Martin Raabe

Cloning, expression and sequences of mouse sterol-carrier protein-x-encoding cDNAs and a related pseudogene

The expression of the sterol-carrier protein 2 (SCP-2)-encoding gene (SCP-2) is unusually complex. At least four SCP-2-related transcripts are detected in mouse liver: two, of 1.6 and 3.0 kb, are expressed to high levels while the other two, of 0.9 and 2.2 kb, reveal relatively low expression. Hybridization with a probe which specifically hybridizes with the rat SCP-2-related cDNA encoding rat SCP-x reveals that the 2.2- and 3.0-kb transcripts encode mouse SCP-x. SCP-x transcripts are expressed predominantly in the liver, but low-level expression can be demonstrated in all tissues analyzed. Isolation and characterization of two overlapping SCP-x cDNAs indicate that the cDNAs are derived from alternatively polyadenylated transcripts spanning approx. 2.2 and approx. 2.9 kb. Nucleotide sequencing reveals that the predicted ORF, which consists of 547 codons, is composed of 143 C-terminal amino acids which are essentially identical with mouse pre-SCP-2 and 404 N-terminal residues which are specific for SCP-x. To date, it is not clear if all SCP-2-related transcripts are transcribed from a single gene. We have isolated a genomic clone containing an SCP-2-related pseudogene which has some of the characteristics expected for a truncated processed pseudogene. Therefore, our results indicate that at least some of the multiple restriction fragments which are detected by Southern hybridization analyses with SCP-x cDNA-derived probes can be explained by cross-hybridization with a pseudogene.

Effects of Genotype and Diet on Cholesterol Efflux into Plasma and Lipoproteins of Normal, Apolipoprotein A-I-, and Apolipoprotein E-Deficient Mice

We investigated the contribution of apoE to cholesterol efflux into plasmas of normal, apoA-I-, and apoE-deficient mice, which were fed with chow- and cholesterol-rich diets. Plasmas of normal and apoA-I-deficient mice contain apoE in pre-beta-migrating VLDL as well as in HDL-like lipoproteins, which have either electrophoretic alpha- or gamma-mobilities. The latter particle resembled gamma-LpE in human plasma also by its mobility on nondenaturing two-dimensional electrophoresis. No apoE-containing lipoproteins were found in plasmas of apoE-deficient mice. When apoA-I- and apoE-deficient mice received both chow- and fat-rich diets, their plasmas released significantly less 3H-cholesterol from radiolabeled fibroblasts than did plasma of normal mice. Removal of apoE from plasmas of normal and apoA-I-deficient mice by anti-apoE immunoaffinity chromatography decreased their cholesterol efflux capacities (per 1 minute/per 1 hour) by 26%/40% (P = 0.0092/0.0007) and 30%/26% (P = 0.0092/0.0003), respectively. Net cholesterol efflux from fibroblasts into apoA-I-deficient plasma was 45% lower compared with plasma of normal mice. Incubation of fibroblasts with apoE-deficient plasma caused net influx of cholesterol. Prior addition of human apoE to or removal of apoB-containing lipoproteins from apoE-deficient plasma restored its ability to cause net cholesterol efflux to 50% of normal plasma. Some of the differences between cholesterol efflux into normal and apoE-deficient plasmas were attributable to the failure of apoE-deficient plasmas to take up cell-derived 3H-cholesterol into gamma-LpE. Compared with normal plasma, both apoA-I-deficient and apoE-deficient plasmas were significantly decreased in their activity to esterify cell-derived 3H-cholesterol. Anti-apoE chromatography decreased significantly cholesterol esterification in normal plasma and apoA-I-deficient plasma but not in apoE-deficient plasma. Taken together, the data provide evidence that apoE is an important contributor to reverse cholesterol transport, partially because of initial uptake of cell-derived cholesterol by gamma-LpE and partially because of the contribution of apoE-containing lipoproteins to esterification of cholesterol in plasma.