When Do Lasses (Longevity Assurance Genes) Become CerS (Ceramide Synthases)?

Ceramide is thekey intermediate in thepathwayof sphingolipid (SL)2 biosynthesis (1) and an important intracellular signaling molecule (2, 3). Ceramide consists of a sphingoid long chain base to which a fatty acid is attached via an amide bond (Fig. 1). When the chemical composition of SLswas first determined in the 1940s (4), stearic acid (C18:0) was identified as the major fatty acid attached to the sphingoid base. Later, with the development of more sensitive techniques (5, 6), it became clear that mammalian SLs contain a wide variety of fatty acids, ranging in length from C14 to C32, that are predominantly saturated and can contain !or "-hydroxyl groups (7, 8). Within the past few years, there has been renewed interest in the functional significance of this fatty acid variability, and thisminireviewwill focus on current advances in our understanding of how the fatty acid composition of ceramide is regulated. In particular, we will discuss a recently discovered family of mammalian ceramide synthase (CerS) genes and emerging evidence that specific ceramides containing distinct fatty acids play important roles in cell growth and apoptosis.

Ceramide is the key intermediate in the pathway of sphingolipid (SL) 2 biosynthesis (1) and an important intracellular signaling molecule (2,3). Ceramide consists of a sphingoid long chain base to which a fatty acid is attached via an amide bond (Fig. 1). When the chemical composition of SLs was first determined in the 1940s (4), stearic acid (C18:0) was identified as the major fatty acid attached to the sphingoid base. Later, with the development of more sensitive techniques (5,6), it became clear that mammalian SLs contain a wide variety of fatty acids, ranging in length from C14 to C32, that are predominantly saturated and can contain ␣or -hydroxyl groups (7,8). Within the past few years, there has been renewed interest in the functional significance of this fatty acid variability, and this minireview will focus on current advances in our understanding of how the fatty acid composition of ceramide is regulated. In particular, we will discuss a recently discovered family of mammalian ceramide synthase (CerS) genes and emerging evidence that specific ceramides containing distinct fatty acids play important roles in cell growth and apoptosis.

