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Originally published In Press as doi:10.1194/jlr.M400276-JLR200 on September 16, 2004

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Journal of Lipid Research, Vol. 45, 2310-2316, December 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology

Amino acids 149 and 294 of human lecithin:cholesterol acyltransferase affect fatty acyl specificity

Yue Zhao, Abraham K. Gebre and John S. Parks1

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

1 To whom correspondence should be addressed. e-mail: jparks{at}wfubmc.edu

We identified two regions of human LCAT (hLCAT) that when mutated separately to the corresponding rat sequence (E149A and Y292H/W294F) and transiently expressed in COS-1 cells increased phospholipase A2 (PLA2) activity by 5.5- and 2.8-fold, respectively, and increased cholesteryl ester (CE) formation by 2.9- and 1.4-fold, respectively, relative to hLCAT using substrate particles containing 1-16:0,2-20:4-sn-glycero-3-phosphocholine (PAPC). In contrast, both activities with 1-16:0,2-18:1-sn-glycero-3-phosphocholine (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 by 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.

Abbreviations: CE, cholesteryl ester; FC, free cholesterol; hLCAT, human LCAT; PAPC, 1-16:0,2-20:4-sn-glycero-3-phosphocholine; PC, phosphatidylcholine; PLA2, phospholipase A2; POPC, 1-16:0,2-18:1-sn-glycero-3-phosphocholine; rHDL, recombinant HDL; rLCAT, rat LCAT

Supplementary key words cholesteryl ester formation • mutagenesis • substrate specificity • recombinant high density lipoprotein • recombinant lecithin:cholesterol acyltransferase • sn-2 fatty acid


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Copyright © 2004 by the American Society for Biochemistry and Molecular Biology.