Analysis of apolipoprotein E7 (apolipoprotein E-Suita) gene from a patient with hyperlipoproteinemia

We have isolated and analyzed apolipoprotein E7 gene from a patient with hyperlipoproteinemia. Apolipoprotein E7 (apolipoprotein E-Suita) is a variant of apolipoprotein E with four additional units of positive charge compared to apolipoprotein E3, which is the major isoform of apolipoprotein E. The heterozygous gene of apolipoprotein E7/3 from the patient was cloned into lambda phage. The cloned apolipoprotein E genes were subcloned into a murine retrovirus shuttle vector and were expressed. Two out of five clones expressed apolipoprotein E7. The analysis of the nucleotide sequence of the exon and exon-intron boundary regions has shown two G-to-A nucleotide substitutions in the 548 and 551 nucleotide positions from the 5'-end of the fourth exon. These two base substitutions change the amino acid residues -Glu-Glu- to -Lys-Lys- at the 244 and 245 positions from the amino-terminus of the mature protein, and give four additional units of positive charge to the molecule.     

Apolipoprotein (apo) E genotypes by polymerase chain reaction and allele-specific oligonucleotide probes: no detectable linkage disequilibrium between apo E and apo CII

Summary Allelic sequence variation in the apolipoprotein (apo) E gene has been analysed by means of synthetic oligonucleotide probes that detect single base pair substitutions in the codons for amino acid positions 112 and 158, substitutions that are responsible for the common isoforms. Use of the polymerase chain reaction procedure to amplify a sequence of 330 base pairs of the human apo E gene has permitted the development of a robust method for apo E genotyping. This technique has been used to determine the apo E genotype in 95 individuals in whom the genotype for an apo CII TaqI restriction fragment length polymorphism has also been determined. No strong linkage disequilibrium between the two gene loci was detected. This suggests that the metabolic effects of variation, in the apo E and apo CII genes, as detected by the polymorphisms used here, would operate in a statistically independent manner.     

There are two gene sequences for human apolipoprotein CI (apo CI) on chromosome 19, one of which is 4 kb from the gene for apo E

A cDNA probe corresponding to the mRNA sequence for apolipoprotein E (apo E) was used to screen two independently-constructed human genomic libraries. Two recombinants (lambda E-2, and lambda E2-1), isolated using the apo E cDNA probe, also contain part or all of the apo CI gene. Hybridisation studies using both apo E and apo CI cDNA probes show that these two genes are in the same orientation and separated by 4 kb.     

Two coding regions closely linked to the rat apolipoprotein E gene: nucleotide sequences of rat apolipoprotein C-I and ECL cDNA.

Restriction fragments isolated from a 17-kb rat genomic DNA clone containing the gene for apolipoprotein (apo) E were radiolabeled and used to screen a rat liver cDNA library. A cDNA clone hybridizing to a 6-kb genomic DNA fragment was isolated and the nucleotide sequence of the cDNA insert determined. The sequence was homologous to the sequence for human apo C-I and was used to derive the corresponding amino acid sequence. Unlike human apo C-I, mature rat apo C-I contains histidine, lacks valine, and has alanine at the C terminus and aspartate as the N terminus. Screening the rat liver cDNA library with a radiolabeled 1.9-kb restriction fragment from the genomic DNA clone containing the rat apo E gene identified another cDNA clone (ECL cDNA). Nucleotide sequencing yielded a derived 75-amino-acid sequence for the ECL protein with a hydrophobicity profile similar to that of rat apo C-I. Northern analysis demonstrated a 0.50-kb band for ECL mRNA. The tissue-specific expression of the gene is similar to that of rat apo C-I. This study indicates that the rat apo C-I and ECL genes are closely linked, about 4.5 and 12 kb downstream of the apo E gene, respectively.     

