Molecular biology of human apolipoproteins B and E and associated diseases of lipoprotein metabolism.
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Amino Acid Sequence↗
Biomedical subjects
V I Zannis
Publications and source records attributed to V I Zannis.
Mutagenesis of the glycosylation site of human ApoCIII. O-linked glycosylation is not required for ApoCIII secretion and lipid binding.
We have used site-directed in vitro mutagenesis to alter the codon ACT of human apoCIII gene, specifying Thr-74, to GCT (Ala-74). The normal and mutant apoCIII genes were then placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus vector and were used for cell transfection and selection of stable cell lines. Blotting analysis of RNA isolated from several independent cell clones showed that both the normal and mutant genes produced apoCIII mRNA in amounts larger than that found in human fetal liver. Pulse-chase analysis of cell clones expressing the normal and mutant apoCIII genes showed that only the normal apoCIII is modified intracellularly to produce a disialated form (apoCIIIs2). Cell clones expressing the normal apoCIII gene secrete exclusively the disialated form, whereas those expressing the mutant gene secrete the unmodified form. The amount of mutant apoCIII protein produced by C127 cell clones expressing the mutant gene was reduced as compared to that produced by the control cells. Density gradient ultracentrifugation analysis of the secreted apoCIII showed that the flotation properties of the secreted normal and mutant proteins were similar. These findings suggest that the intracellular glycosylation of apoCIII is not required for its intracellular transport and secretion. Furthermore, lack of glycosylation has no effect on the relative affinities of apoCIII for plasma very low density lipoproteins and high density lipoproteins.
Animals↗
Alterations of the glutamine residues of human apolipoprotein AI propeptide by in vitro mutagenesis. Characterization of the normal and mutant protein forms.
We have used site-directed mutagenesis to independently alter the Gln residues at positions -1 and -2 of the human apoAI propeptide to Arg residues. The normal and mutated genes were placed under the control of the mouse metallothionein 1 promoter in a bovine papilloma virus (BPV) vector which also carries a copy of the human metallothionein 1A gene. Following transfection of mouse C127 cells [corrected] with the vectors, cell clones resistant to CdCl2 were selected and analyzed for production of apoAI mRNAs and protein. The RNA blotting analysis showed that the steady-state apoAI mRNA levels of cell clones expressing either the normal or the mutant apoAI gene are 3-5-fold higher than that of the liver or HepG2 cells. Two-dimensional gel electrophoresis of radiolabeled apoAI showed that the apoAI-expressing clones secreted mainly the proapoAI form. Furthermore, both mutant proapoAI's differed by one positive charge from the normal apoAI. Secretion of apoAI into the culture medium follows apparent first-order kinetics and gives similar rate constants for the normal and mutant apoAI forms. Separation of secreted apoAI by density gradient ultracentrifugation in the presence of human plasma or HDL shows identical distribution of plasma and nascent (normal and mutant) apoAI. The findings indicate that in the cell system used the modification of either of the two glutamines of the apoAI prosegment does not affect the intracellular transport and secretion of apoAI, and its ability to associate with HDL.
Animals↗
Sequence and expression of Tangier apoA-I gene.
We have isolated and characterized the apoA-I gene from a lambda L47.1 genomic library constructed with DNA obtained from the lymphocytes of a Tangier disease patient. The DNA-derived protein sequence of Tangier apoA-I was found to be identical to normal apoA-I. Transfection of mouse C127 cells with a recombinant vector containing the Tangier apoA-I gene (pSV2-gpt apoA-I) allowed selection of stable clones resistant to aminopterin and mycophenolic acid. Analysis of these clones for apoA-I synthesis showed that the protein secreted by cells expressing the Tangier apoA-I gene was indistinguishable from the apoA-I secreted by HepG2 cells. These experiments establish that the Tangier apoA-I gene is structurally normal. It appears that the molecular basis of Tangier disease is not related to apoA-I structure or regulation of expression, but rather to other factors pertinent to apoA-I and high-density lipoprotein metabolism.
Amino Acid Sequence↗
Expression of the human apolipoprotein A-I gene in rat myogenic L6E9 cells. DNA methylation and regulation of gene activity.