Molecular Identification of Ceramide Synthases
CerS acylates sphinganine (dihydrosphingosine) to form dihydroceramide and sphingosine to form ceramide; sphinganine is produced through the de novo biosynthetic pathway and sphingosine mainly through SL degradation (1,9). Early studies detected CerS activity in microsomal fractions, and the enzyme was shown to utilize a variety of fatty acyl-CoAs although some specificity toward certain CoAs was recognized (10). CerS activity was later localized to the cytoplasmic leaflet of the endoplasmic reticulum (ER) (11,12).
The first clue to the molecular identity of CerS was obtained in yeast, when it was shown that Lag1p and Lac1p were required for the synthesis of C26-ceramide (13,14). 3 Lag1 was originally isolated in a screen for longevity-related genes (15). A paralog, 4 Lac1, was found in a data base screen (16) along with homologs in human, mouse, and Caenorhabditis elegans. The Asc1 gene in tomato was subsequently isolated as a toxin-resistant gene (17); among the toxins tested was fumonisin B1, a CerS inhibitor (18). Homologs of Lag1 and Lac1 have now been found in most species. Six paralogs are known in human and mouse, and were named Lass (longevity assurance) genes; in all species studied to date, at least two genes have been found in every organism.
All of the Lass genes have a distinguishing motif in the C-terminal portion of the protein. This motif has been defined in a narrow but specific manner as the Lag domain, a region of ϳ50 amino acid residues (19), and in a broader manner as the Tram-Lag-CLN8 (TLC) domain, a region of ϳ200 residues also found in other proteins (20). The broader definition is based on two additional proteins, TRAM1 and CLN8; TRAM1 was found in a search for homologs of Lag1 in humans. The exact function of TRAM proteins is not known, but they are thought to be involved in polypeptide transition through the ER (21,22). CLN8 was found by screening for genes causing neuronal ceroid-lipofuscinosis (23), an inherited metabolic disorder (24). Both TRAM1 (13) and CLN8 (25) failed to complement the yeast Lag1/Lac1 knock-out, suggesting that the TLC domain is not sufficient for CerS activity, whereas some human LASS proteins tested were able to complement CerS activity (25,26).
In humans there are 16 TLC domain-containing genes ( Fig.  2): six LASSes, three TRAMs, CLN8, and six other novel genes. All 16 genes are conserved in mouse. When the 16 human proteins are aligned, four distinct branches appear. FAM57A and -B are one subgroup and are closer to CLN8 and the other novel proteins than to LASSes and TRAMs. The TRAM proteins form their own subgroup but are relatively close to the LASSes. The LASSes subdivide into two distinct groups, with LASS1 in its own category and LASSes 2-6 on a separate branch. This is consistent with the fact that LASS1 is much closer to the yeast proteins than the others (27).
A subset of Lass genes is predicted to contain a Homeobox (Hox) domain (19). In humans and mice, all but Lass1 have a Hox domain, in Drosophila only one of the four TLC-containing genes have a Hox domain, and no Hox domains are found in yeast and plant Lass genes. The Hox domain is derived from homeobox proteins, which are sequence-specific transcription factors important in development (28), but there is no evidence that the Hox domain in Lass genes acts as a transcription factor. The fact that many family members lack the Hox domain suggest that it is unlikely to be involved in the catalytic mechanism of CerS.
The six human LASS genes are located on different chromosomes (with the exception of LASS1 and -4, which are located on the same chromosome but at distant locations). Human and mouse orthologs 4 show strong similarity both in sequence and genomic structure, with the two major domains (i.e. Hox and TLC) encoded by the same relative exon in each of the LASS genes. All six genes in human (except, to date, LASS3) encode at least two isoforms, one of which lacks the homeobox domain and part of the TLC domain; the relevance of these short isoforms is currently unknown but may imply similar mechanisms of transcriptional regulation. Fig. 3 shows the genomic organization and protein isoforms of one of the LASS genes, namely LASS2. Recently, a splice variant of LASS5 has been shown to be expressed in lymphoma and other tumor cells and may be involved in tumor recognition by the immune system (29).
The Lass genes appear to encode multi-transmembrane (TM) spanning proteins. The exact number of TM domains, and their topology, has not been resolved experimentally (9,20,25), although a recent study suggested that the yeast proteins, Lag1 and Lac1, contain eight putative TM domains with the N and C termini of the proteins facing the cytoplasm (30). The subcellular location of the CerS proteins (at least those for which experimental data is available) is the ER (27,31,32), consistent with earlier observations (11,12) and similar to the location of Tram proteins (22).
The yeast genes, Lag1 and Lac1, act in an obligate complex with an additional protein, Lip1 (33), an integral ER membrane protein with one predicted TM domain. The Lip1 regions required for CerS activity may be in the membrane or in the lumen of the ER. Mammalian homologs of Lip1 have not been found in data base searches. The activity of mammalian Lass proteins might conceivably be regulated by other TLC family members (34).
The first evidence for specific functional roles of mammalian Lass genes was obtained upon overexpression of LASS1 (formerly known as UOG1), which resulted in a selective increase in C18-ceramide in mammalian cells (31). LASS4 (TRH1) and LASS5 (TRH4) were subsequently shown to selectively utilize C18/20 and C16 acyl-CoAs, respectively (32), LASS6 to produce shorter acyl chain ceramides (C14 and C16) (27), and LASS3 to produce C18-and C24-ceramides (35), although the surprisingly high levels of C18-ceramide synthesis are at vari-ance with other analyses. 5 Verification that mammalian LASS proteins are bona fide ceramide synthases, rather than regulators of endogenous ceramide synthases, was obtained when purified LASS5 was shown to possess CerS activity (36). Together with the CerS activity of the purified Lag1-Lac1-Lip1 complex in yeast (33), this supports the concept that LASS proteins are genuine ceramide synthases, with each mammalian protein utilizing a relatively restricted subset of fatty acyl-CoAs. It is assumed that the six known mammalian LASS proteins account for the synthesis of all known ceramides, but the possibility cannot be excluded that some other proteins, such as ceramidases (37), may also contribute to the synthesis of ceramides with restricted fatty acid composition.

