A lysosomal phospholipase A2, LPLA2, was recently characterized and shown to have substrate specificity for phosphatidylcholine and phosphatidylethanolamine. LPLA2 is ubiquitously expressed but is most highly expressed in alveolar macrophages. Double conditional gene targeting was employed to elucidate the function of LPLA2. LPLA2-deficient mice (Lpla2 ؊/؊ ) were generated by the systemic deletion of exon 5 of the Lpla2 gene, which encodes the lipase motif essential for the phospholipase A2 activity. The survival of the Lpla2 ؊/؊ mice was normal. Lpla2 Previously, a novel enzyme was discovered in the lysosomal fraction of MDCK cells. The enzyme was characterized as having ceramide transacylase and phospholipase A2 activity (3). The enzyme was purified, cloned, and named lysosomal phospholipase A2 (LPLA2) (12). Characteristics of the enzyme include a pH optimum of 4.5 and calcium independence. In addition, phosphatidylcholine and phosphatidylethanolamine are preferentially favored as substrates for the phospholipase A2. The enzyme is highly homologous with lecithin cholesterol acyltransferase and is phylogenetically related to a large group of plant phospholipases (15). Most recently, the phospholipase activity was demonstrated as highly expressed in alveolar macrophages, suggesting a potential role for LPLA2 in the phospholipid catabolism of pulmonary surfactant (1). LPLA2-deficient mice were generated to further evaluate the biological function of LPLA2.In the present study, double conditional targeted mice were generated, and the targeted gene was modified using Cre/loxP and Flp/FRT recombination systems. Lpla2-deficient mice were generated in which exon 5 of the Lpla2 gene, which encodes the lipase motif essential for Lpla2 activity, was systemically deleted. The resultant Lpla2 Ϫ/Ϫ mice showed no LPLA2 activity, developed normally, and were characterized by a marked accumulation of phospholipid in their alveolar macrophages, peritoneal macrophages, and spleens at an early age. Increased lung surfactant phospholipid and splenomegaly were observed in mice by 1 year of age. MATERIALS AND METHODSReagents. Synthetic phospholipids, including 1,2-dioleloyl-sn-glycero-3-phosphorylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), and

Recently, a novel enzyme, 1-O-acylceramide synthase (ACS), was purified and characterized from bovine brain. This enzyme has both calcium-independent phospholipase A 2 and transacylase activities. The discovery of this enzyme led us to propose a new pathway for ceramide metabolism in which the sn-2-acyl group of either phosphatidylethanolamine or phosphatidylcholine is transferred to the 1-hydroxyl group of ceramide. In this study, the partial amino acid sequences from the purified enzyme revealed that the enzyme contains amino acid sequences identical to those of human lecithin:cholesterol acyltransferase-like lysophospholipase (

Lung surfactant is the surface-active agent comprised of phospholipids and proteins that lines pulmonary alveoli. Surfactant stabilizes the alveolar volume by reducing surface tension. Previously, we identified a lysosomal phospholipase A2, termed LPLA2, with specificity toward phosphatidylcholine and phosphatidylethanolamine. The phospholipase is localized to lysosomes, is calcium-independent, has an acidic pH optimum, and transacylates ceramide. Here, we demonstrate that LPLA2 is selectively expressed in alveolar macrophages but not in peritoneal macrophages, peripheral blood monocytes, or other tissues. Other macrophage-associated phospholipase A2s do not show a comparable distribution. LPLA2 is of high specific activity and recognizes disaturated phosphatidylcholine as a substrate. The lysosomal phospholipase A2 activity is six times lower in alveolar macrophages from mice with a targeted deletion of the granulocyte macrophage colonystimulating factor (GM-CSF), a model of impaired surfactant catabolism, compared with those from wild-type mice. However, LPLA2 activity and protein levels are measured in GM-CSF null mice in which GM-CSF is expressed as a transgene under the control of the surfactant protein C promoter. Thus LPLA2 may be a major enzyme of pulmonary surfactant phospholipid degradation by alveolar macrophages and may be deficient in disorders of surfactant metabolism.Previously, in an attempt to identify the enzyme that transacylates ceramide at the 1-hydroxyl position, a novel phospholipase A2 was characterized (1). The phospholipase A2, termed 1-O-acylceramide synthase or lysosomal phospholipase A2 (LPLA2) 1 has the following properties. In the presence of ceramide, the enzyme catalyzes the formation of 1-O-acylceramide by transacylation of fatty acids from the sn-2 position of phosphatidylcholine or phosphatidylethanolamine. In the absence of ceramide or other alcohols as acceptors, the enzyme acts as a traditional phospholipase A2. However, the phospholipase has a pH optimum of 4.5 and is mannose-rich and calcium-independent (2). The phospholipase amino acid sequence is 49% identical to human LCAT (3). The homology with LCAT is highest within the catalytic domain, but the phospholipase does not recognize cholesterol as an acceptor for the fatty acid. The phospholipase colocalizes with other lysosomal proteins in cell fractionates. Upon the initial characterization of this enzyme, the functional role of this phospholipase A2 was not immediately apparent. A role for an acidic phospholipase A2 activity has previously been suggested for the degradation of pulmonary surfactant phospholipids (4). The pulmonary acidic phospholipase A2 activity is also reported to be calcium-independent and inhibited by a transition state analog of arachidonate, MJ33 (5). In rats treated with MJ33 the surfactant phospholipid catabolism was inhibited by ϳ40 -50%, suggesting that the drug-sensitive phospholipase A2 activity contributes significantly to total surfactant degradation (6). In the present paper LPLA2 was stu...

