Recombinant proapoA-I(Lys107del) shows impaired lipid binding associated with reduced binding to plasma high density lipoprotein.

In the present study apoA-I (Lys 107del), a naturally occurring human apoA-I variant with a deletion of Lys 107, was expressed in E. coli to examine the effect of this mutation on lipid binding, cholesterol efflux and lecithin:cholesterol acyltranferase (LCAT) activation. Dimyristoyl phosphatidylcholine (DMPC) binding studies revealed slow interaction of proapoA-I(Lys107del) with DMPC relative to normal proapoA-I. After preincubation with human plasma lipoprotein (d<1.225 g/ml) for 1 h at 37 degrees C, 125I-labeled normal proapoA-I chromatographed as a single peak with the high density lipoprotein (HDL) fraction, whereas 125I-labeled proapoA-I(Lys107del) chromatographed with both HDL and free proapoA-I (26% of the radioactivity). Circular dichroism measurements showed that the alpha-helical content of lipid-bound proapoA-I (Lys107del) was reduced to 64 versus 73% of normal proapoA-I. Non-denaturing gradient gel electrophoresis of reconstituted HDL assembled with either proapoA-I(Lys107del) or normal proapoA-I showed that the mutation led to the formation of a second population of smaller rHDL particles. DMPC/proapoA-I(Lys107del) and normal DMPC/proapoA-I complexes exhibited a similar capacity to promote cholesterol efflux from fibroblasts. ProapoA-I (Lys107del) also activated LCAT similar to wild type proapoA-I and human plasma apoA-I. We conclude that deletion of Lys 107 substantially alters the lipid binding properties of the protein, which correlated with reduced binding to plasma HDL in vitro, but did not affect the capacity of the mutant/lipid complex to promote cholesterol efflux or activate LCAT.

A single amino acid deletion in the carboxy terminal of apolipoprotein A-I impairs lipid binding and cellular interaction.

The carboxy-terminal region of apolipoprotein (apo) A-I has been shown by mutagenesis or synthetic peptides to play an important role in lipid binding. However, the precise functional domain of the C-terminal remains to be defined. In this study, apoA-I Nichinan, a naturally occurring human apoA-I variant with a deletion of glutamic acid 235, was expressed in Escherichia coli to examine the effect of this mutation on the functional domain of apoA-I for lipid binding and related consequences. A dimyristoyl phosphatidylcholine binding study with recombinant (r-) proapoA-I Nichinan showed a significantly slow initial rate of lipid binding. On preincubation with human plasma lipoprotein fractions (d<1.225 g/mL) at 37 degrees C for 1 hour, (125)I-labeled normal r-proapoA-I was chromatographed as a single peak at the high density lipoprotein (HDL) fraction, whereas (125)I-labeled r-proapoA-I Nichinan was chromatographed into the HDL fraction as well as the free r-proapoA-I fraction (23% of radioactivity). Circular dichroism measurements showed that the alpha-helix content of lipid-bound r-proapoA-I Nichinan was reduced, being 62% (versus 73%) of normal r-proapoA-I. Nondenaturing gradient gel electrophoresis of reconstituted HDL particles assembled with r-proapoA-I Nichinan and normal r-proapoA-I showed similar particle size. To study cholesterol efflux, human skin fibroblasts were labeled with [(3)H]cholesterol, followed by incubation with either lipid-free r-proapoA-I or DMPC/r-proapoA-I complex. Fractional cholesterol efflux from [(3)H]cholesterol-labeled fibroblasts to lipid-free r-proapoA-I Nichinan or DMPC/r-proapoA-I Nichinan complexes was significantly reduced relative to that of normal r-proapoA-I or DMPC/r-proapoA-I during the 6-hour incubation. Binding assays of human skin fibroblasts by lipid-free r-proapoA-I showed that r-proapoA-I Nichinan was 32% less bound to fibroblasts than was normal r-proapoA-I. Our data demonstrate that the deletion of glutamic acid 235 at the C-terminus substantially reduces the lipid-binding properties of r-proapoA-I Nichinan, which may cause a reduction in its capacity to interact with plasma membranes as well as to promote cholesterol efflux from cultured fibroblasts.