An 11-kilobase EcoRI fragment containing the human apoA-I gene was cloned in the pSV2gpt vector. The recombinant plasmid was then used to transfect a rat myogenic cell line (L6E9) and HeLa cells. Selection and analysis of cell clones resistant to hypoxanthine/aminopterin/thymidine and mycophenolic acid showed that the apoA-I gene is expressed in L6E9 but not in HeLa cells. The apoA-I mRNA transcribed by either the transfected L6E9 or control human liver cell line, HepG2, has a major transcription initiation site located 26 nucleotides downstream from the CATAAA box. The apoA-I synthesized by the L6E9 cells is secreted into the culture medium and is indistinguishable from the apoA-I secreted by HepG2 cells. These results demonstrate that the L6E9 cells contain the necessary factors required for the expression of the apoA-I gene. Further analysis showed that the endogenous rat apoA-I gene is not expressed in L6E9 cells and exists in a hypermethylated configuration. In contrast, the exogenous human apoA-I gene was found to be hypomethylated in the transfected L6E9 cells and in other apoA-I-producing cells. These findings are consistent with the hypothesis that the expression of the endogenous rat apoA-I gene in L6E9 cells is prevented by hypermethylation, suggesting that compared to other mechanisms which control initiation of transcription, DNA methylation may play a higher hierarchical role in the regulation of expression of eukaryotic genes.
Animals↗
Molecular biology of human apolipoprotein B and related diseases.
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Abetalipoproteinemia↗
Regulation of renin gene expression in hypertensive rats.
A carboxy terminal renin complementary DNA (cDNA) clone from rat kidney was isolated, characterized, and used as a probe for renin messenger RNA (mRNA) quantification in normotensive and hypertensive rats. RNA blotting analysis detected renin mRNA in control kidney and brain. Deoxycorticosterone acetate (DOCA) and high salt (1%) treatment of experimental animals resulted in a greater than 95% decrease in the content of renin mRNA in the kidney, as compared with values in control rats receiving 0.4% NaCl in their diet. In contrast, high salt (1%) treatment alone caused only a twofold decrease in kidney renin mRNA content, as compared with values in controls. DOCA and low salt (0.04%) or low salt (0.04%) treatment alone caused a 1.5-fold increase in the kidney renin mRNA content, as compared with values in control rats. These results indicate that DOCA and salt have a synergistic effect in depressing renin mRNA levels in kidney. Clipping of the left renal artery caused a threefold increase in the steady state level of renin mRNA in the ischemic kidney and a 0.5-fold decrease in the hypertrophied kidney. The data are consistent with the hypothesis that blood pressure and other stimuli regulate the expression of the renin gene in vivo.
Amino Acid Sequence↗
Apolipoprotein gene expression in the rabbit: abundance, size, and distribution of apolipoprotein mRNA species in different tissues.
We have used human apolipoprotein cDNAs as hybridization probes to study the relative abundance and distribution of apolipoprotein mRNAs in rabbit tissues by RNA blotting analysis. The tissues surveyed included liver, intestine, lung, pancreas, spleen, stomach, skeletal muscle, testis, heart, kidney, adrenal, aorta, and brain. We found that liver is the sole or major site of synthesis of apoA-II, apoA-IV, apoB, apoC-I, apoC-II, apoC-III, and apoE, and the intestine is a major site of synthesis of apoA-I, apoA-IV, and apoB. Minor sites of apolipoprotein mRNA synthesis were as follows: apoA-I, liver and skeletal muscle; apoA-IV, spleen and lung; apoB, kidney; apoC-II and apoC-III, intestine. ApoE mRNA was detected in all tissues surveyed with the exception of skeletal muscle. Sites with moderate apoE mRNA (10% of the liver value) were lung, brain, spleen, stomach, and testis. All rabbit mRNAs had forms with sizes comparable to their human counterparts. In addition, hybridization of hepatic and intestinal RNA with human apoA-IV and apoB probes produced a second hybridization band of approximately 2.4 and 8 kb, respectively. Similarly, hybridization of rabbit intestinal RNA with human apoC-II produced a hybridization band of 1.8 kb. The 8 kb apoB mRNA form may correspond to the apoB-48 mRNA, whereas the apoA-IV- and apoC-II-related mRNA species have not been described previously. This study provides a comprehensive survey of the sites of apolipoprotein gene expression and shows numerous differences in both the abundance and the tissue distribution of several apolipoprotein mRNAs between rabbit and human tissues. These findings and the observation of potentially new apolipoprotein mRNA species are important for our understanding of the cis and trans acting factors that confer tissue specificity as well as factors that regulate the expression of apolipoprotein genes in different mammalian species.
Animals↗
Tissue-specific posttranslational modification of rat apoE. Synthesis of sialated apoE forms by neonatal rat aortic smooth muscle cells.
