Eleven residues determine the acyl chain specificity of ceramide synthases.

Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5(299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4(291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.

A rapid ceramide synthase activity using NBD-sphinganine and solid phase extraction.

Ceramides are synthesized by six mammalian ceramide synthases (CerSs), each of which uses fatty acyl-CoAs of different chain lengths for N-acylation of the sphingoid long-chain base. We now describe a rapid and reliable CerS assay that uses a fluorescent N-[6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl) (NBD) sphinganine substrate followed by separation of the NBD-lipid substrate and products using solid phase extraction (SPE) C18 chromatography. SPE chromatography is a quick and reliable alternative to TLC, and moreover, there is no degradation of either NBD-sphinganine or NBD-ceramide. We have optimized the assay for use with minimal amounts of protein in a minimal volume. This assay will prove useful for the analysis of CerS activity, which is of particular importance in light of the growing involvement of CerS in cell regulation and in the pathology of human diseases.

Myristate-derived d16:0 sphingolipids constitute a cardiac sphingolipid pool with distinct synthetic routes and functional properties.

BACKGROUND: Myristate is a novel potential substrate for sphingoid base synthesis. RESULTS: Myocardial sphingoid base synthesis utilizes myristate; these sphingolipids are functionally non-redundant with canonical sphingoid bases. CONCLUSION: d16:0 and d16:1 sphingolipids constitute an appreciable proportion of cardiac dihydrosphingosine and dihydroceramide, with distinct biological roles. SIGNIFICANCE: This pool of sphingolipids may play a heretofore unsuspected role in myocardial pathology or protection. The enzyme serine palmitoyltransferase (SPT) catalyzes the formation of the sphingoid base "backbone" from which all sphingolipids are derived. Previous studies have shown that inhibition of SPT ameliorates pathological cardiac outcomes in models of lipid overload, but the metabolites responsible for these phenotypes remain unidentified. Recent in vitro studies have shown that incorporation of the novel subunit SPTLC3 broadens the substrate specificity of SPT, allowing utilization of myristoyl-coenzyme A (CoA) in addition to its canonical substrate palmitoyl-CoA. However, the relevance of these findings in vivo has yet to be determined. The present study sought to determine whether myristate-derived d16 sphingolipids are represented among myocardial sphingolipids and, if so, whether their function and metabolic routes were distinct from those of palmitate-derived d18 sphingolipids. Data showed that d16:0 sphingoid bases occurred in more than one-third of total dihydrosphingosine and dihydroceramides in myocardium, and a diet high in saturated fat promoted their de novo production. Intriguingly, d16-ceramides demonstrated highly limited N-acyl chain diversity, and in vitro enzyme activity assays showed that these bases were utilized preferentially to canonical bases by CerS1. Functional differences between myristate- and palmitate-derived sphingolipids were observed in that, unlike d18 sphingolipids and SPTLC2, d16 sphingolipids and SPTLC3 did not appear to contribute to myristate-induced autophagy, whereas only d16 sphingolipids promoted cell death and cleavage of poly(ADP-ribose) polymerase in cardiomyocytes. Thus, these results reveal a previously unappreciated component of cardiac sphingolipids with functional differences from canonical sphingolipids.

The complexity of sphingolipid biosynthesis in the endoplasmic reticulum.

Unlike the synthesis of other membrane lipids, sphingolipid synthesis is compartmentalized between the endoplasmic reticulum and the Golgi apparatus. The initial steps of sphingolipid synthesis, from the activity of serine palmitoyltransferase through to dihydroceramide desaturase, take place in the endoplasmic reticulum, but the further metabolism of ceramide to sphingomyelin and complex glycosphingolipids takes place mostly in the Golgi apparatus. Studies over the last decade or so have revealed unexpected levels of complexity in the sphingolipid biosynthetic pathway, mainly due to either the promiscuity of some enzymes towards their substrates, or the tight selectivity of others towards specific substrates. We now discuss two enzymes in this pathway, namely serine palmitoyltransferase (SPT) and ceramide synthase (CerS), and one lipid transport protein, CERT. For SPT and CERT, significant structural information is available, and for CerS, significant information has recently been obtained that sheds light of the roles of the specific ceramide species that are produced by each of the CerS. We consider the mechanisms by which specificity is generated and speculate on the reasons that sphingolipid biosynthesis is so complex. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Acyl chain specificity of ceramide synthases is determined within a region of 150 residues in the Tram-Lag-CLN8 (TLC) domain.