Structural and functional properties of apolipoprotein A-I mutants.

Apolipoprotein (apo) A-I is composed of 243 amino acid residues that fold into amphipathic helixes, and plays a central role in the high density lipoprotein (HDL) metabolism. Familial apoA-I deficiency is a rare metabolic disorder of which three cases have been characterized at a molecular level in western Japan. However, in subjects with apoA-I deficiency, coronary artery disease was not always present. One apo A-I deficiency was compound heterozygous apoA-I mutant for a TATA box mutation and a structural nonsense mutation. To date, screening analysis in our laboratory has identified nine genetically-determined structural mutations of apo A-I. We have also characterized these apo A-I mutations, including apoA-I (Glu235del) Nichinan. Few structural mutations were associated with altered HDL cholesterol levels.

A novel mutant, ApoA-I nichinan (Glu235-->0), is associated with low HDL cholesterol levels and decreased cholesterol efflux from cells.

A novel variant of apolipoprotein (apo) A-I associated with low high density lipoprotein (HDL) cholesterolemia has been identified in a Japanese family during screening for apoA-I variants by isoelectric focusing (IEF) gel analysis. ApoA-I (Glu235-->0) Nichinan was caused by a 3-bp deletion of nucleotides 1998 through 2000 in exon 4 of the apoA-I gene. Four subjects in the family were heterozygous carriers for this mutation; the mean plasma concentrations of apoA-I and HDL cholesterol of affected family members were 30% and 32% lower, respectively, than those of unaffected family members. There were no differences in the levels of very low density lipoprotein and low density lipoprotein cholesterol, triglycerides, and other apolipoproteins between the carriers and the noncarrier family members. In the proband, plasma lecithin:cholesterol acyltransferase activity was normal. Functional consequences of the mutation were examined by expressing the mutated and wild-type proapoA-I cDNAs in Escherichia coli. Cholesterol efflux to recombinant proapoA-I Nichinan from mouse peritoneal macrophages loaded with [3H]cholesterol-labeled acetylated low density lipoprotein was decreased by 54% when compared that of normal recombinant proapoA-I. In vivo turnover studies in normal rabbits demonstrated that the recombinant proapoA-I Nichinan was rapidly cleared (22% faster) compared with normal recombinant proapoA-I. We conclude that apoA-I (Glu235-->0) Nichinan induced a critical structural change in the carboxyl-terminal domain of apoA-I for cellular cholesterol efflux and increased the catabolism of apoA-I, resulting in low HDL cholesterol levels.

Compound heterozygosity for an apolipoprotein A1 gene promoter mutation and a structural nonsense mutation with apolipoprotein A1 deficiency.

Apolipoprotein (apo) A1 plays a central role in the metabolism of HDL. We describe a novel genetic variant of the apoA1 gene identified in a patient with low concentrations of plasma HDL cholesterol. The proband, a 12-year-old Japanese boy, exhibited markedly low levels of both plasma apoA1 and HDL cholesterol. Genomic DNA sequencing of apoA1 genes of the patient showed a compound heterozygosity for an A to C substitution at 27 bp upstream of the transcription start site of 1 apoA1 allele, and a C to T substitution in another allele at residue 84 resulting in aberrant termination. The point mutation at nucleotide position -27 changed ATAAATA of the putative TATA box signal sequence to ATACATA. In addition to this mutation, the patient was heterozygous for a G to A substitution at position -75. Immunoblotting of an isoelectric focusing electrophoresis gel of the proband's plasma showed a trace amount of normal apoA1. No measurable plasma apoA1 and HDL cholesterol in a patient with homozygosity for nonsense mutation at residue 84 has been reported previously. To determine the effects of substitution either at position -27 or -75, plasmids containing the 5'-flanking region of the human apoA1 promoter fused to the CAT reporter gene were constructed and transfected in HepG2 cells. A construct with the A to C substitution at position -27 showed 41. 8+/-4.2%, and G to A substitution at position -75 showed 72.8+/-15. 2% (means+/-SD, n=3) of CAT activities, compared with the wild-type promoter sequence. A construct with the double substitutions at positions -27 and -75 showed only 22.8+/-1.3% (mean+/-SD, n=3) activity relative to the wild type. Our patient is the first case with a TATA box mutation etiologically related to lipoprotein disorders.

