Abstract
In mammalian cells, phosphatidylethanolamine (PtdEtn) is mainly synthesized via the CDP-ethanolamine (Kennedy) pathway and by decarboxylation of phosphatidylserine (PtdSer). However, the extent to which these two pathways contribute to overall PtdEtn synthesis both quantitatively and qualitatively is still not clear. To assess their contributions, PtdEtn species synthesized by the two routes were labeled with pathway-specific stable isotope precursors, d(3)-serine and d(4)-ethanolamine, and analyzed by high performance liquid chromatography-mass spectrometry. The major conclusions from this study are that (i) in both McA-RH7777 and Chinese hamster ovary K1 cells, the CDP-ethanolamine pathway was favored over PtdSer decarboxylation, and (ii) both pathways for PtdEtn synthesis are able to produce all diacyl-PtdEtn species, but most of these species were preferentially made by one pathway. For example, the CDP-ethanolamine pathway preferentially synthesized phospholipids with mono- or di-unsaturated fatty acids on the sn-2 position (e.g. (16:0-18:2)PtdEtn and (18:1-18:2)PtdEtn), whereas PtdSer decarboxylation generated species with mainly polyunsaturated fatty acids on the sn-2 position (e.g. (18:0-20:4)PtdEtn and (18:0-20:5)PtdEtn in McArdle and (18: 0-20:4)PtdEtn and (18:0-22:6)PtdEtn in Chinese hamster ovary K1 cells). (iii) The main PtdEtn species newly synthesized from the Kennedy pathway in the microsomal fraction appeared to equilibrate rapidly between the endoplasmic reticulum and mitochondria. (iv) Newly synthesized PtdEtn species preferably formed in the mitochondria, which is at least in part due to the substrate specificity of the phosphatidylserine decarboxylase, seemed to be retained in this organelle. Our data suggest a potentially essential role of the PtdSer decarboxylation pathway in mitochondrial functioning.
Highlights
The PtdSer decarboxylation pathway for PtdEtn biosynthesis was first described by Borkenhagen et al [5]
Only a Pathway-specific Monitoring of Phosphatidylethanolamine Bio- slight amount of deuterium label was detected in the diacylsynthesis in McA-RH7777 Cells—To get insight into the qualita- glycerol moiety of PtdEtn and PtdCho in McArdle cells tive and quantitative contributions of the CDP-Etn pathway and labeled with d3-Ser for 24 h, which was clear from the fact PtdSer decarboxylation to overall PtdEtn synthesis in mammalian that less than 4% of the PtdCho molecules were detected cells, McA-RH7777 (McArdle) cells were incubated in the pres- with a mass up to 3 mass units heavier than the parental ence of deuterated, pathway-specific precursors
The CDP-Etn pathway and PtdSer decarboxylation are present in all mammalian cells
Summary
The two pathways could serve mainly to “locally” supply certain organelles with PtdEtn for maintaining specific molecular species profiles within these organelles. It is possible that the two pathways yield different molecular species profiles as was shown for the synthesis of PtdCho (CDP-Cho pathway versus PtdEtn methylation) in rat hepatocytes [21]. The development and refinement of mass spectrometry in combination with the availability of deuterated pathwayspecific precursors has opened the possibility of displaying the PtdEtn species synthesized via the CDP-Etn or PtdSer decarboxylation pathways. We report here that McARH7777 cells, when cultured at equimolar concentrations of ethanolamine and serine, prefer the CDP-Etn pathway over PtdSer decarboxylation in a ratio of ϳ2:1, with the decarboxylation route having a preference for the synthesis of long chain, polyunsaturated species
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