We have studied the synthesis, modification, and secretion of rat apoE in primary cultures of neonatal aortic smooth muscle cells and adult rat hepatocytes. The cultures were pulsed with [35S]methionine and the intracellular and secreted apoE were immunoprecipitated and analyzed by two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis and autoradiography. A short pulse (10 min) showed the presence of a major unmodified apoE form in both cultures. This form comigrated on two-dimensional gels with the major rat plasma apoE isoprotein. A longer pulse (15-120 min) resulted in the progressive appearance of intracellularly modified apoE isoproteins in both cultures. The apoE secreted by aortic smooth muscle cells consisted exclusively of sialated apoE isoproteins that were sensitive to neuraminidase treatment. In contrast, the apoE secreted by primary cultures of adult rat hepatocytes, organ cultures of neonatal rat liver, as well as rat plasma apoE, contained several minor modified isoproteins. Nascent apoE secreted by the aortic smooth muscle cell cultures floats in the density range of 1.09 to 1.186 g/ml. We conclude that aortic smooth muscle cells can synthesize and secrete sialated apoE isoproteins associated with nascent lipoproteins floating in the high density lipoprotein region.
Animals↗
The molecular basis of a familial apoE deficiency. An acceptor splice site mutation in the third intron of the deficient apoE gene.
The molecular basis of the familial apoE deficiency was investigated by gene cloning and comparative expression studies of the normal and the deficient apoE gene. For the latter studies the apoE genes were placed under the control of the mouse metallothionein I promoter in a bovine papilloma virus vector. The studies showed that in the normal gene the mouse metallothionein I promoter directs the synthesis of normal apoE mRNA and protein. In contrast, in the deficient apoE gene the same promoter directs the synthesis of two abnormal apoE mRNA forms, which are similar to those observed in the peripheral blood monocyte macrophage cultures of the patient. Restriction analysis of the cloned gene and partial DNA sequence has shown an A to G substitution in the penultimate 3' nucleotide of the third intron of the apoE gene. This single base substitution abolishes the correct 3' splice site thus creating two abnormally spliced mRNA forms. The smaller form contains 53 nucleotides and the larger form contains the entire third intron of the apoE gene. Both of these mRNA species contain chain termination codons within the intronic sequence and code for short apoE peptides that are not detectable by gel electrophoretic techniques. These findings show that this form of familial apoE deficiency results from a point mutation in the 3' splice junction of the third intron of the apoE gene. Furthermore, the expression system we have employed to study apoE deficiency can be utilized to analyze a broad spectrum of genetic diseases associated with mRNA processing mutations.
Animals↗
Effect of egg cholesterol and dietary fats on plasma lipids, lipoproteins, and apoproteins of normal women consuming natural diets.
Nine normal women, 22 to 37 years old, consumed controlled quantities of natural foods to test their responses to dietary cholesterol and saturated fat. All diets contained, as percentage of calories, 14% protein, 31% fat, and 55% carbohydrate. The main sources of polyunsaturated and saturated fats were corn oil and lard, respectively, and egg yolk was used for cholesterol supplementation. All subjects participated in four diet protocols of 15 days duration, and each diet period was separated by 3 weeks without diet control. The first diet (corn) was based on corn oil, had a polyunsaturated to saturated fat ratio (P/S) of 2.14, and contained 130 mg of cholesterol. The second diet (corn+) was identical to the first but contained a total of 875 mg of cholesterol. The third diet (lard) was based on lard, had a P/S ratio of 0.64, and contained 130 mg of cholesterol. The fourth diet (lard+) was identical to the third, but contained 875 mg of cholesterol per day. Changes of the plasma lipid, lipoprotein and apoprotein parameters relative to the corn diet were as follows: the corn+ diet significantly increased total plasma cholesterol, HDL-cholesterol, LDL-cholesterol, and apoB levels; the lard diet significantly increased total cholesterol, HDL-cholesterol, and apoB; and the lard+ diet significantly increased the total cholesterol, HDL-cholesterol, LDL-cholesterol, and apoA-I and apoB levels. There were no significant variations in VLDL-cholesterol, triglyceride, or apoE levels with these diets. The diets affected both the number of lipoprotein particles as well as the composition of LDL and HDL. Compared to the corn diet, cholesterol and saturated fat each increased the number of LDL particles by 17% and 9%, respectively, and the cholesterol per particle by 9%. The combination of saturated fat and cholesterol increased particle number by 18% and particle size by 24%. Switching from lard+ to lard, corn+, or corn diets reduced LDL-cholesterol of the group by 18%, 11%, and 28%, respectively, while a large inter-individual variability was noted. In summary, dietary fat and cholesterol affect lipid and lipoprotein levels as well as the particle number and chemical composition of both LDL and HDL. There is, however, considerable inter-individual heterogeneity in response to diet.