Roles of Ceramides Containing Distinct Fatty Acids
The reason that mammals (and other species such as plants (38)) have multiple CerS genes, whereas most of the other enzymes in the SL biosynthetic pathway exist in only one or two isoforms (9), is not known but implies an important role for ceramides containing specific fatty acids in cell physiology. Support for this notion has been provided by the development of new and more sensitive analytical techniques, particularly mass spectrometry (8,39,40), enabling analysis of the fatty acid composition of ceramides using relatively small amounts of tissue or cells. Mass spectrometric analysis has revealed that spe-  cific ceramides containing a restricted set of fatty acids are generated in response to certain stimuli.
Ceramide has been shown to be generated in intracellular signaling pathways by either degradation of sphingomyelin or via de novo synthesis (3) with the kinetics of appearance and biochemical functionality of each differing (41,42). Both pathways may be activated separately or in parallel depending on the kind of stress or on the cell type (43).
C16-ceramide is one of the major ceramide species in nonneuronal tissues and has been suggested to be the predominant species elevated during apoptosis under a broad spectrum of stimuli (44 -49). For instance, C16-ceramide, derived from sphingomyelin, increases during tumor necrosis factor ␣-induced apoptosis in primary hepatocytes (46), whereas C16-ceramide derived from de novo synthesis is elevated in androgen ablation-induced apoptosis of LNCaP prostate cancer cells (45) and during B cell receptor engagement on Ramos cells (48). C24-ceramide also increases in Ramos cells but not until 24 h after B cell receptor engagement, whereas C16-ceramide is elevated after 6 h (47). A similar temporal pattern was observed in androgen-insensitive DU145 prostate cancer cells in which C16-ceramide was elevated after 24 h and C24-ceramide after 48 h (49). Changes in C16-and C24ceramide levels via the de novo pathway have also been observed in bone marrow-derived dendritic cells (50). In apparent contrast, tumor cells, such as head and neck squamous cell carcinoma (51), Sarcoma 180, B16 melanoma, and Lewis lung carcinoma cells (52), contain high levels of C16-ceramide, suggesting that C16-ceramide might also be able to promote cell growth. It is therefore not clear if C16-ceramide is pro-apoptotic, or alternatively, whether it is the delayed elevation of C24-ceramide or another ceramide species that is involved in cell death. A possible role of C18-ceramide in growth arrest was also suggested because levels of C18-ceramide were decreased in head and neck squamous cell carcinoma samples, and overexpression of LASS1 increased C18-ceramide levels and inhibited cell growth (51). Thus, at present, it is difficult to draw definitive conclusions concerning the role of particular ceramides in defined physiological and pathophysiological events. However, the availability of the Lass genes and the possibility to experimentally manipulate their expression, will permit dissection of the precise roles of specific ceramide species.
How might ceramides containing different fatty acids impact upon cell physiology? One or more biophysical properties of the membrane lipid bilayer could be influenced by the fatty acid composition of ceramide (53). Alternatively, specific ceramides could directly interact with downstream components in signaling pathways. In both scenarios, regulation of the activity of the Lass genes (for instance by transcriptional or post-translational mechanisms) would be an important and novel mode of controlling the fatty acid composition of ceramides. However, very little is known about the relationship between levels of specific ceramides and the activity of Lass genes in different cells. Early work implied post-translational modification of CerS (39,54), and the restricted tissue distribution of Lass genes (32, 55) is  consistent with multiple levels of regulation of CerS. For instance, the mRNA of Lass3 is expressed at high levels in skin (32), which contains very long acyl chain ceramides that are involved in maintaining the water permeability barrier function of skin (56).

Perspectives
The unexpected discovery of a family of genes involved in regulating the fatty acid composition of ceramides is likely to lead to a new level of understanding of the complexity of ceramide biology (3,57). Many open questions remain concerning the function and mode of regulation of the Lass genes themselves. For instance, what is the role of the TLC domain and why are there so many TLC domain-containing proteins (Fig. 2) with only some appearing to meet the criteria to be characterized as genuine ceramide synthases? What are the functions of the other family members? Do they act as regulators of the CerS proteins or do they have unrelated functions? What are the functions of the splice variants (Fig. 3) and might they act in a dominant-negative fashion to modify the activity of full-length Lass genes? Finally, when do Lasses become CerS (Fig. 4)? What are the critical regions in the LASS proteins that confer ceramide synthase activity? What are the regions responsible for acyl-CoA and sphingoid base selectivity and how are these regions conserved or how do they differ between the different LASS proteins? Irrespective of the answers to these and other questions, all the available evidence strongly implies that the key attribute of Lass genes is their ceramide synthase activity rather than their role in longevity. This being the case, we propose renaming the six Lass genes as CerS1-6 ( Fig. 4), to more accurately reflect their known function.