Subunit composition of the mammalian serine-palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases Museer A. Lonea,1, Andreas J. Hülsmeiera, Essa M. Saiedb,c, Gergely Karsaia, Christoph Arenzc, Arnold von Eckardsteina, and Thorsten Hornemanna,1 aInstitute of Clinical Chemistry, University Hospital Zurich, University of Zurich, Zurich-8091, Switzerland; bInstitute for Chemistry, Suez Canal University, Ismailia, Egypt; and cInstitute for Chemistry, Humboldt Universität zu Berlin, 12489 Berlin, Germany Edited by Howard Riezman, University of Geneva, Geneva, Switzerland, and accepted by Editorial Board Member David J. Mangelsdorf, May 20, 2020 (received for review February 11, 2020) Sphingolipids (SLs) are chemically diverse lipids that have impor- SPT activity is metabolically controlled by negative feedback tant structural and signaling functions within mammalian cells. SLs regulation. This mechanism is well understood in yeast, where are commonly defined by the presence of a long-chain base (LCB) SPT activity is regulated by two phosphoproteins, Orm1 and that is normally formed by the conjugation of L-serine and Orm2 (9, 10). Another protein, Tsc3, is required for maximal palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reac- SPT activation (11). SPT together with Orm1 and Orm2, Tsc3, tion is mediated by the enzyme serine-palmitoyltransferase (SPT). and the phosphoinositide phosphatase Sac1 form a multisubunit However, SPT can also metabolize other acyl-CoAs, in the range of complex (10). Orthologs of these proteins are also found in C14 to C18, forming a variety of LCBs that differ by structure and mammals, but their roles in regulating SL metabolism are less function. Mammalian SPT consists of three core subunits: SPTLC1, well understood. Mammalian cells express three ORM orthologs SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously (ORMDL1, 2, and 3) but lack the regulatory phosphorylation expressed, SPTLC3 expression is restricted to certain tissues only. sites of yeast Orm proteins (10, 12). The polypeptides ssSPTa The influence of the individual subunits on enzyme activity is not and ssSPTb are functional orthologs of Tsc3 that appear to clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, modulate SPT activity and substrate affinity (13–15). While ssSPTa BIOCHEMISTRY we investigated the role of each subunit on enzyme activity and promotes canonical C18 LCB synthesis, ssSPTb is associated with the LCB product spectrum. We showed that SPTLC1 is essential for increased synthesis of C20 LCBs. In mice, a single gain-of-function activity, whereas SPTLC2 and SPTLC3 are partly redundant but mutation in ssSPTb (H56L) was shown to increase C20 LCB for- differ in their enzymatic properties. SPTLC1 in combination with mation in the brain, leading to retinopathy and central neuro- SPTLC2 specifically formed C18, C19, and C20 LCBs while the com- degeneration (14). However, the role of the individual SPT subunits bination of SPTLC1 and SPTLC3 yielded a broader product spec- trum. We identified anteiso-branched-C18 SO (meC18SO) as the with respect to SPT enzyme activity and substrate affinity has not primary product of the SPTLC3 reaction. The meC18SO was syn- yet been addressed systematically. thesized from anteiso-methyl-palmitate, in turn synthesized from In the present study, using SPTLC1-, SPTLC2-, and SPTLC3- a precursor metabolite generated in the isoleucine catabolic path- deficient cell models, we demonstrated that SPTLC1 is essential way. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins. Significance serine-palmitoyltransferase | long-chain base | omega-3-methyl- Sphingolipids (SLs) are complex lipids that constitute hundreds sphingosine of subspecies. All SLs share a long-chain base (LCB) as a de- fining structural component. LCBs are formed by serine- phingolipids (SLs) share the presence of a long-chain base palmitoyltransferase (SPT) in the first and rate-limiting step S(LCB) backbone as a common structural element. LCBs are of SL de novo synthesis. SPT consists of three subunits that show aliphatic amino alcohols and formed in the first and rate-limiting tissue-specific expression. In presence of the SPTLC3 subunit, the step of SL de novo synthesis (SI Appendix, Fig. S1). This reaction enzyme forms a spectrum of straight and branched LCBs with is catalyzed by the enzyme serine-palmitoyltransferase (SPT). distinct biochemical and biophysical properties. This alters the composition of cellular membranes and might influence the SPT is a pyridoxal 5‐phosphate (PLP)-dependent α‐oxoamino- dynamics of membrane-related transport and signaling events. transferase that consists of three core subunits—SPTLC1, SPTLC2, — SPTLC3 is particularly abundant in skin, and changes in SPT ac- and SPTLC3 that share a mutual homology. SPTLC2 is 68% tivity are related to dermal pathologies. Genetic variants of identical to SPTLC3 (84% similarity), whereas SPTLC1 is