Phosphatidylcholine formation by LPCAT1 is regulated by Ca(2+) and the redox status of the cell.
BACKGROUND
Unsaturated fatty acids are susceptible to oxidation and damaged chains are removed from glycerophospholipids by phospholipase A(2). De-acylated lipids are then re-acylated by lysophospholipid acyltransferase enzymes such as LPCAT1 which catalyses the formation of phosphatidylcholine (PC) from lysoPC and long-chain acyl-CoA.


RESULTS
Activity of LPCAT1 is inhibited by Ca(2+), and a Ca(2+)-binding motif of the EF-hand type, EFh-1, was identified in the carboxyl-terminal domain of the protein. The residues Asp-392 and Glu-403 define the loop of the hairpin structure formed by EFh-1. Substitution of D(392) and E(403) to alanine rendered an enzyme insensitive to Ca(2+), which established that Ca(2+) binding to that region negatively regulates the activity of the acyltransferase amino-terminal domain. Residue Cys-211 of the conserved motif III is not essential for catalysis and not sufficient for sensitivity to treatment by sulfhydryl-modifier agents. Among the several active cysteine-substitution mutants of LPCAT1 generated, we identified one to be resistant to treatment by sulfhydryl-alkylating and sulfhydryl-oxidizer agents.


CONCLUSIONS
Mutant forms of LPCAT1 that are not inhibited by Ca(2+) and sulfhydryl-alkylating and -oxidizing agents will provide a better understanding of the physiological function of a mechanism that places the formation of PC, and the disposal of the bioactive species lysoPC, under the control of the redox status and Ca(2+) concentration of the cell.
Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr, Way, Oakland, CA 94609, USA. esoupene@chori.org
Eric
E
Soupene
Frans A
FA
Kuypers
D008243:1-Acylglycerophosphocholine O-Acyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000214:Acyl Coenzyme A / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D000478:Alkylation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D055162:Biocatalysis; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D007477:Ions / Q000737:chemistry; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010713:Phosphatidylcholines / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000214:Acyl Coenzyme A; D007477:Ions; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D008243:1-Acylglycerophosphocholine O-Acyltransferase; C526203:Lpcat1 protein, mouse; D003545:Cysteine; D000409:Alanine; D002118:Calcium