Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers

ApoA-I(R160L) Oslo and apoA-I(P165R) are naturally occurring apolipoprotein (apo) A-I variants that are associated with low HDL-cholesterol in heterozygous carriers. We characterized the capacity of these variants to bind lipid, to activate lecithin:cholesterol acyltransferase (LCAT), and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. During cholate dialysis, normal apoA-I and both variants associated completely with dipalmitoylphosphatidylcholine (DPPC) and formed rLpA-I of identical size. However, both apoA-I(P165R) and apoA-I(R160L) Oslo showed a reduced capacity to clear a turbid emulsion of dimyristoylphosphatidylcholine (DMPC). Compared to normal apoA-I, the LCATcofactor activity of apoA-I(P165R) and apoA-I(R160L) Oslo as defined by the ratio of V max to app K m was reduced significantly by 62% and 29%, respectively (here and throughout the text, the apparent K m is given as Michaelis-Menten kinetics do not take particle binding into account and therefore would result in errors with an interfacial enzyme such as LCAT; V max estimates are not affected by this error). ApoAI/DPPC complexes induced biphasic cholesterol efflux from SMCs with a fast and a slow efflux component. Compared to rLpA-I reconstituted with wild type apoA-I, rLpA-I with apoA-I(P165R) or apoA-I(R160L) Oslo were significantly less effective in promoting cholesterol efflux from SMCs in incubations of 10 min duration but equally effective in incubations of 6 h duration. Lipid-free apoA-I did not induce efflux of biosynthetic cholesterol from SMCs but induced hydrolysis of cholesteryl esters and cholesterol efflux from acetyl-LDL-loaded mouse peritoneal macrophages. In the lipid-free form, both apoA-I variants promoted normal cholesterol efflux from murine peritoneal macrophages. We conclude that amino acid residues arginine 160 and proline 165 of apoA-I contribute to the formation of a domain that is very important for initial lipid binding and contributes to LCAT-activation and promotion of initial cholesterol efflux but not to the stabilization of preformed rLpA-I. —Daum, U., T. P. Leren, C. Langer, A. Chirazi, P. Cullen, P. H. Pritchard, G. Assmann, and A. von Eckardstein. Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L) Oslo and apoA-I(P165R), that are associated with hypoalphalipoproteinemia in heterozygous carriers. J. Lipid Res. 1999. 40: 486–494. Supplementary key words familial hypoalphalipoproteinemia • apoAI variants • cholesterol efflux • LCAT • reverse cholesterol transport The concentration of high density lipoprotein (HDL) cholesterol is inversely correlated with the risk of myocardial infarction (reviewed in ref. 1). Nonsynonymous mutations in the gene encoding apolipoprotein (apo) A-I are a rare cause of familial low HDL-cholesterol levels, i.e., hypoalphalipoproteinemia (reviewed in refs. 2, 3). Although structural apoA-I variants do not account for the common form of familial hypoalphalipoproteinemia seen in survivors of myocardial infarction (2), they are nevertheless invaluable tools in elucidating structure–function relationAbbreviations: apo, apolipoprotein; DMEM, Dulbecco’s modified Eagle’s minimum essential medium; DMPC, dimyristoylphosphatidylcholine; DPPC, dipalmitoylphosphatidylcholine; FAFA, fatty acid-free albumin; FER, fractional esterification rate; FCS, fetal calf serum; HDL, high density lipoprotein; HPLC, high performance liquid chromatography; IEF, isoelectric focusing; LCAT, lecithin:cholesterol acyltransferase; LDL, low density lipoprotein; PBS, phosphate-buffered saline; rLpA-I, reconstituted lipoproteins containing apoA-I; SMC, smooth muscle cell. 1 To whom correspondence should be addressed. at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom