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A more recent version of this article appeared on December 1, 2004

Papers In Press, published online ahead of print September 16, 2004
J. Lipid Res., doi:10.1194/jlr.M400276-JLR200
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Submitted on July 20, 2004
Revised on August 27, 2004
Accepted on September 7, 2004

Amino acids 149 and 294 of human lecithin: Cholesterol acyltransferase (hLCAT) affect phospholipase A2 and acyltransferase fatty acyl specificity

Yue Zhao, Abraham K. Gebre, and John S. Parks

Department of Pathology, Wake Forest University, Winston-Salem, NC 27157

Corresponding Author: jparks{at}wfubmc.edu

Human LCAT (hLCAT) prefers sn-2 18 carbon fatty acyl phosphatidylcholine (PC) substrates (1-16:0, 2-18:1-sn-glycero-3-phosphocholine, POPC) for cholesteryl ester (CE) synthesis, whereas rat LCAT (rLCAT) prefers sn-2 20 carbon substrates (1-16:0, 2-20:4-sn-glycero-3-phosphocholine, PAPC). We identified two regions of hLCAT, which when mutated separately to the corresponding rat sequence (E149A and Y292H/W294F) and transiently expressed in COS-1 cells, increased phospholipaseA2 (PLA2) activity 5.5 and 2.8-fold, respectively, and CE formation 2.9 and 1.4-fold, respectively, using substrate particles containing PAPC. In contrast, both activities with POPC substrate were similar among the three LCAT proteins. The triple mutant (E149A/Y292H/W294F) had increased PLA2 activity with PAPC similar to that observed with the E149A mutation alone; however, unlike E149A, the triple mutant demonstrated a 50% decrease in activity with POPC for both PLA2 activity and CE formation, suggesting an interaction between the two regions of LCAT. Additional mutagenesis studies demonstrated that W294F, but not Y292H, increased PLA2 activity 3-fold with PAPC without affecting activity with POPC. The E149A/W294F double mutation mimicked the LCAT activity phenotype of the triple mutant (more activity with PAPC, less with POPC). In conclusion, separate mutation of two amino acids in hLCAT to the corresponding rat sequence increases activity with PAPC, whereas the combined mutations increase PAPC and decrease POPC activity, suggesting that these amino acids participate in the LCAT PC binding site and affect fatty acyl specificity.


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