H.B. Brewer

The amino acid sequence of human APOA-I, an apolipoprotein isolated from high density lipoproteins.

Abstract The complete amino acid sequence of human A-I has been determined by manual and automated Edman degradation of intact and peptide fragments of A-I. A-I is a single chain protein of 243 residues with the following amino acid composition: Asp16, Asn5, Thr10, Ser15, Glu27, Gln19, Pro10, Gly10, Ala19, Val13, Met3, Leu37, Tyr7, Phe6, Trp4, Lys21, His5, and Arg16. The amino acid sequence contains no linear segments of hydrophobic or hydrophilic residues. A detailed correlation of the amino acid sequence, conformation, and self association of A-I will add further insight into the molecular mechanisms involved in protein-protein and protein-lipid interactions.

Human apolipoprotein A-I and C-III genes reside in the p11 → q13 region of chromosome 11

Apolipoprotein (apo) A-I is a major protein of high density lipoproteins (HDL). The gene for apoA-I has been localized to the p11 leads to q13 region of chromosome 11 by filter hybridization analysis of mouse-human hybrid cell cDNAs containing chromosome 11 translocations utilizing a cloned human apoA-I cDNA probe. The known linkage of apoA-I and apoC-III also permitted the simultaneous assignment of the apoC-III gene to the same region on chromosome 11. Comparison with previously established gene linkages on the mouse and human genome suggests that apoA-I + apoC-III may be linked to the esterase A4 and uroporphyrinogen synthase genes which are present on the long arm of human chromosome 11. The localization of the apoA-I + apoC-III genes in the p11----q13 region of chromosome 11 represents a definitive chromosomal assignment of a human apolipoprotein gene, and will now enable more detailed analysis of the geneomic organization and linkages of the apolipoprotein genes.

Human plasma proapoa-I: Isolation and amino-terminal sequence

Human apoA-I is synthesized as preproapoA-I, a 267 amino acid precursor apolipoprotein. PreproapoA-I initially undergoes intracellular co-translational proteolytic cleavage into proapoA-I. ProapoA-I is secreted from the cell and was isolated from thoracic duct lymph in the apoA-I1 isoform position. The amino-terminal sequence of proapoA-I isolated from human lymph revealed the presence of 6 additional amino acids, Arg-His-Phe-Trp-Gln-Gln, on the amino-terminal end of apoA-I consistent with the proapoA-I sequence determined by nucleic acid sequence analysis of cloned apoA-I. Our results indicate that proapoA-I is present in human plasma, and undergoes post-translational proteolytic cleavage to mature plasma apoA-I.

Analysis of the apoC-II gene in apoC-II deficient patients

Apolipoprotein C-II (apoC-II), a 79 amino acid protein, is a cofactor for lipoprotein lipase, the enzyme which catalyzes the lipolysis of triglycerides on plasma chylomicrons and VLDL. Patients with apoC-II deficiency have marked elevations in plasma triglycerides, chylomicrons, VLDL, and a type I hyperlipoproteinemia. In order to evaluate the molecular defect in apoC-II deficiency, genomic DNA was analyzed using Southern Blot from 2 independent apoC-II deficient patients and compared to normal controls. Restriction digests of genomic DNA were performed with five different enzymes and the restriction fragments analyzed utilizing a 354 base pair nick-translated apoC-II probe for hybridization following Southern blotting. The restriction fragments varied from 0.8 to 21 Kb, and the pattern with normal DNA was identical to that of the two apoC-II deficient patients. The present study reveals that the apoC-II gene is present in patients with apoC-II deficiency. In addition, no insertional or deletional polymorphism was detected in the apoC-II gene of apoC-II deficient patients.

The human apolipoprotein A-II gene is located on chromosome 1

Apolipoprotein (apo) A-II is a major constituent of high density lipoproteins (HDL). The gene for apoA-II has been localized to the p21----qter region of chromosome 1 in man by Southern blot hybridization analysis of DNA from human-mouse cell hybrids using a cloned human apoA-II cDNA probe. The regional assignment was established using two hybrids carrying a reciprocal translocation involving chromosomes 1 and 2. Comparison with previously established gene loci on chromosomes 1 suggests that apoA-II may reside in a conserved linkage group with renin and peptidase C. On the other hand, apoA-II is not linked to the apoA-I gene, which has been localized previously to chromosome 11.

Human ApoB-100 gene resides in the p23----pter region of chromosome 2.

Human apolipoprotein (apo) B-100 is the major apolipoprotein of low density lipoproteins (LDL) and the principal ligand for interaction with the LDL receptor. The gene for apoB-100 has been localized to the p23----pter region of chromosome 2 by filter hybridization analysis with radiolabelled apoB-100 cDNA probes and human-mouse somatic cell hybrids containing chromosome 2 translocations. Other genes at the end of the short arm of chromosome 2 include acid phosphatase, proopiomelanocortin complex, malate dehydrogenase, and N-myc, the latter gene has been previously localized to the same bands (2p23----pter) as the apoB-100 gene. The localization of the apoB-100 gene to the p23----pter region of chromosome 2 completes the genomic organizational relationship of the LDL receptor and the two apolipoprotein ligands for the LDL receptor, apoE and apoB-100; the LDL receptor and apoE having been previously localized to chromosome 19.

