Studies of plasmalogen biosynthesis and regulation in animal cells: Insights using somatic cell mutants

Plasmalogens are a major subgroup of animal cells membrane phospholipids that contain a unique vinyl ether linkage at the sn-1 position of the glycerol backbone. They constitute 18% of the total phospholipid mass in humans, but we know little about how a cell regulates plasmalogen levels and there are questions concerning the biosynthetic pathway(s) used for plasmalogen biosynthesis in animal cells. In an effort to better address these issues I developed a new selection protocol designed to isolate novel animal cell mutants that are defective in the later steps of the plasmalogen biosynthesis pathway. A mutagenized population from the NRel-4 cell line (a derivative of the Chinese hamster cell line, CHO-K1) was treated with a fluorescent plasmalogen precursor, sn-1-O-[9(1'-pyrenyl)]nonylglycerol (pAG) followed by exposure to long wavelength (>300 nm) UV light. This killed most of the cells due to the generation of reactive oxygen.; A clonal cell line, pAG.103, that survived this selection, was further characterized. This cell line was less able to synthesize plasmenylethanolamine (the primary plasmalogen in this cell line) even when grown in the presence of alkylglycerol. The synthesis of the non-vinyl phosphatidylethanolamine was also affected, although less so. Enzymatic characterization showed that ethanolamine kinase activity was reduced by 60% compared to the parent strain. Choline kinase activity was also slightly decreased (25% reduction) suggesting that ethanolamine kinase and choline kinase activities may reside on the same protein in CHO cells. The data obtained from the pAG.103 cell line demonstrated the dependence of plasmalogen biosynthesis upon the cytidinediphosphoethanolamine (CDP-ethanolamine) pathway and that ethanolamine kinase may be a rate-limiting step for plasmalogen biosynthesis. Additional studies, examining the proposal that plasmenylethanolamine can also be synthesized through head-group exchange with serine phospholipids, showed that this is not an option in CHO cells.; The influence of ethanolamine supply on the ethanolamine plasmalogen biosynthesis was also investigated in CHO cells. The data showed that plasmalogen biosynthesis could and plasmalogen levels could be modified by changes in ethanolamine levels in the medium.