Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family.

Sphingolipids (SLs) are ubiquitous in eukaryotic membranes, where they are structural components of biological membranes, participate in protein sorting and cell signaling via membrane rafts, act as apoptotic and anti-apoptotic secondary messengers, and serve as precursors for other essential lipid biosynthetic pathways like the Kennedy pathway (1-6). Sphingolipid synthases (SLS) transfer a phosphoryl head group (Figure 1A) from phosphatidylinositol (PI), phosphatidylethanolamine (PE) or phosphatidylcholine (PC) to ceramide (CM) to generate inositol phosphorylceramide (IPC), ethanolamine phosphorylceramide (EPC), or sphingomyelin (SM) with release of diacylglycerol (DAG). These reactions can occur in the ER, Golgi, and the plasma membrane (7, 8), thus trafficking of substrates and enzymes represents another important facet of sphingolipid biogenesis and function (8-11). Figure 1 A, schematic of phospholipid head groups phosphorylinositol (PI), phosphotidylethanolamine (PE) and phosphotidylcholine (PC), which are transferred by SLS to ceramide (CM) yielding inositol phosphorylceramide (IPC), ethanolamine phosphorylceramide (EPC) ... Figure 1B shows a topology model for the SLS family, which is predicted to contain 6 transmembrane helices (10, 12). Sequence analyses revealed that conserved motifs of Ser, Gly, His (SGH) and His, Asp (H-X3-D) originally identified in lipid phosphate phosphatases (LPPs), glucose-6-phosphatase and other integral membrane phosphomonoester hydrolyzing enzymes (13) were also present in the mammalian SMS (10, 14), fungal IPC synthase (15), and the trypanosomal SLS family (12). Collectively, these proteins comprise the phosphatidic acid phosphatase (PAP2) superfamily, PFAM Pf01569. In the following, the combination of His and Asp residues present in these motifs will be referred to as the HHD triad. Interestingly, chloroperoxidase also contain this motif (13, 16) and thus provides a model for positioning of active site residues around a covalently bound phosphoryl group (17). The role of the HHD triad in catalysis was initially implicated by site-directed mutagenesis in the LPPs and later in the SMSs (18). Transmembrane Hidden Markov Modeling (7, 12, 19) and biochemical fusion reporter studies (7, 20, 21) also suggested that the conserved motifs would be present in extra-membrane loops, which were predicted to reside on the luminal side of the membrane bilayer. Eukaryotic genomes typically contain multiple, highly conserved paralogs of SLS that allow the synthesis of a diverse array of sphingolipids (7, 22). For example, human sphingomyelin synthase 1 (hSMS1, UniProt {"type":"entrez-protein","attrs":{"text":"Q86VZ5","term_id":"44888473"}}Q86VZ5) is an SM synthase (7), while hSMS2 (60% identity with hSMS1, UniProt {"type":"entrez-protein","attrs":{"text":"Q8NHU3","term_id":"44888519"}}Q8NHU3) is a bi-functional SM/EPC synthase (7, 23) and hSMSr (34% identity with hSMS1, UniProt {"type":"entrez-protein","attrs":{"text":"Q96LT4","term_id":"44888529"}}Q96LT4) is an EPC synthase (21). Recently, we determined the enzymatic specificities of each of the set of four Trypanosoma brucei SLS paralogs, which have >90% primary sequence identity [Figure S1 of Supporting Information and (12)]. Among these, TbSLS1 is an IPC synthase and TbSLS2 is an EPC synthase, while TbSLS3 and TbSLS4 are bi-functional SM/EPC synthases (24). The presence of the full suite of catalytic specificities in a single organism offers unique possibilities for studies of structure, function and cellular physiology (11, 25). Upon consideration of the primary sequences of the enzymes from trypanosomes and other species, it is particularly striking that the marked discrimination between substrates by charge (IP versus PE/PC) and size of the zwitterionic head group (PE versus PC) must be provided by relatively few changes in residues (Figure 1C). Efforts to study the mechanism of action and to identify the residues contributing to the selectivity observed in phosphosphingolipid synthesis have been generally hindered by issues arising in the production of integral membrane proteins for unambiguous functional analysis. For example, difficulties in establishing homologous transgenic lines, heterologous expression hosts, or in obtaining sufficient quantities of purified proteins for in vitro studies all constrained prior studies of the trypanosomal SLS paralogs. To overcome the latter limitation, we utilized a cell-free translation system developed for the translation and purification of integral membrane proteins (24, 26). This approach facilitated the analysis of mutations in the TbSLS family. From this work, the requirement for the HHD triad in catalysis has been established in the trypanosomal SLS family. Moreover, three residues in the sequences proximal to the HHD triad are shown to have an essential role in establishing substrate selectivity in the entire T. brucei SLS family. The role of these residues in substrate-specific reaction mechanisms is proposed, and similarities and differences of the trypanosomal SLS with the broader family of SLS and their relationship to the larger PAP2 superfamily are discussed.