Rabbit apolipoprotein A-I mRNA and gene. Evidence that rabbit apolipoprotein A-I is synthesized in the intestine but not in the liver

In order to study the tissue-specific expression of rabbit apolipoprotein (apo) A-I, a 923-base-pair clone, pRBA-502, complementary to rabbit apo A-I mRNA was identified from a rabbit intestinal cDNA library by hybrid-select translation and immunoprecipitation methods. Northern blot and dot-blot hybridization, utilizing 32P-labeled pRBA-502, revealed that the rabbit apo A-I gene is expressed in the intestine, not in the liver and that rabbit apo A-I mRNA is about 950 nucleotides in length. The entire nucleotide sequence of pRBA-502 has been determined and the complete amino acid sequence of the corresponding apo A-I has been deduced. The mRNA codes for a protein comprising 265 amino acids. Amino acids 1-18 and 19-24 of the primary translation product represent the presegment and prosegment, respectively, of apo A-I. Matured rabbit apo A-I contains 241 amino acids and has a molecular mass of 27612 Da. Using pRBA-502 as a probe, a 15.5-kb genomic fragment, which contains the entire apo A-I gene, was isolated from a rabbit liver genomic library. Sequence analysis of the gene shows that the 200 base pairs of the 5' upstream flanking region of the rabbit and human apo A-I genes showed 78% sequence homology. Like the human apo A-I gene, the rabbit apo A-I gene is interrupted by three intervening sequences. Except for two nucleotides in the fourth exon, the coding sequence of the rabbit liver apo A-I gene is identical to that of pRBA-502. Our data showed that the lack of expression of apo A-I gene in rabbit liver is not due to the alternation of rabbit liver apo A-I gene sequence and suggest that the expression of apo A-I gene in rabbit liver is regulated by a trans-acting regulating element(s).     

Analysis of apolipoprotein E5 gene from a patient with hyperlipoproteinemia

We have isolated and analyzed apolipoprotein E5 gene from a patient with hyperlipoproteinemia. Apolipoprotein E5 is a variant of apolipoprotein E with two additional units of positive charge and smaller apparent molecular weight than apolipoprotein E3, which is the major isoform of apolipoprotein E. The heterozygous gene of apolipoprotein E5/3 from the patient was cloned into lambda phage. The cloned apolipoprotein E genes were subcloned into a murine retrovirus shuttle vector and were expressed. One out of four clones expressed apolipoprotein E5. The analysis of the nucleotide sequence of the exons and exon-intron boundary regions has shown a G to A substitution in the 18th nucleotide from the 5'-end of the third exon. This single base substitution changes the amino acid residue Glu to Lys at the third position from the amino-terminus of the mature protein, and gives two additional units of positive charge to the molecule.     

The baboon gene for apolipoprotein A-I: characterization of a cDNA clone and identification of DNA polymorphisms for genetic studies of cholesterol metabolism

We have isolated and sequenced a baboon apolipoprotein A-I (ApoA-I) clone from a liver cDNA library using a human cDNA hybridization probe. This baboon cDNA contains the entire ApoA-I coding region (801 bp, 267 aa), a 3' untranslated region, and a poly(A)tail. Among comparisons with apoAI sequences from other species, the baboon cDNA is most similar to that of the cynomolgus macaque (99.2% homologous) and least similar to the rat sequence (72.6% homologous). A high frequency of nonsynonymous substitutions are observed by alignment of baboon and human apoAI cDNAs, but comparisons of hydrophilicity profiles show that protein structure is conserved by substitutions of aa with similar properties. A polymorphic PstI cleavage site was identified by Southern blot analysis and subsequently mapped to the 5' end of the baboon apoAI gene. To identify effects of apoAI allelic variation on cholesterol metabolism, we used immunoblotting to compare the distributions of ApoA-I among lipoprotein size classes in baboons from each genotype under basal and atherogenic diets. We observed an increase of ApoA-I in high density lipoprotein (size class 1) particles after atherogenic diets in homozygotes for one allele, as compared to slight decreases in the other genotypes.     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Polymorphism of the apolipoprotein E gene and risk of multiple sclerosis in ethnic Russians

Multiple sclerosis (MS) is a multifactorial disease of the central nervous system with pronounced hereditary predisposition. The purpose of the study was to test the assumption on the involvement of the apolipoprotein E gene (APOE) polymorphism in exon 4 in the development of MS in ethnic Russians. Samples independently collected in Moscow (106 MS cases and 189 control healthy volunteers), Sverdlovsk oblast (54 and 109, respectively), and the Republic of Bashkortostan (119 and 285, respectively) were examined. Genotypes for 2059C/T and 2197C/T polymorphisms of the APOE gene, which determine the amino acid substitutions C112R and R158C in apolipoprotein E, were analyzed by polymerase chain reaction followed by restriction analysis of amplificates. No statistically significant differences in genotype or allele frequencies were found between the control group and the group of MS cases. The APOE*4 allele is not associated with the risk of MS in ethnic Russians.     