more SPTLC3 are associated with metabolic conditions such as distinct, sharing ∼21% identity (45% similarity) with SPTLC2 and dyslipidemia and atherosclerosis. SPTLC3 (1). The PLP-binding motif is present in SPTLC2 and SPTLC3 but not in SPTLC1. While SPTLC1 and SPTLC2 are Author contributions: M.A.L. and T.H. designed research; M.A.L., A.J.H., and G.K. per- ubiquitously expressed, SPTLC3 expression is restricted to specific formed research; E.M.S. and C.A. contributed new reagents/analytic tools; M.A.L., A.J.H., and G.K. analyzed data; and M.A.L., A.v.E., and T.H. wrote the paper. tissues, such as placenta, skin, and some glands (2–6). LCBs vary structurally within and across species. In mammals, The authors declare no competing interest. the most abundant LCB is sphingosine (SO; d18:1), which repre- This article is a PNAS Direct Submission. H.R. is a guest editor invited by the Editorial Board. sents ∼60% of the total LCBs in human plasma (7). The remaining Published under the PNAS license. LCBs differ with respect to chain length, desaturation, and hy- 1To whom correspondence may be addressed. Email: [email protected] or droxylation (reviewed in ref. 8). Plants and fungi, including yeast, [email protected]. mostly form phytosphingosine (phytoSO; t18:0), which is also pre- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ sent at low levels in humans. Insects mainly form short-chain LCBs doi:10.1073/pnas.2002391117/-/DCSupplemental. in the range of C14 to C16 (8). www.pnas.org/cgi/doi/10.1073/pnas.2002391117 PNAS Latest Articles | 1of8 Downloaded by guest on October 4, 2021 for formation of an active enzyme. In contrast, SPTLC2 and the absence of SPTLC1, we observed a concomitant loss of SPTLC3 are partly redundant but differ in function and substrate SPTLC2, whereas deletion of SPTLC2 did not influence SPTLC1 specificity. Furthermore, we identified a novel, methyl-branched levels (Fig. 1A). This suggested that SPTLC2 is unstable in the LCB as a specific product of human SPTLC3. Methyl-branched absence of SPTLC1, whereas SPTLC1 is stable on its own. LCBs are the major forms in lower invertebrates, such as Cae- Next, SPT activity was measured by the time-dependent in- 15 norhabditis elegans (16), but have not been reported in humans corporation of isotope-labeled L-serine (2,3,3-D3, N) into the until now. de novo formed LCBs. As one of the deuteriums is lost during the conjugation reaction (18), the de novo synthesized LCBs had Results an additional mass of +3 Da. In HAP1 wild-type (WT) cells, we SPTLC1 Is Indispensable for De Novo SPT Function. To investigate the observed a significant formation of C18SO+3, which was com- role of individual subunits on SPT activity, we generated SPTLC1 pletely abrogated in the absence of either SPTLC1 or SPTLC2 and SPTLC2 knockout (KO) HAP1 cell lines using a CRISPR/ (Fig. 1B). Reconstitution of SPTLC1 and STPLC2 expression in Cas9 approach. HAP1 cells are haploid and derived from chronic the respective null background restored enzyme activity (Fig. myelogenous leukemia (CML) cells (17). Natively, HAP1 cells 1C). SPTLC1 expression in the SPTLC1 KO cells rescued en- express SPTLC1 and SPTLC2 but not SPTLC3 (SI Appendix, Fig. dogenous SPTLC2 expression (Fig. 1D). Expression of SPTLC3 S2A). The CRISPR/Cas9 induced loss of SPTLC1 and SPTLC2 in the absence of SPTLC2 resulted in an active SPT enzyme but was confirmed by Western blot analysis (Fig. 1A). Surprisingly, in with significantly lower formation of C18SO+3 compared with C18SO C18SA A B 120 SPTLC2 + + - SPTLC1 + - + [kDa] 80 100 Calnexin 70 HAP1 cells SPTLC1 55 40 70 nd SPTLC2 55 pmoles/10 0 SPTLC1 + + - + SPTLC2 + + + - Myriocin - + - - C D WT SPTLC1 KO 100 C18SO C18SA ** Vector + + - - - SPTLC1-V5 - - + - - SPTLC2-V5 - - - + - SPTLC3-V5 - - - - + [kDa] 100 HAP1 cells Calnexin 50 70 70 Anti-V5 55 70 nd SPTLC1 55 nd pmoles/10 70 0 SPTLC2 Vector + - - - - + - - - 55 SPTLC1 - + - - - - + - - SPTLC2 - - + - + - - + - SPTLC3 - - - + + - - - + SPTLC1 KO SPTLC2 KO E F IP V5-tag IP V5-tag 1.5 Vector + - - - + - - - C16SO SPTLC1-V5 - + - - - + - - SPTLC2-V5 C17SO - - + - - - + - C19SO SPTLC3-V5 - - - + - - - + [kDa] 1.0 70 C20SO Anti-V5 55 HAP1 cells SPTLC1 55 0.5 70 SPTLC2 55 nd ndnd pmoles/10 0 Wild type SPTLC2 KO Vector SPTLC2 SPTLC3 Fig. 1. SPT composition and activity in SPTLC1- and SPTLC2-deficient HAP1 cells. (A) Western blot showing CRISPR/Cas9-mediated loss of SPTLC1 and SPTLC2 expression in the respective HAP1 KO lines. The loss of SPTLC1 led to a concomitant loss of SPTLC2 expression. Calnexin served as a loading control. (B) SPT 15 activity in SPTLC1- and SPTLC2-2 deficient cells compared with HAP1 WT cells. SPT activity was measured by the incorporation of (2,3,3-D3, N)-L-serine into de novo formed LCBs. One deuterium is lost during the conjugation reaction, which results in a mass shift of +3 Da for the de novo formed LCBs. No LCBs were formed in presence of the SPT inhibitor myriocin.