In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl-CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and -5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N terminus) and half of CerS2 (from the C terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 toward C16-CoA; likewise, the putative sixth transmembrane domain (at the C terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity toward C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity toward C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.

Ceramide synthases: roles in cell physiology and signaling.

Ceramide synthases (CerS) are integral membrane proteins of the endoplasmic reticulum. Six mammalian CerS have been described, with each utilizing fatty acyl CoAs of relatively defined chain lengths for N-acylation of the sphingoid long chain base. In this chapter, we review the main functional features of the CerS proteins, discuss their fatty acid specificity, kinetics, tissue distribution and mode of inhibition, as well as possible posttranslational modifications. We then address the reason that mammals contain six distinct CerS, whereas most other enzymes in the sphingolipid biosynthetic pathway only occur in one or two isoforms. Finally, we discuss the putative roles of CerS and the ceramide derived from the CerS, in signaling pathways and in development of disease.

Impaired epidermal ceramide synthesis causes autosomal recessive congenital ichthyosis and reveals the importance of ceramide acyl chain length.

The barrier function of the human epidermis is supposed to be governed by lipid composition and organization in the stratum corneum. Disorders of keratinization, namely ichthyoses, are typically associated with disturbed barrier activity. Using autozygosity mapping and exome sequencing, we have identified a homozygous missense mutation in CERS3 in patients with congenital ichthyosis characterized by collodion membranes at birth, generalized scaling of the skin, and mild erythroderma. We demonstrate that the mutation inactivates ceramide synthase 3 (CerS3), which is synthesized in skin and testis, in an assay of N-acylation with C26-CoA, both in patient keratinocytes and using recombinant mutant proteins. Moreover, we show a specific loss of ceramides with very long acyl chains from C26 up to C34 in terminally differentiating patient keratinocytes, which is in line with findings from a recent CerS3-deficient mouse model. Analysis of reconstructed patient skin reveals disturbance of epidermal differentiation with an earlier maturation and an impairment of epidermal barrier function. Our findings demonstrate that synthesis of very long chain ceramides by CerS3 is a crucial early step for the skin barrier formation and link disorders presenting with congenital ichthyosis to defects in sphingolipid metabolism and the epidermal lipid architecture.

Ceramide kinase-like (CERKL) interacts with neuronal calcium sensor proteins in the retina in a cation-dependent manner.

PURPOSE: CERKL encodes for a ceramide kinase (CERK)-like protein. CERKL mutations are associated with severe retinal degeneration. Several studies have been conducted to prove a biochemical similarity between CERK and CERKL enzymatic activities. However, so far there has been no evidence that CERKL phosphorylates ceramide or any other lipid substrate in vitro or in vivo. The purpose of this work was to characterize CERKL's function by identification of CERKL-interacting proteins in the mammalian retina. METHODS: CERKL-interacting proteins were identified implementing the Ras-recruitment system (RRS) on a bovine retina cDNA library. Co-immunoprecipitation (co-IP) in transfected cells and in photoreceptor outer segments was used to verify the identified interactions. Serial deletion constructs were used to map the interacting sites. CERKL's kinase activity was tested by a CERK activity assay. RESULTS: We identified an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including guanylate cyclase activating protein 1 (GCAP1), GCAP2, and recoverin. These interactions were confirmed by co-IP experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in photoreceptor outer segments. We found that CERKL-GCAP interaction is cation dependent and is mediated by CERKL's N-terminal region and by GCAPs cation-binding domains (EF-hands 2-4). CONCLUSIONS: This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to proteins involved in the photoresponse and Ca(2+) signaling, providing important clues for future research required in this direction.