Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are gener ally low, but can increase rapidly and transiently when cells are exposed t o mitogenic agents and other stimuli. This increase is largely due to incre ased activity of sphingosine kinase (SK), the enzyme that catalyses its for mation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activit y and possible activation mechanisms. The enzyme was purified to homogeneit y from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chro matography. This resulted in a purification of over 10(5)-fold from the ori ginal placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E, coli-de rived SK purified to homogeneity. To establish whether post-translational m odifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E . coli, where such modifications would not occur. The premise for this stud y was that posttranslational modifications are likely to cause conformation al changes in the structure of SK, which may result in detectable changes i n the physico-chemical or catalytic properties of the enzyme. Thus the enzy mes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all c ases, both the native and recombinant SKs displayed remarkably similar prop erties, indicating that post-translational modifications are not required f or basal activity of human SK.