Restriction isotyping of human apolipoprotein E by gene amplification and cleavage with HhaI

We have used restriction isotyping (restriction enzyme isoform genotyping) for rapid typing of common apolipoprotein E isoforms (E2, E3, E4). ApoE restriction isotyping used oligonucleotides to amplify apolipoprotein E gene sequences containing amino acid positions 112 and 158. The amplification products were digested with HhaI and subjected to electrophoresis on polyacrylamide gels. Each of the isoforms was distinguished by a unique combination of HhaI fragment sizes that enabled unambiguous typing of all homozygotic and heterozygotic combinations. HhaI cleaves at GCGC encoding 112arg (E4) and 158arg (E3, E4), but does not cut at GTGC encoding 112cys (E2, E3) and 158cys (E2).     

Primary structure of Beijing duck apolipoprotein A-1

The primary structure of Beijing duck apolipoprotein A-1 was determined by sequencing peptide fragments derived from tryptic and endoproteinase Asp-N digestion of the protein, and alignment with homologous chicken apo A-1. All of the peptide fragments were isolated by high-pressure liquid chromatography (HPLC) with a Vydac C18 column using a trifluoroacetic acid (TFA) buffer system. The N-terminus of the protein was determined to be aspartic acid by directly sequencing 52 residues of the intact protein. The C-terminus was alanine. The protein contains 240 amino acid residues. By analysis of the whole protein and its tryptic peptides, a six amino acid (Arg-Tyr-Phe-Trp-Gln-His) prosegment was determined. No cross-reactivity between duck and human apo A-1 with a goat antiserum against human apo A-1 was found. Sequence analysis of apo A-1 of other species indicates that amino acid substitutions in rat are more extensive than in other mammals. Isoleucine residues in apo A-1 are inversely correlated to the homology of human to other species, except dog.     

Structure and evolution of the apolipoprotein multigene family

We present the complementary DNA and deduced amino acid sequence of rat apolipoprotein A-II (apoA-II), and the results of a detailed statistical analysis of the nucleotide and amino acid sequences of all the apolipoprotein gene sequences published to date: namely, those of human and rat apoA-I, apoA-II and apoE, rat apoA-IV, and human apoC-I, C-II and C-III. Our results indicate that the apolipoprotein genes have very similar genomic structures, each having a total of three introns at the same locations. Using the exon/intron junctions as reference points, we have obtained an alignment of the coding regions of all the genes studied. It appears that the mature peptide regions of these genes are almost completely made up of tandem repeats of 11 codons. The part of mature peptide region encoded by exon 3 contains a common block of 33 codons, whereas the part encoded by exon 4 contains a much more variable number of internal repeats of 11 codons. These genes have apparently evolved from a primordial gene through multiple partial (internal) and complete gene duplications. On the basis of the degree of homology of the various sequences, and the pattern of the internal repeats in these genes, we propose an evolutionary tree for the apolipoprotein genes and give rough estimates of the divergence times between these genes. Our results show that apoA-II has evolved extremely rapidly and that apoA-I and apoE also have evolved at high rates but some regions are better conserved than the others. The rate of evolution of individual regions seems to be related to the stringency of their functional requirements.     

Structure and expression of mouse apolipoprotein E gene

The mouse apolipoprotein E gene was isolated from a genomic library by screening with a cDNA probe. DNA including apolipoprotein E gene plus segments 2.5 kilobases upstream and 0.3 kilobase downstream of the coding region was transfected into NIH3T3 cells. The cells expressed the same-size apolipoprotein E mRNA and protein as those produced by mouse endogenously. The nucleotide sequence of the gene plus 5' and 3' flanking regions (one kilobase each) was determined. The sequence of the mouse apoliprotein E gene was highly homologous to that of the rat gene, not only in the coding regions but also in the non-coding and intron regions. The mouse and the human apolipoprotein E genes were homologous in the 5' proximal flanking region up to about 200 nucleotides as well as in the four exons. This proximal region was highly conserved for the genes of mouse, rat and human; the relative positions of the "TATA box" and the two copies of "GC box" were identical.     

