Deletion of amino acids Glu146-->Arg160 in human apolipoprotein A-I (ApoA-ISeattle) alters lecithin:cholesterol acyltransferase activity and recruitment of cell phospholipid.
Human apolipoprotein A-I (apoA-I) has an important role in the efflux of cholesterol from peripheral cells, the first step in reverse cholesterol transport. Deletion of amino acids Glu146-->Arg160 in apoA-I (apoA-ISeattle) removes a large section of a lipid binding helix and is associated in vivo with an atherogenic lipoprotein profile characterized by a deficiency in high-density lipoproteins (HDL). In the present study, we asked whether apoA-ISeattle had normal ability to recruit lipids from cells and to form nascent high-density lipoprotein (HDL) particles and whether the altered secondary structure affected lecithin:cholesterol acyltransferase (LCAT) activity. Wild-type apoA-I and apoA-ISeattle expressed in transfected Chinese hamster ovary cells formed nascent HDL particles with similar density distribution and protein-to-lipid ratio. Phospholipid subclass distribution of apoA-ISeattle nascent HDL demonstrated a significant increase in sphingomyelin and phosphatidylethanolamine compared to wild type. ApoA-ISeattle nascent HDL had a unique size distribution compared to wild-type nascent HDL; large (9-20 nm) particles predominated while there were virtually no small (7.5 nm) particles. LCAT reactivity was impaired by apoA-ISeattle nascent HDL where cholesterol esterification was only half that of wild-type complexes. The apoA-ISeattle conformation on nascent HDL was studied with a panel of monoclonal antibodies (Mabs) specific for apoA-I. Mabs that recognize the putative LCAT activation site, residues 95-122, had normal reactivity. As expected, the Mabs that recognized residues 141-164 were unreactive because of the 146-160 deletion; in addition, there was low reactivity with a Mab that recognizes residues 220-242. The data suggest that apoA-I residues 146-160 and/or 220-242 partake in normal LCAT activation and that cooperative interactions between helices may be important for maximal cholesterol esterification.
Lindholm
E M
EM
Department of Molecular and Nuclear Medicine, Life Sciences Division MS 1-220, Lawrence Berkeley National Laboratory, University of California, Berkeley, California 94720, USA.
Bielicki
J K
JK
Curtiss
L K
LK
Rubin
E M
EM
Forte
T M
TM
D000818:Animals; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D000939:Epitopes / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008075:Lipoproteins, HDL / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D010316:Particle Size; D010743:Phospholipids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D002785:Sterol O-Acyltransferase / Q000737:chemistry / Q000378:metabolism*
D016632:Apolipoprotein A-I; D000939:Epitopes; D008075:Lipoproteins, HDL; D010743:Phospholipids; D011994:Recombinant Proteins; D018698:Glutamic Acid; D001120:Arginine; D002785:Sterol O-Acyltransferase