The localization of the gene for apolipoprotein C-II to chromosome 19

Human apolipoprotein (apo) C-II, a 79 amino acid protein, functions as a cofactor for lipoprotein lipase, the enzyme which catalyzes the hydrolysis of plasma triglycerides. The chromosomal location of apoC-II has been determined by filter hybridization analysis of human-mouse hybrid cells. Southern blots of DNA from 21 human-mouse hybrid cells were hybridized with a 190 base pair nick translated probe prepared from a Hinf I digest of an apoC-II cDNA clone. Without exception, ApoC-II segregated with chromosome 19 thus establishing synteny with the apoE and LDL receptor genes known to be localized to this chromosome. The localization of the apoC-II gene to chromosome 19 will permit more detailed analysis of the genomic organization and linkages of the apolipoprotein genes.

Human proapoA-ITangier: isolation of proapoA-ITangier and amino acid sequence of the propeptide.

The metabolic defect in Tangier disease is an increased catabolism of apoA-ITangier. The plasma concentration of proapoA-ITangier (apoA-I1 isoform) is increased in patients with Tangier disease. ProapoA-ITangier has been purified to homogeneity, and the amino acid sequence of the propeptide determined by automated Edman degradation. The propeptide sequence was Arg-His-Phe-Trp-Gln-Gln which is identical to the propeptide sequence of normal proapoA-I. These studies indicate that the increase in plasma proapoA-ITangier is not due to a structural defect in the propeptide sequence of proapoA-ITangier and a defect in conversion of proapoA-ITangier to mature apoA-ITangier. The increased catabolism of apoA-ITangier is due to a primary structural defect in mature apoA-ITangier.

Human Apolipoprotein B

Human liver apo B-100 has been cloned in plasmid and phage λgt-11 expression vectors and apo B-100 cDNA clones were identified by screening with monospecific anti-apo B antibodies and synthetic oligonucleotides based on peptides isolated and sequenced from apo B-100. Overlapping cDNA clones containing the entire apo B-100 mRNA were isolated and sequenced. All peptides previously isolated from apo B-100 mRNA were identified and their locations defined. Northern blot analysis utilizing radiolabeled cDNA probes revealed that the apoB-100 mRNA is 14.1 kb in size. On the contrary, two distinct molecular species of apo B mRNA are being produced by the small intestine. The larger-molecular-weight species migrate at the same position as liver apo B-100 mRNA. The smaller species is 7.5 kb in size, and most likely codes for apo B-48. Hybridization and nucleic acid sequence studies were carried out to define common domains and apo B-100 mRNA-specific domains. Since apo B is a single-copy gene, we propose a mechanism for the generation of apo B-100 mRNA and apo B-48 mRNA through differential splicing of the precursor apo B RNA transcript. We have also localized the apo B gene to the p23 → pter region of the short arm of chromosome 2 by filter hybridization analysis of human-mouse hybrid cell DNAs. We have also initiated studies on the gene organization and expression of apo B in patients with abetalipoproteinemia. Studies on two unrelated kindreds revealed no major deletions or insertions and that the apo B-100 mRNA and apo B-100 protein are present in the liver cells of abetalipoproteinemia patients.

Genomic Structure, Biosynthesis, and Processing of Preproapolipoprotein C-II

Apolipoprotein C-II plays a major role in lipid metabolism as a cofactor for lipoprotein lipase, the enzyme involved in the hydrolysis of plasma triglycerides. Patients with deficiency of apo C-II have marked elevations of plasma triglyceride-rich lipoproteins and are at increased risk of pancreatitis. Apolipoprotein C-II has been cloned, and the complete genomic structure elucidated. The apo C-II gene consists of four exons interrupted by three introns and encodes a 22-amino-acid signal peptide that undergoes cotranslational cleavage. The posttranslational processing of apo C-II was analyzed by two-dimensional gel electrophoresis followed by immunoblotting of apo C-II isoforms in the media of Hep G2 cells and in plasma. Four major isoforms have been identified and designated apo C-II-2, apo C-II-1, apo C-II-1/2, and apo C-II0. Neuroaminidase studies have shown that apo C-II-2 and apo C-II-1 are sialic-acid-containing glycoproteins. There is a relative enrichment of these two isoforms of apo C-II in Hep G2 cell media, but they represent minor apo C-II isoforms in normal fasting plasma. Apolipoprotein C-II0, the major plasma isoform of apo C-II, is a proprotein that undergoes proteolytic cleavage of the amino-terminal hexapeptide to form mature apo C-II (apo C-II-1/2)-Amino acid composition and amino-terminal analysis of apo C-II-1/2 confirms the loss of the six terminal amino acids of apo C-II0. In summary: (1) apo C-II has been cloned, and its complete genomic sequence determined; (2) apo C-II is synthesized as preproapo C-II, which undergoes cleavage of a 22-amino-acid signal peptide to form proapo C-II; (3) proapo C-II is glycosylated to generate the sialic-acid-containing glycoproteins apo C-II-2 and apo C-II-1; (4) apo C-II-2 and apo C-II-1 are deglycosylated to form apo C-II0; (5) apo C-II0, the major plasma isoform of apo C-II, is a proprotein; (6) proteolytic processing of apo C-IIo results in the loss of six amino terminal residues to form apo C-II-1/2, the mature apo C-II isoform. A better understanding of the structural relationship of the various plasma isoforms of apo C-II will help to elucidate the mechanisms involved in normal as well as defective processing of apo C-II.