1-O-Hexadecyl-2-desoxy-2-amino-sn-glycerol, a substrate for human sphingosine kinase

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Abstract

The substrate specificity of human sphingosine kinase was investigated using a bacterially expressed poly(His)-tagged protein. Only the D-erythro isomer of the sphingoid bases, sphinganine and sphingenine, was effectively phosphorylated. Long chain 1-alkanols, alkane-1,2-diols, 2-amino-1-alkanol or 1-amino-2-alkanol and short chain 2-amino-1,3-alkanediols were very poor substrates, indicating that the kinase is recognizing the chain length and the position of the amino and secondary hydroxy group. A free hydroxy group at carbon 3 is not a prerequisite, however, since 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol was an efficient substrate with an apparent Km value of 3.8 μM (versus 15.7 μM for sphingenine). This finding opens new perspectives to design sphingosine kinase inhibitors. It also calls for some caution since it cannot be excluded that this ether lipid analogue is formed from precursors that are frequently used in research on platelet activating factor or from phospholipid analogues which are less prone to degradation.

Introduction

The phosphorylation of sphingoid bases, followed by the cleavage of the phosphorylated base, are the last steps in the degradation of sphingolipids [1], [2]. In addition, the phosphorylation reaction might be responsible for the generation of bioactive sphingenine-1-phosphate [3]. Amongst LEU2-tagged transposon gene disruption transformants of a yeast strain lacking sphingosine-phosphate lyase, some transformants, selected for growth on sphingenine, were found to be defective in the phosphorylation of sphinganine [4]. Further characterization of the clones at the DNA level revealed an open reading frame (YOR171c; renamed to LCB4) which encodes a long chain base kinase. A search in the yeast genome indicated the presence of a related kinase, coded by YLR260w (renamed LCB5) [4]. Based on the deduced amino acid sequences of both yeast kinases, the human databases were searched [5] in order to find ESTs, STSs or genomic sequences coding for sphingosine kinase. The sequences recovered appear to belong to at least three different kinases (P.P. Van Veldhoven, unpublished data). The kinase, derived from one of these cDNAs, was expressed in bacteria and further characterized with regard to substrate specificity.

Section snippets

Materials

The different sphinganine and sphingenine stereoisomers, and other lipids, including the isomers of 2-amino-3-phenyl-1-propanol and 2-amino-3-phenyl-1,3-propanediol and their derivatives, were obtained from Sigma, Aldrich, or Toronto Research Chemicals. Hexadecyl-1-phosphate was from Novabiochem, D-erythro-sphingenine-1-phosphate from Toronto Research Chemicals, and 1-O-hexadecyl-2-desoxy-2-amino-sn-glycerol, 1-O- and 3-O-octadecyl-sn-glycerol were from Bachem. N-Acetyl-sphingenine was prepared

Results and discussion

By homology to the yeast sphingosine kinases [4], a cDNA coding for a human kinase was delineated containing an ORF of 1152 bp. The deduced amino acid sequence displayed 28% identity to the yeast enzymes [4] and 85% to the mouse kinase [17] which was reported during our work. Five domains, characteristic for lipid kinases, as initially described by Kohama et al. [17], are present. Following deposition in the databank (AJ245504), other groups reported on the cloning of the same human cDNA [18],

Acknowledgements

This work was supported by grants from the ‘Interuniversitaire Attractiepolen (IUAP-P4/23)’ and the (FWO-Vlaanderen G.0240.98). The authors like to thank K. De Greef and G. Van der Hoeven for technical help.

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