Sphingolipids are comprised of a backbone sphingoid base that may be phosphorylated, acylated, glycosylated, bridged to various headgroups through phosphodiester linkages, or otherwise modified. Organisms usually contain large numbers of sphingolipid subspecies and knowledge about the types and amounts is imperative because they influence membrane structure, interactions with the extracellular matrix and neighboring cells, vesicular traffic and the formation of specialized structures such as phagosomes and autophagosomes, as well as participate in intracellular and extracellular signaling. Fortunately, "sphingolipidomic" analysis is becoming feasible (at least for important subsets such as all of the backbone "signaling" subspecies: ceramides, ceramide 1-phosphates, sphingoid bases, sphingoid base 1-phosphates, inter alia) using mass spectrometry, and these profiles are revealing many surprises, such as that under certain conditions cells contain significant amounts of "unusual" species: N-mono-, di-, and tri-methyl-sphingoid bases (including N,N-dimethylsphingosine); 3-ketodihydroceramides; N-acetyl-sphingoid bases (C2-ceramides); and dihydroceramides, in the latter case, in very high proportions when cells are treated with the anticancer drug fenretinide (4-hydroxyphenylretinamide). The elevation of DHceramides by fenretinide is befuddling because the 4,5-trans-double bond of ceramide has been thought to be required for biological activity; however, DHceramides induce autophagy and may be important in the regulation of this important cellular process. The complexity of the sphingolipidome is hard to imagine, but one hopes that, when partnered with other systems biology approaches, the causes and consequences of the complexity will explain how these intriguing compounds are involved in almost every aspect of cell behavior and the malfunctions of many diseases.

Fabry disease is a lysosomal storage disorder that results in an accumulation of globotriaosylceramide in vascular tissue secondary to a deficiency in ␣-galactosidase A. The glycolipid-associated vasculopathy results in strokes and cardiac disease, but the basis for these complications is poorly understood. Recent studies in the ␣-galactosidase A-knockout mouse suggested that a decrease in nitric oxide (NO) bioavailability may play a role in the abnormal thrombosis, atherogenesis, and vasorelaxation that are characteristic of these mice. To understand better the association between impaired NO bioavailability and glycolipid accumulation, we studied ␣-galactosidase A-knockout mice or primary cultures of their aortic endothelial cells. Treatment of knockout mice with a potent inhibitor of glucosylceramide synthase reversed accumulation of globotriaosylceramide but failed to normalize the defect in vasorelaxation. Basal and insulin-stimulated endothelial NO synthase (eNOS) activities in endothelial cells derived from knockout mice were lower than those observed from wild-type mice; normalization of glycolipid only partially reversed this reduction in eNOS activity. The loss of eNOS activity associated with a decrease in high molecular weight caveolin oligomers in endothelial cells and isolated caveolae, suggesting a role for glycolipids in caveolin assembly. Finally, concentrations of ortho-tyrosine and nitrotyrosine in knockout endothelial cells were markedly elevated compared with wild-type endothelial cells. These findings are consistent with a loss of NO bioavailability, associated with eNOS uncoupling, in the ␣-galactosidase A-knockout mouse.

A phospholipase A 2 was identified from MDCK cell homogenates with broad specificity toward glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. The phospholipase has the unique ability to transacylate short chain ceramides. This phospholipase is calcium-independent, localized to lysosomes, and has an acidic pH optimum. The enzyme was purified from bovine brain and found to be a water-soluble glycoprotein consisting of a single peptide chain with a molecular weight of 45 kDa. The primary structure deduced from the DNA sequences is highly conserved between chordates. The enzyme was named lysosomal phospholipase A2 (LPLA 2 ) and subsequently designated group XV phospholipase A 2 . LPLA 2 has 49 percent of amino acid sequence identity to lecithin cholesterol acyltransferase and is a member of the αβ-hydrolase superfamily. LPLA 2 is highly expressed in alveolar macrophages. A marked accumulation of glycerophospholipids and extensive lamellar inclusion bodies, a hallmark of cellular phospholipidosis, is observed in alveolar macrophages in LPLA 2 −/− mice. This defect can also be reproduced in macrophages that are exposed to cationic amphiphilic drugs such as amiodarone. In addition, older LPLA 2 −/− mice develop a phenotype similar to human autoimmune disease. These observations indicate that LPLA 2 may play a primary role in phospholipid homeostasis, drug toxicity, and host defense.