Characterization of lecithin:cholesterol acyltransferase expressed in a human lung cell line.

Lecithin:cholesterol acyltransferase (LCAT) is a key enzyme for the transfer of mammalian cholesterol from peripheral tissues to the liver. In patients deficient in LCAT, serum cholesterol levels rise and can lead to corneal opacity, proteinuria, anemia, and kidney failure. As early as 1968, relatively low volume transfusion of normal plasma was shown to temporarily correct the abnormal lipoprotein profiles in LCAT-deficient patients. However, despite the cloning, study, and extensive expression of LCAT in mammalian cell lines, there is still no viable, clinical therapy for LCAT deficiency. The current study was initiated to provide a source of recombinant human LCAT for enzyme replacement therapy. Accordingly, human LCAT has been cloned and expressed for the first time in a human cell line. The recombinant LCAT secreted by these cells was purified by phenyl-Sepharose chromatography, analyzed to determine the nature of its glycosylation, and tested for its enzymatic properties. The activity and basic kinetic parameters for the enzyme were determined using both a fluorescent water-soluble substrate and a macromolecular (proteoliposome) substrate. The enzymatic properties and the carbohydrate components of the recombinant LCAT were all sufficiently similar to those of the circulating human plasma enzyme, suggesting that this source of LCAT may be appropriate for use in some form of enzyme replacement therapy.

Characterization of the rat cytoplasmic C1-tetrahydrofolate synthase gene and analysis of its expression in liver regeneration and fetal development.

The eukaryotic trifunctional enzyme, C(1)-tetrahydrofolate (THF) synthase, interconverts folic acid derivatives between various oxidation states and is critical for normal cellular function, growth, and differentiation. Using a rat C(1)-THF synthase cDNA and synthetic oligonucleotides, the rat C(1)-THF synthase gene was isolated and characterized. The gene consists of 28 exons and spans 67.5 kbp. Primer extension, RNase protection, and rapid amplification of cDNA ends (RACE) experiments indicate the presence of multiple transcription start points (tsp) within a 250-bp window located between 50 and 300 bp upstream from the start codon. The 5' flanking region is devoid of a TATA consensus sequence motif, but putative regulatory elements, including NF-kappabeta, HNF-4alpha1, RARalpha1, C/EBP, and PPAR are present in the promoter region. The 5' flanking region also contains two sets of tetranucleotide repeats and two short interspersed nuclear elements (SINES). The initial 2500 bp of 5' flanking sequences of the rat and mouse cytoplasmic C(1)-THF synthase genes share 70% identity. However, comparison with the human gene from the Human Genome Data Bank revealed no significant homology in the 5' flanking region. The gene structure characterization led to the identification of a pseudogene that is 94% identical to the C(1)-THF synthase gene and probably diverged 10-12 million years ago. In addition, the gene expression patterns of C(1)-THF synthase were investigated during liver regeneration and liver and kidney organogenesis, two highly regulated events. In both processes, C(1)-THF synthase expression correlated with increased nucleotide metabolism. This pattern suggests that the gene is regulated in response to changes in the demand for folate-dependent one-carbon units.