A novel homozygous missense mutation in the apo A-I gene with apo A-I deficiency.

We analyzed the genetic defect in a 67-year-old Japanese male patient with apolipoprotein (apo) A-I and high density lipoprotein (HDL) deficiencies, corneal opacities, and coronary artery disease. The plasma concentrations of apoA-I and HDL cholesterol were 2.9 to 7.3 mg/dL and 0.08 to 0.19 mmol/L, respectively. The lecithin:cholesterol acyltransferase (LCAT) activity and cholesterol esterification rate were <40% of normal control values. LCAT mass was 550% of normal control. Sequence analysis of polymerase chain reaction-amplified DNA of the proband's apoA-I gene showed a homozygous T-to-A transition resulting in the substitution of Val 156 with Glu (apoA-I Oita). Direct sequencing of samples obtained from other family members showed that the brother was homozygous, whereas the son was a heterozygous carrier of apoA-I Oita. The heterozygote for apo A-I Oita showed nearly 60% of normal apoA-I and normal HDL cholesterol levels. In vivo turnover studies in rabbits demonstrated that the variant apoA-I was rapidly cleared from plasma compared with normal human apoA-I. Our data suggest that the Val156Glu substitution is associated with apoA-I and HDL deficiency, partial LCAT deficiency, and corneal opacities and that Val156 of apoA-I may play an important role in apoA-I function.

Effects of monatepil, a novel calcium antagonist with alpha 1-adrenergic blocking activity, on the low-density lipoprotein receptor in human skin fibroblasts.

To investigate the mechanisms of the hypolipidemic effect of monatepil, a new class of calcium antagonists with alpha 1-adrenergic blocking activity, we examined the effects of the drug on low-density lipoprotein (LDL) receptor activity and the level of LDL receptor mRNA present in cultured human skin fibroblasts. At concentrations of 2 x 10(-5) M, monatepil increased the binding (248 +/- 43%; mean +/- SD), internalization (374 +/- 18%), and degradation (145 +/- 2%) of 125I-LDL in human skin fibroblasts (n = 3, p < 0.05). Treatment of human skin fibroblasts with 2 x 10(-5) M of monatepil for 6 hours resulted in an increase in LDL receptor mRNA to 163% of the control level (n = 2), as shown by Northern blot analysis. Our results suggest that the hypolipidemic clinical effects of monatepil may be due to increased LDL receptor activity.

[A case of sarcoidosis with cranial mononeuritis multiplex and peripheral polyneuropathy].

A 67-year-old woman with sarcoidosis, cranial mononeuritis multiplex and peripheral neuropathy was reported. The initial presentation of her sarcoidosis was a subcutaneous nodule of left knee joint on June, 1990. The number of subcutaneous nodules increased. Sarcoidosis was diagnosed by biopsy of the subcutaneous nodule. Paresthesia of the face and limbs appeared from November, 1991 and one month later, left abducens palsy and hearing disturbances were added. Alternate day therapy of 20mg prednisolone was effective in improvement of symptoms. The case of sarcoidosis with cranial mononeuritis multiplex and peripheral polyneuropathy was rarely found in 6 to 25% of patients with sarcoid neuropathy in the previous reports. Angiotensin-converting enzyme (ACE) in cerebro-spinal fluid not only raised on exacerbation also lowered with improvement of symptoms after steroid therapy. The ACE, which had a selective activity in the central nervous system, seemed to contribute to the index of treatment for sarcoidosis.