Apolipoprotein E genotyping in women with recurrent pregnancy loss: An in silico and experimental hybrid study

The role of apolipoprotein E gene polymorphisms in the pathogenesis of recurrent pregnancy loss remains controversial. Therefore, our objective was to investigate the association between recurrent pregnancy loss and apolipoprotein E gene polymorphisms among northwest Iranian women, and also to predict the impact of these nonsynonymous single nucleotide polymorphisms on structure and function of apolipoprotein E protein. The subjects of our current study consisted of 100 women that have had two or more consecutive idiopathic first trimester miscarriages, and one hundred healthy women from the same geographical areas were used as a control group. After DNA extraction, we used a polymerase chain reaction–restriction fragment length polymorphism to genotype of the apolipoprotein E gene. In addition, we predicted the possible effects of amino acid substitutions at codons 112 and/or 158 on the structure and function of apolipoprotein E protein using Polymorphism Phenotyping online software v2. Our results showed that the rate of apolipoprotein E ε4 carriers and the frequency of the ε4 allele in the case group were statistically and significantly higher than those in the control group (P < 0.05). Therefore, our data support the association of the Apo ε4 allele with RPL; however, in silico analysis predicted that the amino acid substitution at residue 112 (Apo ε4 allele) is a benign mutation. Accordingly, further studies are required to elucidate the mechanism(s) underlying the link between RPL pathogenesis and the Apo ε4 allele.     

A 10-bp deletion in the apolipoprotein epsilon gene causing apolipoprotein E deficiency and severe type III hyperlipoproteinemia.

Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2. We identified a 30-year-old male German of Hungarian ancestry with severe type III HLP and apo E deficiency. The disease was expressed in an extreme phenotype with multiple cutaneous xanthomas. Apo E was detectable only in trace amounts in plasma but not in the different lipoprotein fractions. Direct sequencing of PCR-amplified segments of the apo epsilon gene identified a 10-bp deletion in exon 4 (bp 4037-4046 coding for amino acids 209-212 of the mature protein). The mutation is predictive for a reading frameshift introducing a premature stop codon (TGA) at amino acid 229. By western blot analysis, we found small amounts of a truncated apo E in the patient's plasma. Family analysis revealed that the proband was homozygous--and 10 of 24 relatives were heterozygous--for the mutation. Heterozygotes had, as compared to unaffected family members, significantly higher triglycerides (TG), very low-density lipoprotein (VLDL) cholesterol and a significantly higher VLDL cholesterol-to-serum TG ratio, which is indicative of a delayed remnant catabolism. We propose that the absence of a functionally active apo E is the cause of the severe type III HLP in the patient and that the mutation, even in a single dose in heterozygotes, predisposes in variable severity to the phenotypic expression of the disease.     

Role of apolipoprotein E and B gene variation in determining response of lipid, lipoprotein, and apolipoprotein levels to increased dietary cholesterol.

A large segment of the population is modifying its dietary cholesterol intake to achieve a healthier life-style. However, all individuals do not respond equally. We have investigated the effects that that two physiologically important polymorphisms in the apolipoprotein (apo) E and B genes have on the responses of plasma lipid, lipoprotein, and apolipoprotein levels to a high-cholesterol diet. Over a 6-wk period, individuals were prescribed two diets, one consisting of 300 mg dietary cholesterol/d for 3 wk and one consisting of 1,700 mg dietary cholesterol/d for 3 wk. Total cholesterol, low-density-lipoprotein cholesterol (LDL-C), and apo B levels were significantly increased on the high-cholesterol diet. Average total cholesterol (numbers in parentheses are SDs) went from 167.6 (23.4) mg/dl on the low-cholesterol diet to 190.8 (36.2) mg/dl on the high-cholesterol diet; LDL-C went from 99.9 (24.8) mg/dl to 119.2 (33.4) mg/dl, and apo B went from 74.9 (24.5) mg/dl to 86.8 (29.5) mg/dl. In 71 individuals, the frequencies of the apo epsilon 2, epsilon 3, and epsilon 4 alleles were .09, .84, and .07, respectively. The frequency of the longer, apo B signal peptide allele (5'beta SP27) was .68. Apo epsilon 2/3 individuals had significantly lower LDL-C levels than did epsilon 3/3 homozygotes, on both the low-cholesterol diet (LDL-C lower by 21 mg/dl) and the high-cholesterol diet (LDL-C lower by 27 mg/dl). Average triglyceride levels were significantly different among apo B signal peptide genotypes, with the 5'beta SP27/37 homozygotes having the lowest levels (70 mg/dl). When individuals were switched from the low-cholesterol diet to the high-cholesterol diet, in no case were the average responses in lipid levels significantly different among apo E or B genotypes. Therefore, these gene loci do not have a major effect on the response of lipid levels to increased dietary cholesterol.     