Adult↗
The complete sequence and structural analysis of human apolipoprotein B-100: relationship between apoB-100 and apoB-48 forms.
We have isolated and sequenced overlapping cDNA clones covering the entire sequence of human apolipoprotein B-100 (apoB-100). DNA sequence analysis and determination of the mRNA transcription initiation site by S1 nuclease mapping showed that the apoB mRNA consists of 14,112 nucleotides including the 5' and 3' untranslated regions which are 128 and 301 nucleotides respectively. The DNA-derived protein sequence shows that apoB-100 is 513,000 daltons and contains 4560 amino acids including a 24-amino-acid-long signal peptide. The mol. wt of apoB-100 implies that there is one apoB molecule per LDL particle. Computer analysis of the predicted secondary structure of the protein showed that some of the potential alpha helical and beta sheet structures are amphipathic, whereas others have non-amphipathic neutral to apolar character. These latter regions may contribute to the formation of the lipid-binding domains of apoB-100. The protein contains 25 cysteines and 20 potential N-glycosylation sites. The majority of cysteines are distributed in the amino terminal portion of the protein. Four of the potential glycosylation sites are in predicted beta turn structures and may represent true glycosylation positions. ApoB lacks the tandem repeats which are characteristic of other apolipoproteins. The mean hydrophobicity the mean value of H1 and helical hydrophobic moment the mean value of microH profiles of apoB showed the presence of several potential helical regions with strong polar character and high hydrophobic moment. The region with the highest hydrophobic moment, between amino acid residues 3352 and 3369, contains five closely spaced, positively charged residues, and has sequence homology to the LDL receptor binding site of apoE. This region is flanked by three neighbouring regions with positively charged amino acids and high hydrophobic moment that are located between residues 3174 and 3681. One or more of these closely spaced apoB sequences may be involved in the formation of the LDL receptor-binding domain of apoB-100. Blotting analysis of intestinal RNA and hybridization of the blots with carboxy apoB cDNA probes produced a single 15-kb hybridization band whereas hybridization with amino terminal probes produced two hybridization bands of 15 and 8 kb. Our data indicate that both forms of apoB mRNA contain common sequences which extend from the amino terminal of apoB-100 to the vicinity of nucleotide residue 6300. These two messages may have resulted from differential splicing of the same primary apoB mRNA transcript.
Adult↗
Intracellular modifications of human apolipoprotein E.
We have used pulse-chase techniques to study the synthesis, intracellular modification, and secretion of human apolipoprotein E by cultures of HepG2 cells and peripheral blood human monocyte macrophages. We have found that modified apoE isoproteins are detectable intracellularly after 14 min of pulse, and their relative concentration increases linearly over a 2-h pulse-chase period. At the same time, the relative concentration of unmodified apoE decreases. All the major modified apoE isoproteins appear simultaneously, and they correspond to the sialo apoE forms apooEs2, apoEs4, and apoEs6. ApoE secretion is first detected after a 30-min pulse. Secreted apoE consists of 92% of the same sialated apoE forms observed intracellularly. ApoE sialation and secretion were not affected by treatment of the HepG2 cultures with tunicamycin, whereas monensin inhibited both the intracellular sialation and secretion of this protein. These findings suggest that apoE is secreted in the form of three major isoproteins generated by intracellular modification of this protein with one or more O-linked oligosaccharide chains containing sialic acid.
Apolipoproteins E↗
Complementary DNA derived structure of the amino-terminal domain of human apolipoprotein B and size of its messenger RNA transcript.
In this paper the sequence of a 5.2-kilobase (kb) cDNA covering the amino-terminal domain of human apolipoprotein B-100 (apoB-100) is reported. The cDNA-derived protein sequence provides the primary structure of 1748 amino acids. This segment of apoB-100 is more hydrophilic than hydrophobic and contains short stretches of predicted helical and beta structures that are interrupted by beta turns. Blotting analysis of RNA isolated from fetal human and adult monkey tissues and various human cell lines showed synthesis of apoB mRNA by liver and intestine and by cells of hepatic (HepG2) and intestinal (Caco-2) origin. The isolation and characterization of overlapping cDNA clones, which provide a nearly full-length copy of human apoB-100, are also reported. From the length of these clones the size of the cytoplasmic apoB mRNA is estimated to be 14.0 kb and codes for a protein of approximately 512,000 daltons.