Expression of G alpha i2 mimics several aspects of LPS priming in a murine macrophage-like cell line.

Priming of macrophages with low concentrations of lipopolysaccharide (LPS) enhances the ability of substances that act through heterotrimeric G proteins to stimulate immune cell functions. Although LPS-induced alterations in the expression and functions of G proteins of the alpha i family have been reported in hematopoietic cells, their effects on subsequent steps in LPS priming of macrophages have not been defined. To study the role of G alpha i2 in priming of macrophages by LPS, we expressed a mutant, activated form of alpha i2 (alpha i2Q205L) in P388D1 cells, and compared its effects on PAF-dependent C alpha signalling and arachidonic acid release to those in cells treated with LPS. In control P388D1 cells, treatment with LPS (100 ng/ml) for 1 hr increased the amount of alpha i2 protein 2-fold. Both LPS treatment and expression of alpha i2Q205L increased the rate of PAF-induced C alpha influx across the cell membrane and arachidonic acid release, although neither altered release of C alpha from intracellular stores by PAF. Expression of alpha i2Q205L is sufficient to mimic the effects of LPS on the PAF-induced C alpha i signal and enhanced arachidonic acid release. Consequently, although increasing the expression of alpha i2 may not be the sole mechanism by which LPS enhances signalling by PAF, increased alpha i2 expression can account for the alterations in PAF-induced C alpha i regulation, and arachidonic acid release in LPS-primed P388D1 cells.

Characterization of fatty acid elongation system in porcine neutrophil microsomes.

Microsomes purified from porcine neutrophils containing the fatty acid chain-elongation system for long- and very-long-chain fatty acyl-CoAs, and several enzymatic characters for the elongation of palmitoyl-CoA (16:0-CoA) and arachidoyl-CoA (20:0-CoA) were examined. The heat-inactivation profile for the elongation of 16:0-CoA was different from that of 20:0-CoA, suggesting the presence of different enzyme systems for palmitoyl-CoA and arachidoyl-CoA. Contrary to the elongation system of brain microsomes, the successive synthesis of lignoceric acid (24:0) from 20:0-CoA at 60 microM was not prominent under normal conditions in the neutrophil microsomes. The synthesis of behenic acid (22:0) was slightly inhibited by 0.5 mM N-ethylmaleimide (NEM) present in the assay mixture, whereas the pre-treatment of microsomes with 0.5 mM NEM largely inhibited the synthesis of 22:0 from 20:0-CoA. The synthesis of 24:0, however, was enhanced by 0.5 mM NEM in the elongation of 20:0-CoA and the rate of 24:0 synthesis became dominant over the synthesis of 22:0. These results suggested that the elongation enzyme for very-long-chain fatty acyl-CoA, especially for 20:0-CoA elongation to 22:0 in the neutrophil microsomes contained NEM-sensitive sulfhydryl groups in the active center and the mechanism for the synthesis of 24:0 through successive elongation from 20:0-CoA was different from that of 22:0, as the former was enhanced by NEM whereas the latter was strongly inhibited.

Decrease of palmitoyl-CoA elongation in platelets and leukocytes in the patient of hereditary methemoglobinemia associated with mental retardation.

Effect of the deficiency of NADH-cytochrome b5 reductase on fatty acid elongation was studied in the platelets and leukocytes taken from a patient of hereditary methemoglobinemia associated with mental retardation. The activity of fatty acid elongation was determined by measuring the incorporation of [2-14C]malonyl-CoA into palmitoyl-CoA. The de novo biosynthesis of fatty acids was blocked by the addition of phosphotransacetylase, and the elongation system could be assayed in the homogenates separated from de novo biosynthesis. As compared to normal subjects approximately 40% decrease of fatty acid elongation was observed both in the platelets and leukocytes from the patient.