Nucleotide sequence and structure of the human apolipoprotein E gene.

Abstract

The gene for human apolipoprotein E (apo-E) was selected from a library of cloned genomic DNA by screening with a specific cDNA hybridization probe, and its structure was characterized. The complete nucleotide sequence of the gene as well as 856 nucleotides of the 5' flanking region and 629 nucleotides of the 3' flanking region were determined. Analysis of the sequence showed that the mRNA-encoding region of the apo-E gene consists of four exons separated by three introns. In comparison to the structure of the mRNA, the introns are located in the 5' noncoding region, in the codon for glycine at position -4 of the signal peptide region, and in the codon for arginine at position +61 of the mature protein. The overall lengths of the apo-E gene and its corresponding mRNA are 3597 and 1163 nucleotides, respectively; a mature plasma protein of 299 amino acids is produced by this gene. Examination of the 5' terminus of the gene by S1 nuclease mapping shows apparent multiple transcription initiation sites. The proximal 5' flanking region contains a "TATA box" element as well as two nearby inverted repeat elements. In addition, there are four Alu family sequences associated with the apo-E gene: an Alu sequence located near each end of the gene and two Alu sequences located in the second intron. This knowledge of the structure permits a molecular approach to characterizing the regulation of the apo-E gene.Apolipoprotein E (apo-E) is a component of various classes of plasma lipoproteins in all mammals that have been studied (for review, see refs. 1 and 2). It is a single chain polypeptide (Mr, 34,000) of 299 amino acids (3) that is synthesized initially with an 18-residue signal peptide that is removed cotranslationally (4,5). The amino acid sequence as well as the mRNA nucleotide sequence are known for both the human (3, 6) and rat (7) species. The major site of synthesis is the liver, but relatively abundant levels of apo-E mRNA have been detected in many extrahepatic tissues, including the brain and the adrenals (8). In addition, apo-E is produced by mouse peritoneal macrophages, as well as human monocytederived macrophages (9).A major function of apo-E is its mediation of the cellular uptake of specific lipoproteins through an interaction with apo-B,E(LDL) receptors on extrahepatic and hepatic cell surfaces and with distinct hepatic apo-E receptors (for review, see ref. 10). The receptor binding domain of human apo-E has been determined to be an arginine-and lysine-rich region in the vicinity of residues 140 and 160 (11,12). Variant forms of apo-E with single amino acid substitutions in this region show decreased receptor binding activity (13)(14)(15) and are associated with type III hyperlipoproteinemia and accelerated cardiovascular disease (for review, see refs. 16 and 17). Apolipoprotein E with normal receptor binding activity is found in two common isoforms, the E3 and E4 phenotypes, with either cysteine or arginine, respectively, at residue position 112 (13).Because of the central role that apo-E plays i...

Apolipoprotein E and Complement C3 Polymorphism and Their Role in the Response to Gemfibrozil and Low Fat Low Cholesterol Therapy

“…The amino-terminal domain of apolipoprotein E, around arginine 158, contains the region that binds to the cysteine rich repeats on the LDL and LDL receptor related protein/a 2 -macrpglobulin-receptor (2) which bears homologous repeats. The human apolipoprotein E gene spans 3.7 Χ 10 3 bases including four exons (3,4) and is located on chromosome 19 (4) in a gene family that is also close to the locus of the complement factor C3 gene (5). Apolipoprotein E polymorphic with three common isoforms, ε2 (112cys, ISScys), ε3 (112cys, 158arg, the most common) and ε4 (112arg, 158arg), which are determined by separate alleles at the apolipoprotein Ε locus.…”

Apolipoproteína E e a doença de Alzheimer

“…O gene apoE humano está localizado no braço longo do cromossomo 19 (19q13.2) (Olaisen et al, 1982;Das et al, 1985;Paik et al, 1985) A apoE é uma glicoproteína com 317 aminoácidos e é uma das principais proteínas presentes no plasma humano, para onde é exportada após sua síntese no fígado (Lin et al, 1986;Elshourbagy et al, 1985) (Weisgraber et al, 1981;Rall et al, 1982a,b;Weisgraber et al, 1982): o alelo ε2 possui o aminoácido cisteína nas duas posições da proteína (aminoácidos 112 e 158), o ε3 tem a cisteína na 112 e uma arginina na 158 (sendo este o alelo mais comum da apoE), enquanto que o alelo ε4 possui argininas nestas duas posições (Figura 2). As outras variantes da apoE são chamadas de ε1, ε5 e ε7, mas elas são extremamente raras.…”

Direct sequencing of affinity‐captured amplified human DNA application to the detection of apolipoprotein E polymorphism

“…In our method, this was circumvented by carrying out two consecutive PCR amplifications with a nested set of primers in the second amplification [l]. The primers ( fig.2) were chosen from the known sequence of the apo E gene [18]. The primary primers Pl and P4 were 328 base pairs apart on the apo E gene.…”