Contribution of hepatic lipase, lipoprotein lipase, and cholesteryl ester transfer protein to LDL and HDL heterogeneity in healthy women.

Hepatic lipase (HL) and cholesteryl ester transfer protein (CETP) have been independently associated with low density lipoprotein (LDL) and high density lipoprotein (HDL) size in different cohorts. These studies have been conducted mainly in men and in subjects with dyslipidemia. Ours is a comprehensive study of the proposed biochemical determinants (lipoprotein lipase, HL, CETP, and triglycerides) and genetic determinants (HL gene [LIPC] and Taq1B) of small dense LDL (sdLDL) and HDL subspecies in a large cohort of 120 normolipidemic, nondiabetic, premenopausal women. HL (P<0.001) and lipoprotein lipase activities (P=0.006) were independently associated with LDL buoyancy, whereas CETP (P=0.76) and triglycerides (P=0.06) were not. The women with more sdLDL had higher HL activity (P=0.007), lower HDL2 cholesterol (P<0.001), and lower frequency of the HL (LIPC) T allele (P=0.034) than did the women with buoyant LDL. The LIPC variant was associated with HL activity (P<0.001), HDL2 cholesterol (P=0.034), and LDL buoyancy (P=0.03), whereas the Taq1B polymorphism in the CETP gene was associated with CETP mass (P=0.002) and HDL3 cholesterol (P=0.039). These results suggest that HL activity and HL gene promoter polymorphism play a significant role in determining LDL and HDL heterogeneity in healthy women without hypertriglyceridemia. Thus, HL is an important determinant of sdLDL and HDL2 cholesterol in normal physiological states as well as in the pathogenesis of various disease processes.

Probing the 121-136 domain of lecithin:cholesterol acyltransferase using antibodies.

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of plasma lipoprotein cholesterol in mammals as part of the reverse cholesterol transport pathway. Studies of the natural mutations of LCAT revealed a region that is highly sensitive to mutations (residues 121-136) and it is highly conserved in six animal species. The purpose of these studies was to investigate the reactivity of wild type and several mutated forms of LCAT, with a series polyclonal antibodies to further characterize this specific domain (residues 121-136). Two polyclonal antibodies directed against the whole enzyme, one against human plasma LCAT and the other against purified recombinant LCAT, and one site specific polyclonal antibody, directed against the 121-136 region of LCAT, were employed. All three antibodies reacted with a recombinant form of purified LCAT; however, only the polyclonal antibodies directed against the whole enzyme were able to recognize the LCAT when it was adsorbed to a hydrophobic surface in a solid phase immunoassay, or when bound to HDL in a sink immunoassay. These findings indicate that the epitope(s) of the 121-136 region are not accessible to antibodies under these conditions. Three mutant forms of LCAT, representing alterations in the 121-136 region, were also examined for their immunoreactivity with the same panel of antibodies and compared to the wild-type enzyme. These studies demonstrate that in its native configuration the 121-136 region of LCAT is likely to reside on a surface of LCAT. Furthermore, mutations within this region appear to markedly impact the exposure of epitopes at additional sites. These findings suggest that the 121-136 region could play an important role in enzyme interaction with its hydrophobic lipoprotein substrates as mutations within this region appear to alter enzyme conformation, catalytic activity, and the specificity of LCAT.

Biochemical and compositional analyses of recombinant lecithin:cholesterol acyltransferase (LCAT) obtained from a hepatic source.

Lecithin:cholesterol acyltransferase (LCAT) is an important plasma glycoprotein which plays a central role in lipid metabolism. This protein is responsible for generation of cholesteryl esters in plasma and it has been proposed to play a pivotal role in the reverse cholesterol transport pathway. Structural and functional studies of LCAT have employed various expression systems for production of recombinant LCAT (rLCAT). However, recent studies have shown some differences in the oligosaccharide structure and composition of rLCAT. In this study, we have generated a new hepatic based expression system using McArdle-RH7777 (Mc-7777) cells to produce a recombinant protein most similar to human plasma LCAT. The expressed glycoprotein was compared to the LCAT expressed in previously characterized baby hamster kidney (BHK) cells. Both proteins were compared on the basis of their carbohydrate structure and composition as well as their functional properties. Although the functional properties of both glycoproteins were similar, the carbohydrate structure was significantly different. While BHK-LCAT contained bi-, tri-, and tetraantennary structures, Mc-7777 LCAT presented only biantennary oligosaccharide structures. The difference in glycosylation pattern of rLCAT from Mc-7777 and BHK cells underlines the importance of appropriate expression system, both in vivo and in vitro.

Separation of liposomes from plasma components using fast protein liquid chromatography.

We describe an efficient method for separating liposomes (large unilamellar vesicles, 120-150 nm diameter) from plasma lipoproteins employing fast protein liquid chromatography (FPLC). This method resolves very low density lipoprotein (VLDL), low-density lipoprotein, high-density lipoprotein, and other plasma components. Selective detection of liposomes (large unilamellar vesicles, 120-150 nm diameter) was achieved using either radiolabeled or fluorescent lipid probes. The liposomes were found to coelute with the earliest FPLC-eluting lipoprotein fraction, VLDL. The remaining plasma lipoprotein and protein components eluted at later times and were resolved from liposomes and VLDL. In order to separate VLDL from liposomes, we selectively precipitated the VLDL fraction from plasma using tungstophosphoric acid and magnesium chloride, prior to separation by FPLC. Furthermore, we demonstrate that this technique can be used to separate liposomes from lipoproteins in plasma samples collected after intravenous administration of liposomes to mice. This technique has wide application in studies of liposome stability in blood and, in particular, for the characterization of liposomal drug carrier systems.

T-->G or T-->A mutation introduced in the branchpoint consensus sequence of intron 4 of lecithin:cholesterol acyltransferase (LCAT) gene: intron retention causing LCAT deficiency.

Previous mutations associated with lecithin:cholesteryl acyltransferase (LCAT) deficiency syndromes have been identified in the coding regions of the LCAT gene. However, recently, an intron mutation was found in a family in which three sisters presented with fish-eye disease (FED). The probands were shown to be heterozygotes for a mutation in intron 4. The respective T-->C nucleotide substitution, 22 bases upstream of the 3'-splice site, causes a null allele as the result of complete intron retention. Since the natural mutation occurs in a putative branchpoint consensus sequence of the intron, it was hypothesized that the point mutation may disrupt the splicing of the pre-mRNA. To further study the functional significance of the above thymine residue in the branchpoint sequence, we introduced other nucleotides at this position, i.e., LCAT Int-4 MUT-1 (T-->G) and LCAT Int-4 MUT-2 (T-->A). After stable transfection of the mutated pNUT-LCAT minigenes into BHK cells, we could detect neither LCAT activity nor LCAT protein in the culture medium of the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines, as was previously described for the natural mutation. To determine the effects of the introduced mutations on pre-mRNA splicing, total RNA from transfected BHK cells was used for RT-PCR analysis. All BHK cell lines were shown to transcribe the integrated LCAT minigenes. However, the sizes of these LCAT messengers indicated that intron 4 was retained in the pNUT-LCAT Int-4 MUT-1 and pNUT-LCAT Int-4 MUT-2 cell lines. Subsequent sequence analysis of the RT-PCR products demonstrated that the unspliced intronic sequences contained the introduced mutations. In conclusion, the observed retention of intron 4 of the LCAT gene is the result of the specific loss of a thymine residue two bases upstream of the branchpoint adenosine residue in the putative branchpoint consensus sequence. The results confirm that a single base change in the branchpoint consensus sequence of an intron can cause human disease although this sequence is poorly conserved in mammals.