Identification of N-acylphosphatidylserine molecules in eukaryotic cells.

A comprehensive qualitative and quantitative characterization of lipids in the context of systems biology is crucial to a complete understanding cellular physiology and pathology, and is the rationale behind the Lipid Maps Consortium (www.lipidmaps.org) (1, 2). One of the major aims of the project is to discover novel lipids, with emphasis on those from mouse RAW264.7 macrophage tumor cells. There is ample biochemical and genomic evidence indicating the existence of novel lipids. For instance, radiochemical experiments with high levels of 32Pi indicate the presence of numerous unidentified minor phospholipid species at levels of 0.1 % or less in the total lipids of both prokaryotic and eukaryotic cells (3, 4). In addition, genomic analyses suggest the existence of proteins of unknown function, distantly related in their primary sequences to well-characterized enzymes of lipid metabolism (5). These predicted proteins might be involved in the biosynthesis of some of the minor unknown lipids. State-of-the-art, high-resolution mass spectrometry (MS) represents a powerful initial approach to the identification and structural characterization of novel lipids. Although MS has long been employed for the characterization of lipid molecules (6, 7), the introduction of electrospray ionization (ESI) (8) and matrix-assisted laser desorption ionization (MALDI) (9) dramatically improved the applicability of MS for lipid analysis. These two soft ionization techniques, together with instrumentation developments, have allowed intact, labile lipid molecules to be analyzed directly by MS with exceedingly high sensitivity and molecular specificity (6, 10, 11), including the detection of trace biosynthetic intermediates (12). By combining large-scale phospholipid pre-fractionation procedures with high-resolution ESI/MS/MS analysis, we have now discovered a family of novel N-acyl phosphatidylserine (N-acyl-PS) molecules in mouse brain, pig brain, mouse RAW264.7 macrophage tumor cells and yeast. The proposed structures of these N-acyl-PS species were confirmed by comparison with a synthetic standard. In 1970, Nelson provided preliminary evidence for the presence of N-acyl-PS in sheep red blood cells. However, mass spectrometry and NMR were not used to characterize this material, and no follow studies were published. Donahue et al. reported the presence of N-acyl-PS as a major component of Rhodopseudomonas sphaeroides phospholipids in 1982 (13), but Schmid et al. later demonstrated that the proposed R. sphaeroides N-acyl-PS was actually phosphatidyl-Tris, arising by phosphatidyl group transfer to the Tris buffer present in the growth medium (14). To our knowledge, no subsequent reports of N-acyl-PS as a component of biological membranes have appeared. We suggest that N-acyl-PS may function as a precursor of N-acyl L-serine in animal cells, a bioactive signaling lipid recently isolated from bovine brain (15). The biosynthesis, metabolism and function of N-acyl-PS may be analogous to that of N-acyl phosphatidylethanolamine (N-acyl-PE), the precursor of the mammalian endocannabinoid N-arachidonyl ethanolamine (16–18). The unequivocal identification of a family of N-acyl-PS molecular species in brain, macrophages and yeast set the stage for the complete elucidation of its biosynthesis, turnover and function.