Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
1-O-Hexadecyl-2-desoxy-2-amino-sn-glycerol, a substrate for human sphingosine kinase
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 The sequences recovered appear to belong to at least three different kinases (P.P. Van Veldhoven, unpublished data).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.
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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.
References (29)
- et al.
J. Biol. Chem.
(1998) - et al.
J. Biol. Chem.
(1991) - et al.
Bioorg. Med. Chem. Lett.
(1999) - et al.
FEBS Lett.
(1981) - et al.
Biochim. Biophys. Acta
(2001) - et al.
FEBS Lett.
(1994) - et al.
J. Biol. Chem.
(1998) - et al.
Genomics
(1996) - et al.
J. Biol. Chem.
(1998) - et al.
FEBS Lett.
(2000)
Gene
Anal. Biochem.
Biochim. Biophys. Acta
Adv. Lipid Res.
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