Apolipoprotein A-I (R151C)Paris is defective in activation of lecithin: cholesterol acyltransferase but not in initial lipid binding, formation of reconstituted lipoproteins, or promotion of cholesterol efflux.

ApoA-I(R151)Paris is a natural apolipoprotein (apo) A-I variant that is associated with low levels of high-density lipoprotein cholesterol (HDL-cholesterol) and the partial deficiency of lecithin:cholesterol acyl-transferase (LCAT) in the plasma of heterozygous carriers. We compared the abilities of recombinant normal apoA-I and recombinant apoA-I(R151C)Paris to clear an emulsion of dimyristoylphosphatidylcholine (DMPC), to form reconstituted lipoproteins with dipalmitoylphosphatidylcholine (DPPC), to activate LCAT, and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or of exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. Recombinant apoA-I(R151C)Paris occurred in monomeric and dimeric forms at a ratio of 60:40. Normal apoA-I and apoA-I(R151C)Paris cleared DMPC emulsions at equal rates. Both isoforms associated completely with DPPC during cholate dialysis. Normal apoA-I formed one single particle with a mean diameter of 9.3 nm, whereas apoA-I(R151)Paris gave rise to three particles with mean diameters of 9.3 nm (containing 74% of apoA-I), 10.6 nm, and 12.1 nm, respectively. Compared to normal apoA-I, apoA-I(R151C)Paris had a reduced LCAT-cofactor activity with a 60% lower Vmax/Km ratio due to a 50% higher affinity constant, Km. During incubations for 10 min and 360 min, normal apoA-I/DPPC complexes and apoA-I(R151C)Paris/DPPC complexes were equally efficient in releasing biosynthetic cholesterol from SMCs. In the lipid-free form, apoA-I(R151C)Paris induced normal hydrolysis of cholesteryl esters and normal cholesterol efflux from lipid-loaded mouse-peritoneal macrophages. In conclusion, in addition to its ability to form homo- and heterodimers, apoA-I(R151C)Paris is characterized by defective LCAT-cofactor activity but by normal lipid binding and cholesterol-efflux-promoting abilities.

Multiple dysfunctions of two apolipoprotein A-I variants, apoA-I(R160L)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 LCAT-cofactor activity of apoA-I(P165R) and apoA-I(R160L)Oslo as defined by the ratio of Vmax to appKm was reduced significantly by 62% and 29%, respectively (here and throughout the text, the apparent Km 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; Vmax estimates are not affected by this error). ApoA-I/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.

A natural apolipoprotein A-I variant, apoA-I (L141R)Pisa, interferes with the formation of alpha-high density lipoproteins (HDL) but not with the formation of pre beta 1-HDL and influences efflux of cholesterol into plasma.

ApoA-I(L141R)Pisa is a naturally occurring apolipoprotein A-I variant that causes virtual absence of HDL in hemizygotes and hypoalphalipoproteinemia with half-normal levels of HDL-cholesterol in heterozygotes. In this study we analyzed the distribution of HDL subclasses in plasmas of four hemizygotes for this mutation. We also investigated the abilities of these plasmas to esterify cholesterol and to promote cholesterol efflux. Residual apoA-I-containing lipoproteins in plasmas of hemizygotes for apoA-I(L141R)Pisa correspond to pre beta 1-LpA-I and small alpha-LpA-I. Unlike normal pre beta 1-LpA-I, pre beta 1-LpA-I of apoA-I(L141R)Pisa hemizygotes was not converted into a larger alpha-migrating particle. Plasmas of apoA-I(L141R)Pisa hemizygotes were significantly reduced in their activity to esterify cholesterol in either endogenous or exogenous lipoproteins. Cholesterol efflux capacity was significantly lower than that of normal plasma. Efflux of [3H] cholesterol from radiolabeled fibroblasts into apoB-depleted plasma of normal probands was biphasic with fast cholesterol efflux occurring in the first minute. Thereafter, cholesterol efflux was slow and unsaturable. After incubation with radiolabeled fibroblasts, efflux values of [3H]cholesterol into apoB-depleted plasma from normal controls and from apoA-I(L141R)Pisa hemizygotes were indistinguishable at 1 min. Longer incubations with apoB-free plasma from apoA-I(L141R)Pisa hemizygotes did not, however, lead to the unsaturable increase in cholesterol efflux that was observed during incubations with apoB-free plasma of normolipidemic probands. Pre beta 1-LpA-I of apoA-I(L141R)Pisa hemizygotes took up significantly less cell-derived [3H]cholesterol than pre beta 1-LpA-I of normal donors. We conclude that apoA-I(L141R)Pisa interferes with the formation of lipid-rich alpha-HDL but not with that of lipid-poor pre beta 1-LpA-I. Very low concentrations of alpha-HDL in plasmas of apoA-I(L141R)Pisa hemizygotes combined with reduced LCAT activity cause a decrease of the slow, unspecific, and LCAT-dependent components of cholesterol efflux into plasma.

Heterozygosity for apolipoprotein A-I(R160L)Oslo is associated with low levels of high density lipoprotein cholesterol and HDL-subclass LpA-I/A-II but normal levels of HDL-subclass LpA-I.

We studied a Norwegian patient and his family, who presented with low HDL-cholesterol. DNA sequence analysis of the apoA-I gene revealed heterozygosity for a mutation in the apoA-I gene that causes a leucine for arginine replacement at residue 160. Compared to unaffected family members, heterozygous carriers of apoA-1 (R160L)Oslo had 60-70% lower mean levels of HDL-cholesterol, 50-60% lower mean levels of apoA-I and 70-80% lower levels of apoA-II. Moreover, the serum concentration of the apoA-II-containing HDL-subclass LpA-I/A-II was decreased by 70% whereas the concentration of the apoA-II-free HDL-subclass LpA-I did not differ from that in unaffected family members. The decrease of LpA-I/A-II was associated with the lack of large LpA-I/A-II. ApoA-I(R160L)Oslo was present at increased concentrations relative to normal apoA-I in plasma, HDL3, and LpA-I. However, only trace amounts of the variant isoform were detectable in immunopurified LpA-I/A-II. Pre beta1-LpA-I contained normal and variant apoA-I isoforms. We conclude that the failure of apoA-I(R160L)Oslo to form LpA-I/A-II causes low HDL-cholesterol in heterozygous carriers of this apoA-I variant.