Point mutagenesis of positively charged amino acids of cholesteryl ester transfer protein: conserved residues within the lipid transfer/lipopolysaccharide binding protein gene family essential for function.
The cholesteryl ester transfer protein (CETP) binds to plasma lipoproteins and transfers neutral lipids between them. Previous studies showed that lipoprotein binding involves ionic interactions between CETP and lipoproteins, with increased binding of CETP to lipoproteins carrying increased negative charge. In order to understand the molecular determinants of lipoprotein binding in CETP, site-directed mutagenesis was carried out on positively charged amino acids within and outside regions of conserved sequence in the putative family of lipid transfer/lipopolysaccharide (LPS) binding proteins (LT/LBP). Within the conserved regions, two mutant proteins, K233A and R259D, were well secreted by the transfected cells but showed markedly reduced cholesteryl ester transfer activity. Separating the bound from free CETP by gel filtration after incubation with HDL, HDL binding by K233A was found to be impaired, suggesting that the binding deficiency of the mutant may be responsible for decreased transfer activity. Kinetic analysis showed a marked increase in the apparent Km but no change in Vmax, consistent with a lipoprotein binding defect. Thus, within CETP, K233 and R259 play an essential role in cholesteryl ester transfer activity probably by mediating binding of CETP to lipoproteins. Sequence alignment of CETP, phospholipid transfer protein, LPS binding protein, and bactericidal permeability-inducing protein showed that K223 and R259 were strictly conserved as positively charged amino acids, suggesting a common function within the LT/LBP gene family.
Jiang
X C
XC
Department of Medicine, Columbia University, New York, New York 10032, USA.
Bruce
C
C
Cocke
T
T
Wang
S
S
Boguski
M
M
Tall
A R
AR
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D053480:Cholesterol Ester Transfer Proteins; D004563:Electrochemistry; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D000596:Amino Acids; D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; D002352:Carrier Proteins; D053480:Cholesterol Ester Transfer Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D000099231:Lipopolysaccharide-Binding Protein; C021623:bactericidal permeability increasing protein; C030363:lipid transfer protein