Role of glutamic acid residues 154, 155, and 165 of lecithin:cholesterol acyltransferase in cholesterol esterification and phospholipase A2 activities

HOME

HELP

FEEDBACK

SUBSCRIPTIONS

Role of glutamic acid residues 154, 155, and 165 of lecithin:cholesterol acyltransferase in cholesterol esterification and phospholipase A₂ activities

Jingchuan Wang^a, Jeanine A. DeLozier^a, Abraham K. Gebre^a, Peter J. Dolphin^b, and John S. Parks^a
^a Department of Comparative Medicine, Wake Forest University, School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157
^b Department of Biochemistry, Dalhousie University, Halifax, Nova Scotia, B3H 4H7, Canada

Correspondence to: John S. Parks.

Previous studies have shown that cholesterol esterificationactivity by lecithin:cholesterol acyltransferase (LCAT) is progressivelyinhibited as up to three acidic acid residues are chemicallymodified. The purpose of this study was to determine whetherthree glutamic acid residues in LCAT (154, 155, and 165), thatalign exactly with three acidic acid residues (270, 271, and281) in the amphipathic phospholipid binding region of apoE,were necessary for enzymatic activity. Site-directed mutagenesiswas used to generate mutant constructs of LCAT in which glutamicacid residues 154, 155, and 165 were replaced with glutamineor lysine. Media harvested from transiently transfected COScells was used as a source of LCAT for cholesterol esterificationand phospholipase A ₂ (PLA₂) assays. Cholesterol esterificationfor all mutant constructs (11–26 nmol CE/h/µg) wassimilar to or greater than that of wild type LCAT (16 nmol CE/h/µg),except for a triple mutant, in which glutamic acid residues154, 155, and 165 were changed to lysines (5 nmol CE/h/µg).PLA₂ activity followed a similar trend. There was a significantdecrease in the cholesterol esterification to PLA₂ activityratio when residue 165 was mutated from its wild type negativecharge (E) to an uncharged (Q) or positive (K) charged residue(10.2 vs. 6.0 vs. 4.3, respectively).

We conclude that glutamic acid residues 154, 155, and 165 individuallyor collectively are not necessary for LCAT activity and thatresidue 165 may be in a region of LCAT that is involved withcholesterol binding or is sensitive to cholesterol binding atthe active site of the enzyme.—Wang, J., J. A. DeLozier,A. K. Gebre, P. J. Dolphin, and J. S. Parks. Role of glutamicacid residues 154, 155, and 165 of lecithin:cholesterol acyltransferasein cholesterol esterification and phospholipase A₂ activities.J. Lipid Res. 1998. 39: 51–58.

Supplementary key words: COS cells, apolipoprotein E, apolipoprotein A-I, recombinantHDL mutagenesis, glutamic acid, LCAT, enzyme activity

Add to CiteULike CiteULike Add to Complore Complore Add to Connotea Connotea Add to Del.icio.us Del.icio.us Add to Digg Digg Add to Reddit Reddit Add to Technorati Technorati What's this?

This article has been cited by other articles:

F. Peelman, B. Vanloo, J.-L. Verschelde, C. Labeur, H. Caster, J. Taveirne, A. Verhee, N. Duverger, J. Vandekerckhove, J. Tavernier, et al.
Effect of mutations of N- and C-terminal charged residues on the activity of LCAT
J. Lipid Res., April 1, 2001; 42(4): 471 - 479.
[Abstract] [Full Text]

P. G. Frank and Y. L. Marcel
Apolipoprotein A-I: structure;-function relationships
J. Lipid Res., June 1, 2000; 41(6): 853 - 872.
[Abstract] [Full Text]

M. Lee, P. Uboldi, D. Giudice, A. L. Catapano, and P. T. Kovanen
Identification of domains in apoA-I susceptible to proteolysis by mast cell chymase: implications for HDL function
J. Lipid Res., June 1, 2000; 41(6): 975 - 984.
[Abstract] [Full Text]

F. Peelman, B. Vanloo, O. Perez-Mendez, A. Decout, J.-L. Verschelde, C. Labeur, N. Vinaimont, A. Verhee, N. Duverger, R. Brasseur, et al.
Characterization of functional residues in the interfacial recognition domain of lecithin cholesterol acyltransferase (LCAT)
Protein Eng. Des. Sel., January 1, 1999; 12(1): 71 - 78.
[Abstract] [Full Text] [PDF]