Role of the apolipoprotein E polymorphism in determining normal plasma lipid and lipoprotein variation.

The structural gene locus for apolipoprotein E (apo E) is polymorphic. Three common alleles (epsilon 2, epsilon 3, epsilon 4) code for three major isoforms in plasma and determine six apo E phenotypes that may be identified by isoelectric focusing on polyacrylamide. To establish what fraction of the inherited variation in a normal plasma lipid and lipoprotein profile is attributable to the segregation of the common alleles at the apo E gene locus, we have estimated the average apo E allelic effects on plasma cholesterol (C), triglycerides, very low-density lipoprotein (VLDL)-C, VLDL-apo B, low-density lipoprotein (LDL)-C, LDL-apo B, and high-density lipoprotein (HDL)-C in a representative sample of normolipidemic individuals from Ottawa, Canada. Data from published studies were also analyzed by the same statistical procedures. As much as 16% of the genetic variance (8.3% of the total variance) for LDL-C could be accounted for by the apo E gene locus. After correction for differences in age, sex, height, and weight, it was found that the epsilon 2 allele lowered and the epsilon 4 allele raised total cholesterol, LDL-C, and LDL-apo B. No other gene has been identified that contributes as much to normal cholesterol variability. Analysis of these data and those of others also indicates that the apo E locus imparts a differential susceptibility to a variety of factors that promote hyperlipidemia. The hypothesis is proposed that the epsilon 2 allele protects against coronary heart disease (CHD) and, hence, gives a reproductive advantage that is balanced by a predisposition to CHD when the epsilon 2 is combined with a second, independent causative factor to give a reproductive disadvantage. A similar mechanism is proposed for the maintenance of the epsilon 4 allele in the population.     

Distribution of apolipoprotein A-I, C-II, C-III, and E mRNA in fetal human tissues. Time-dependent induction of apolipoprotein E mRNA by cultures of human monocyte-macrophages

Recently developed molecular probes for human apolipoprotein (apo) genes have been used to study the specificity of human tissue expression of the apo A-I, apo C-II, apo C-III, and apo E genes. We have found that apo E mRNA was present in all tissues examined. On the basis of total RNA concentration the relative abundance of apo E mRNA expressed as a percentage of the liver value is as follows: adrenal gland and macrophages, 74-100%; gonads and kidney, 12-15%; spleen, brain, thymus, ovaries, intestine, and pancreas, 3-9%; heart, 1.5%; stomach, striated muscle, and lung, less than 1%. The relative concentration of apo E mRNA in cultures of human peripheral blood monocyte-macrophages increases dramatically as a function of time in culture, and after 5 days, it compares to that of liver. The human tissues shown to synthesize apo E mRNA were also examined for their ability to synthesize apo A-I, apo C-II, and apo C-III mRNA. The relative abundance of apo A-I, apo C-III, and apo C-II mRNA expressed as a percentage of the liver value is as follows: apo A-I, intestine, 50%; apo A-I, pancreas and gonads, 12%; apo A-I, kidney, 4%; apo A-I, adrenal, 2.5%; apo A-I, ovaries and heart, 1%; apo A-I, stomach and thymus, less than 1%; apo C-III, intestine, 62%; apo C-III, pancreas, 7%; apo C-II, intestine, 3%; apo C-II, pancreas, less than 1%. The knowledge of tissue specificities in the synthesis of apolipoproteins is important for our understanding of the regulation of apolipoproteins and lipoprotein metabolism.