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the Fc␥RIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERKtransfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERKtransfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.The second messengers produced by membrane lipids through agonist stimulation include not only glycerolipids but also sphingolipids. Sphingolipids are comprised of lipids that contain a long chain sphingoid base. Sphingolipids, in addition to being structural components of membranes, regulate cell-cell and cell-substrate interactions, proliferation, and differentiation. Members of this diverse group of lipids have emerged as a novel class of signaling molecules that also regulate phagocytosis. The mechanisms by which sphingolipids exert these effects remain incompletely defined. More than a decade ago, it was found that ceramide can be phosphorylated to ceramide 1-phosphate (C1P) 1 (1-3).C1P is found in brain synaptic vesicles, and it is thought to play a role in regulating the secretion of neurotransmitters by promoting the fusion of vesicle membranes (4). Ceramide kinase activity exists in HL-60 cells where C1P is derived from ceramide released from sphingomyelin (5). Human ceramide kinase (hCERK) was recently cloned based on the homology to two isoforms of mice and human sphingosine kinase (6). The expressed kinase displayed specific ceramide phosphorylating activity. BLAST search analyses using the hCERK sequence revealed that a series of putative CERKs exist in a variety of cellular organisms, including plants, nematodes, insects, and vertebrates. CERKs represent a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases (6, 7).C1P shares struc...

Purpose: To elucidate the clinical and epidemiologic characteristics of optic neuritis in Japan. Design: Multicenter cross-sectional, observational cohort study. Participants: A total of 531 cases of unilateral or bilateral noninfectious optic neuritis identified in 33 institutions nationwide in Japan. Methods: Serum samples from patients with optic neuritis were tested for antieaquaporin-4 antibodies (AQP4-Abs) and antiemyelin oligodendrocyte glycoprotein antibodies (MOG-Abs) using a cell-based assay and were correlated with the clinical findings. Main Outcome Measures: Antibody positivity, clinical and radiologic characteristics, and visual outcome. Results: Among 531 cases of optic neuritis, 12% were AQP4-Ab positive, 10% were MOG-Ab positive, 77% were negative for both antibodies (double-negative), and 1 case was positive for both antibodies. Pretreatment visual acuity (VA) worsened to more than a median 1.0 logarithm of the minimum angle of resolution (logMAR) in all groups. After steroid pulse therapy (combined with plasmapheresis in 32% of patients in AQP4-Abepositive group), median VA improved to 0.4 logMAR in the AQP4-Abepositive group, 0 logMAR in the MOG-Abepositive group, and 0.1 logMAR in the double-negative group. The AQP4-Abepositive group showed a high proportion of females, exhibited diverse visual field abnormalities, and demonstrated concurrent spinal cord lesions on magnetic resonance imaging (MRI) in 22% of the patients. In the MOG-Abepositive group, although posttreatment visual outcome was good, the rates of optic disc swelling and pain with eye movement were significantly higher than those in the AQP4-Abepositive and double-negative groups. However, most cases showed isolated optic neuritis lesions on MRI. In the double-negative group, 4% of the patients had multiple sclerosis. Multivariate logistic regression analysis of all participants identified age and presence of antibodies (MOG-Ab and AQP4-Ab) as significant factors affecting visual outcome. Conclusions: The present large-scale cohort study revealed the clinicoepidemiologic features of noninfectious optic neuritis in Japan. Antieaquaporin-4 antibodyepositive optic neuritis has poor visual outcome. In contrast, MOG-Ab positive cases manifested severe clinical findings of optic neuritis before treatment, but few showed concurrent lesions in sites other than the optic nerve and generally showed good treatment response with favorable visual outcome. These findings indicate that autoantibody measurement is useful for prompt diagnosis and proper management of optic neuritis that tends to become refractory.