On the importance of plasmalogen status in stimulated arachidonic acid release in the macrophage cell line RAW 264.7
Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany Street, Boston, Massachusetts 02118, USA. Biochimica et Biophysica Acta
(Impact Factor: 4.66).
05/2008; 1781(4):213-9. DOI: 10.1016/j.bbalip.2008.01.007
We examined the dependence of stimulated arachidonic acid release on plasmalogens using the murine, macrophage cell line 264.7 and two plasmalogen-deficient variants, RAW.12 and RAW.108. All three strains responded to unopsinized zymosan to release arachidonic acid from phospholipid stores. Arachidonic acid release appeared to be dependent on calcium-independent phospholipase A(2) activation (iPLA(2)); bromoenol lactone, a specific inhibitor of calcium-independent iPLA(2), blocked arachidonic acid release with an IC(50) of approximately 2 x 10(-7)M. Propanolol, an inhibitor of phosphatidate phosphatase, and RHC-80267, an inhibitor of diglyceride lipase, had no effect on arachidonic acid release. Arachidonic acid release in the variants displayed similar magnitude, kinetics of response and sensitivity to the inhibitors when compared to the parent strain. Arachidonic acid was released from all major phospholipid head group classes with the exception of sphingomyelin. In wild-type cells, arachidonic acid released from the ethanolamine phospholipids was primarily from the plasmalogen form. However, in the plasmalogen-deficient cells release from the diacyl species, phosphatidylethanolamine, was increased to compensate. Restoration of plasmalogens by supplementation of the growth medium with the bypass compounds sn-1-hexadecylglycerol and sn-1-alkenylglycerol had no effect on arachidonic acid release. In summary, plasmalogen status appears to have no influence on the zymosan A stimulated release of arachidonic acid from the RAW 264.7 cell line.
Available from: Charles E Bell
- "In mammals, lysoplasmalogens are formed from plasmalogens by the action of calcium-independent PLA 2 (iPLA 2 ) enzymes, which cleave the acyl chain at sn-2 to release a free fatty acid . A number of iPLA2 enzymes , including those from alveolar cells  and macrophages , are selective for plasmalogen substrates, and activated by extracellular signals . At submicellar concentrations, lysoplasmalogens affect the dynamics of cell membranes by increasing membrane fluidity . "
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ABSTRACT: Lysoplasmalogenase catalyzes hydrolytic cleavage of the vinyl-ether bond of lysoplasmalogen to yield fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. We recently purified lysoplasmalogenase from rat liver microsomes and identified the protein as TMEM86B, an integral membrane protein that is a member of the YhhN family found in numerous species of eukaryotes and bacteria. To test the hypothesis that bacterial YhhN proteins also function as lysoplasmalogenase enzymes, we cloned the Lpg1991 gene of Legionella pneumophila, which encodes a 216 amino acid YhhN protein, and expressed it in Escherichia coli as a C-terminal-GFP-His8-fusion. Membranes were solubilized and the fusion protein was purified by nickel-affinity chromatography, cleaved with Tobacco Etch Virus protease, and subjected to a reverse nickel column to purify the un-tagged LpYhhN. Both the fusion protein and un-tagged LpYhhN exhibit robust lysoplasmalogenase activity, cleaving the vinyl-ether bond of lysoplasmalogen with a Vmax of 12 and a Km of 45μM. LpYhhN has no activity on diradyl plasmalogen, 1-alkenyl-glycerol, and monoacylglycerophospho-ethanolamine or monoacylglycerophospho-choline; the pH optimum is 6.5-7.0. These properties are very similar to mammalian TMEM86B. Sequence analysis suggests that YhhN proteins contain eight transmembrane helices, an N-in/C-in topology, and about 5 highly conserved amino acid residues that may form an active site. This work is the first to demonstrate a function for a bacterial YhhN protein, as a vinyl ether bond hydrolase specific for lysoplasmalogen. Since L. pneumophila does not contain endogenous plasmalogens, we hypothesize that LpYhhN may serve to protect the bacterium from lysis by lysoplasmalogen derived from plasmalogens of the host.
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