipids [1]. The reaction requires pyridoxal 5′-phosphate as the cofactor and results in the cleavage of the phosphorylated sphingoid base at the C2–C3 carbon–carbon bond, yielding a long-chain aldehyde and ethanolamine phosphate products. The major phosphorylated sphingoid base in humans is S1P, which is synthesized in most cell types and circulates in blood and lymph at high concentrations [2]. S1P signals through a family of five known G protein-coupled membrane receptors that regulate cell survival, migration, and complex processes such as vascular maturation and lymphocyte trafficking [3–5]. SPL activity in tissues and endothelium are required to regulate circulating S1P levels and maintain the chemical gradient that facilitates S1P-mediated lymphocyte egress from peripheral lymphoid organs and thymus [6,7]. Inhibition of SPL through genetic or pharmacological approaches has been shown to block lymphocyte trafficking, indicating that SPL may be a useful target for immune modulation [8]. The SPL gene and gene product are highly conserved throughout evolution and are essential in vertebrates and invertebrates, although its deletion in plants did not lead to deleterious effects [9,10]. Studies in model organisms have demonstrated that SPL expression is necessary for normal development, survival, and tissue homeostasis. The requirement for SPL in mammals is primarily attributed to its regulation of S1P. However, in Leishmania major, a null mutation of SPL blocks infectivity of the parasite due to ethanolamine depletion, demonstrating that product regulation may be critical for some physiological processes [11]. The standard SPL assay employs a tritiated dihydrosphingosine 1-phosphate substrate labeled at C4 and C5 [12]. The assay is radioactive, time consuming, and the substrate is commercially available from only a single source. We previously developed a fluorescent assay employing an ω-labeled NBD–S1P substrate [13]. Although this assay is convenient and involves less exposure to hazardous materials than 4,5-[3H]-dihydro-S1P, the 7-nitrobenz-2-oxa-1,3-diazole (NBD) fluorophore has the disadvantages of being photolabile and polar [14]. The fluorophore consisting of a dipyrrometheneboron difluoride chelate (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), which is referred to under the trademark BODIPY, possesses several distinctive and useful characteristics, including photochemical and chemical stability, efficient partitioning into membranes, and high molar absorption coefficient and fluorescence quantum yield, which represent significant improvements over the NBD fluorophore [15]. In this study, we have developed a BODIPY-labeled SPL substrate and demonstrated that it can be used effectively to measure SPL activity in biological samples.