Serine-palmitoyltransferase (SPT) catalyzes the rate-limiting step of the de novo synthesis of sphingolipids. SPT is considered to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Here we report the identification of a novel, third, SPT subunit (SPTLC3) that shows 68% homology to the SPTLC2 subunit. Quantitative real-time PCR revealed that SPTLC3 expression is highly variable between different human tissues and cell lines. The highest expression was observed in placenta tissue and human trophoblast cell lines. The overexpression of SPTLC3 in Hek293 cells, which otherwise have very little endogenous SPTLC3, led to a 2-to 3-fold increase in cellular SPT activity. Silencing of SPTLC3 expression in HepG2 cells or human trophoblast cells by transfecting SPTLC3-specific siRNA resulted in a significant reduction of cellular SPT activity. The expression of two SPT isoforms could be a cellular mechanism to adjust SPT activity to tissue-specific requirements of sphingolipid synthesis.Sphingolipids are a ubiquitously distributed class of lipids that can be found in all higher organisms. Sphingoid bases confer important structural properties to membranes and to their partition into microdomains (membrane rafts) and modulate the activities of various enzymes such as protein kinases, protein phosphatases, and phospholipases in cells or cell-free systems (1). They are involved in many cellular events, including proliferation, differentiation, senescence, apoptosis, and inflammatory response (2). De novo sphingolipid biosynthesis is initiated by the condensation of L-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. This pyridoxal 5-phosphate (PLP) 2 -dependent reaction is catalyzed by the serinepalmitoyltransferase (SPT, EC 2.3.1.50). SPT is believed to be a heterodimer and intracellularly bound to the outer membrane of the endoplasmic reticulum (3, 4). The two SPT subunits SPTLC1 (55 kDa) and SPTLC2 (65 kDa) show a mutual similarity of ϳ20% and are highly conserved among species. Although both subunits seem to be required for enzyme activity, SPTLC2 is considered to be the catalytic subunit due to the presence of a PLP binding site (3).SPT activity was detected in various tissues, including brain, lung, liver, kidney, and muscle (5) and is essential for embryonic development, because homozygous SPTLC1 and SPTLC2 knock-out mice die during embryogenesis (6).In contrast to the membrane-bound mammalian isoform a soluble, homodimeric SPT isoform (sSPT) was found in the sphingolipid producing prokaryote Sphingomonas paucimobilis (7,8). A third 10-kDa subunit was identified in yeast, but no mammalian homologue has yet been described (9). Here we report the identification and initial characterization of a previously unknown third SPT subunit (SPTLC3) in mammalian cells. MATERIALS AND METHODSGeneral-All Chemicals, unless otherwise stated, were purchased from Sigma. The direct Topo Cloning Vector pcDNA3.2 was from Invitrogen. Protein A-agarose was from Roche Applied Science. Anti-V5 tag monoclonal antibodies ...

Aims/hypothesis Sphingolipid synthesis is typically initiated by the conjugation of L-serine and palmitoyl-CoA, a reaction catalysed by serine palmitoyltransferase (SPT). SPT can also metabolise other acyl-CoAs (C 12 to C 18 ) and other amino acids such as L-alanine and glycine, giving rise to a spectrum of atypical sphingolipids. Here, we aimed to identify changes in plasma levels of these atypical sphingolipids to explore their potential as biomarkers in the metabolic syndrome and diabetes.Methods We compared the plasma profiles of ten sphingoid bases in healthy individuals with those of patients with the metabolic syndrome but not diabetes, and diabetic patients (n=25 per group). The results were verified in a streptozotocin (STZ) rat model. Univariate and multivariate statistical analyses were used. Results Deoxysphingolipids (dSLs) were significantly elevated (p ¼ 5 Â 10 À6 ) in patients with the metabolic syndrome (0.11±0.04 μmol/l) compared with controls (0.06±0.02 μmol/l) but did not differ between the metabolic A. Othman and M. F. Rütti contributed equally to this study. Diabetologia (2012) 55:421-431 DOI 10.1007/s00125-011-2384 syndrome and diabetes groups. Levels of C 16 -sphingosinebased sphingolipids were significantly lowered in diabetic patients but not in patients with the metabolic syndrome but without diabetes (p=0.008). Significantly elevated dSL levels were also found in the plasma and liver of STZ rats. A principal component analysis revealed a similar or even closer association of dSLs with diabetes and the metabolic syndrome in comparison with the established biomarkers. Conclusions/interpretation We showed that dSLs are significantly elevated in patients with type 2 diabetes mellitus and non-diabetic metabolic syndrome compared with healthy controls. They may, therefore, be useful novel biomarkers to improve risk prediction and therapy monitoring in these patients.

BackgroundSphingoid bases are formed from the precursors L-serine and palmitoyl-CoA-a reaction which is catalyzed by the serine-palmitoyltransferase (SPT). SPT metabolizes, besides palmitoyl-CoA also other acyl-CoAs but shows also variability towards the use of other amino acid substrates. The enzyme is also able to metabolize alanine, which results in the formation of an atypical deoxy-sphingoid base (DSB). This promiscuous activity is greatly increased in the case of the sensory neuropathy HSAN1, and pathologically elevated DSB levels have been identified as the cause of this disease. Clinically, HSAN1 shows a pronounced similarity to the diabetic sensory neuropathy (DSN), which is the most common chronic complication of diabetes mellitus. Since serine and alanine metabolism is functionally linked to carbohydrate metabolism by their precursors 3-phosphoglycerate and pyruvate, we were interested to see whether the levels of certain sphingoid base metabolites are altered in patients with diabetes.ResultsIn a case-control study we compared plasma sphingoid base levels between healthy and diabetic individuals. DSB levels were higher in the diabetic group whereas C16 and C18 sphingoid bases were not significantly different. Plasma serine, but not alanine levels were lower in the diabetic group. A subsequent lipoprotein fractionation showed that the DSBs are primarily present in the LDL and VLDL fraction.ConclusionOur results suggest that DSBs are a novel category of plasma biomarkers in diabetes which reflect functional impairments of carbohydrate metabolism. Furthermore, elevated DSB levels as we see them in diabetic patients might also contribute to the progression of the diabetic sensory neuropathy, the most frequent complication of diabetes.

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 C 16 -sphinganine and C 16 -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 C 16 -sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C 16 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 C 16 -sphingoid bases and that sphingolipids with a C 16 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 palmitoylCoA 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 ␣-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 compose...

Serine palmitoyltransferase (SPT) catalyzes the condensation of L-serine and palmitoyl-CoA, which is the ratelimiting step in the de novo synthesis of sphingolipids. SPT activity is commonly measured by monitoring the incorporation of radiolabeled L-serine into 3-ketodihydrosphingosine.