Adult↗
Structure, evolution, and tissue-specific synthesis of human apolipoprotein AIV.
Apolipoprotein AIV (apoAIV) is a protein of the lipid transport system found associated with chylomicrons, high-density lipoprotein (HDL), and the lipoprotein-free fraction of the plasma. The gene coding for the human apoAIV is closely linked with the genes coding for apolipoproteins AI (apoAI) and CIII (apoCIII). In this paper a nearly full-length apoAIV cDNA clone has been isolated by screening an adult human liver DNA library using a human apoAIV gene probe. In-frame translation of the cDNA sequence in this clone indicated that the human apoAIV consists of 396 amino acid residues including a 20 residue long signal peptide. The coding region of this cDNA sequence contains 15 nucleotide repeats, 11 of which code for amino acid repeats with potentials of forming amphipathic helices. Alignment and comparison of the human and rat apoAIV amino acid sequences indicated a five-residue deletion near the carboxy terminus of the rat protein. This comparison also indicated that these proteins are 61.8% homologous, suggesting that the rate of evolution of apoAIV is 65 accepted point mutations (PAMs) per 100 residues per 100 million years. The rates of evolution of certain amino acid repeats in apoAIV are higher than the rate of evolution of the entire protein. However, the corresponding, computer-generated, secondary structures and hydropathy profiles of these repeats are very similar between the human and rat apoAIV. The relative steady-state levels of apoAIV mRNA in various human and monkey tissues were determined by hybridization blotting analysis of total RNA from these tissues using a human apoAIV cDNA probe.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Intra- and extracellular modifications of apolipoproteins.
This chapter outlined the methods used to study intra- and extracellular modifications of apolipoproteins. These and other related studies have shown that several of the apolipoproteins undergo a series of intra- and extracellular modifications as follows: All apolipoproteins studied contain an 18-26 long signal peptide which is cleaved cotranslationally by the signal peptidase of the rough endoplasmic reticulum. ApoE is further modified intracellularly with carbohydrate chains containing sialic acid and is secreted in the modified form designated apoEs. The modified apoE is subsequently desialated in plasma. ApoA-I is secreted in a proapoA-I form, which consists of 249 amino acids. The N-terminal hexapeptide of proapoA-I is cleaved extracellularly by a proapoA-I to plasma apoA-I converting protease. This cleavage generates the plasma apoA-I form which consists of 243 amino acids. Other known apolipoprotein modifications include the modification of apoB, apoC-III, and apoD with carbohydrate chains that contain sialic acid and the proteolytic cleavage of the proapoA-II segment. At the present time we are able to distinguish several isoprotein forms for a particular apolipoprotein. In addition, we began to understand the biochemical changes which lead to a few of these isoproteins. Future research should be directed toward a better understanding not only of the structure but most importantly of the physiological significance of the different apolipoprotein forms.
Amino Acid Sequence↗
Characterization of the apolipoprotein A-I-C-III gene complex.
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Apolipoprotein A-I↗
Genetic polymorphism in human apolipoprotein E.
This chapter provides the methodologies employed to study the polymorphism of human apoE. These and other related studies have advanced our understanding of the structure and function of this protein as follows: The complex array of human apoE observed by two-dimensional gel electrophoresis results from genetic variation and posttranslational modification. The genetic polymorphism of apoE is explained by the existence of three common alleles (epsilon 4, epsilon 3, epsilon 2) at a single structural gene locus. Combinations of above alleles can generate three homozygous (E4/4, E3/3, E2/2) and three heterozygous (E4/3, E3/2, E4/2) apoE phenotypes. The apoE phenotype E2/2 is found in 91% of patients with type III hyperlipoproteinemia and can be used as a molecular marker for the diagnosis of this disease. However, other rare or common apoE phenotypes have been observed in patients with type III HLP. ApoE originating from E2/2 phenotype (Arg 158 to Cys 158 substitution) has reduced affinity for the LDL receptor. This property of apoE2 can account partially for the accumulation of apoE-rich lipoprotein remnants in the plasma of patients with type III HLP. However, other genetic or environmental factors are necessary for the phenotypic expression of the disease.
Apolipoproteins E↗