The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C₁₆-sphinganine and C₁₆-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C₁₆-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C₁₆ backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C₁₆-sphingoid bases and that sphingolipids with a C₁₆ backbone constitute a significant proportion of human plasma sphingolipids.Sphingolipids comprise a class of bioactive lipids that contribute to plasma membrane and plasma lipoprotein formation and exert a broad range of cellular signaling functions such as cell proliferation, endocytosis, and the response of cells to inflammatory and apoptotic stress signals (1–4).Sphingolipids are derived from the aliphatic amino alcohol sphingosine, which is formed from the precursors l-serine and palmitoyl-CoA. The condensation of serine with palmitoyl-CoA is catalyzed by the enzyme serine palmitoyltransferase (SPT)³ (EC 2.3.1.50) and leads to the intermediate 3-ketodihydrosphingosine. 3-Ketodihydrosphingosine is then rapidly converted to dihydrosphingosine (sphinganine) and dihydroceramide. The desaturation of dihydroceramide generates ceramide, and the breakdown of ceramide by ceramidase finally forms sphingosine. The sphingosine backbone of ceramide is usually O-linked to a polar head group such as phosphocholine or carbohydrates and amide-linked to an acyl group. The combination of the sphingosine backbone with different head groups, in particular with various oligosaccharides, leads to a complex variety of different sphingolipid metabolites (5, 6). Moreover, it was shown recently that SPT is also able to use l-alanine as an alternative substrate, thereby generating the atypical sphingoid base 1-deoxysphinganine (7).SPT belongs to the family of pyridoxal phosphate-dependent α-oxoamine synthases. Other members of this family include 5-aminolevulinic acid synthase, 2-amino-3 ketobutyrate ligase, and 8-amino-7-oxononanoate synthase (8). SPT is ubiquitously expressed, and enzyme activity has been detected in all tissues tested so far including brain, lung, liver, kidney, and muscle (9). SPT is essential for embryonic development, and homozygous SPT knock-out mice are not viable (10). SPT has been believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. The two subunits SPTLC1 and SPTLC2 show a similarity at AA level of ∼20% and are highly conserved among species. Although both subunits seem to be required for enzyme activity, only the SPTLC2 subunit contains a pyridoxal phosphate binding motif (8, 11).Recently, we identified and cloned a novel third SPT subunit (SPTLC3) (12). The SPTLC3 sequence shows 68% homology to the SPTLC2 subunit and also includes a pyridoxal phosphate consensus motive. The SPTLC3 gene is present in mammals, birds, and some lower vertebrates like fish (Danio rerio) and frog (Xenopus laevis) but not in invertebrate lineages. The SPTLC3 mRNA has been detected in most human tissues with a particularly high expression in placenta (12), indicating a special role for SPTLC3 during development and pregnancy. By using immunoprecipitation, native gel analysis, cross-linking studies, and size exclusion chromatography, it was demonstrated that the native SPT enzyme contains all three subunits and forms a protein complex with a molecular mass of about 460 kDa (13). However, because SPTLC2 and SPTLC3 are encoded by two distinct genes and expressed within the same cell types, we assume a distinct function for the two subunits. One of these differences might be altered substrate affinity or enzymatic activity. This issue is addressed in the present study.