Human and Murine Serine‐Palmitoyl‐CoA Transferase
Open Access
- 1 October 1997
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 249 (1) , 239-247
- https://doi.org/10.1111/j.1432-1033.1997.00239.x
Abstract
Serine palmitoyltransferase (SPT, EC 2.3.1.50) is the key enzyme in sphingolipid biosynthesis. It catalyzes the pyridoxal‐5’‐phosphate‐dependent condensation of L‐serine and palmitoyl‐CoA to 3‐oxo‐sphinganine. Human expressed‐sequence‐tag (EST) clones are similar to the two yeast genes for synthesis of long‐chain bases, LCB1 and LCB2, which are believed to encode two subunits of SPT [Buede, R., Pinto, W. J., Lester, R. L. & Dickson, R. C. (1991) J. Bacteriol. 173, 4325–5332; Nagiec, M. M., Baltisberger, J. A., Wells, G. B., Lester, R. L. & Dickson, R. C. (1994) Proc. Natl Acad. Sci. USA 91, 7899–7902]. We have cloned and characterized two complete human and murine cDNA sequences named hLCB1 & mLCB1 and hLCB2 & mLCB2, respectively, similar to the yeast LCB1 and LCB2 genes. Human embryonic kidney cells (HEK 293) transfected with murine sequences of LCB1 (mLCB1) and LCB2 (mLCB2) independently and in coexpression showed an overexpression of the transcripts on the mRNA and protein level. The enzymatic activity of cells expressing mLCB2 alone or coexpressed with mLCB1 was three times higher than the activity of untransfected HEK cells. mLCB1 expression was not required for the synthesis of 3‐oxo‐sphinganine in mammalian cells. Transcription/translation in vitro yielded mLCB1 (53 kDa) and mLCB2 (63 kDa). The two proteins do not contain a signal peptide nor are they glycosylated. The endogenous and overexpressed SPT activity were both sensitive to common SPT inhibitors. Labeling studies with [1‐14C]palmitic acid indicated that cell lines transfected with mLCB2 preferentially use the excess sphingoid bases for glucocerebroside and galactocerebroside synthesis. Our results provide conclusive genetic and biochemical evidence that the human and murine LCB2 genes described here encode serine palmitoyltransferase. Further studies will be required to unravel the function of the LCB1 gene in mammalian cells.Keywords
This publication has 37 references indexed in Scilit:
- Sphingolipid synthesis: Identification and characterization of mammalian cDNAs encoding the Lcb2 subunit of serine palmitoyltransferaseGene, 1996
- Serine Palmitoyltransferase Is the Primary Target of a Sphingosine-like Immunosuppressant, ISP-1/MyriocinBiochemical and Biophysical Research Communications, 1995
- Heme biosynthesis in mammalian systems: Evidence of a schiff base linkage between the pyridoxal 5′‐phosphate cofactor and a lysine residue in 5‐aminolevulinate synthaseProtein Science, 1993
- Biosynthesis of sphingolipids: Dihydroceramide and not sphinganine is desaturated by cultured cellsBiochemical and Biophysical Research Communications, 1992
- Sphingolipid biosynthesis in cultured neuronsEuropean Journal of Biochemistry, 1991
- 2-Amino-3-ketobutyrate CoA ligase of Escherichia coli: stoichiometry of pyridoxal phosphate binding and location of the pyridoxyllysine peptide in the primary structure of the enzymeBiochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1990
- Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferaseGene, 1988
- Inhibition of Sphingolipid Synthesis by Cycloserine In Vitro and In VivoJournal of Neurochemistry, 1984
- Metabolism of Sphingosine Bases, V. Biosynthesis of Dihydrosphingosinein vitroHoppe-Seyler´s Zeitschrift Für Physiologische Chemie, 1968
- Metabolism of Sphingosine Bases, IV. 2-Amino-1-hydroxyoctadecane-3-one (3-oxodihydrosphingosine), the Common Intermediate in the Biosynthesis of Dihydrosphingosine and Sphingosine and in the Degradation of DihydrosphingosineHoppe-Seyler´s Zeitschrift Für Physiologische Chemie, 1967