Rosemary Ronan

[10] Isolation and characterization of apolipoproteins A-I, A-II, and A-IV

A number of different analytical techniques are now available for the isolation of apoA-I, apoA-II, and apoA-IV. The choice of a particular technique is dependent on the instrumentation available, and the quantity of isolated apolipoprotein required. The isolation and characterization of the separate isoforms and the precursor isoproteins of the individual apolipoproteins are detailed, and methods for the evaluation of the purity of the separate apolipoproteins presented. A method for the evaluation of apolipoproteins in plasma is now available which permits the identification of structural variants of plasma apolipoproteins in patients with dyslipoproteinemias.

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.

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.

Amino acid sequence of human plasma apolipoprotein C-III from normalipidemic subjects

The complete amino acid sequence of human plasma apolipoprotein C‐III (apoC‐III) isolated from normal subjects is described. ApoC‐III is a linear polypeptide chain of 79 amino acids. Tryptic digestion of intact apoC‐III produced 5 major peptides, while tryptic digestion of the citraconylated protein yielded two peptides. The complete amino acid sequence of apoC‐III was determined by the automated Edman degradation of the intact protein as well as the various tryptic peptides. Phenylthiohydantoin amino acids were identified by high‐performance liquid chromatography and chemical ionization mass spectrometry. The amino acid sequence of apoC‐III isolated from normalipidemic subjects is identical to the apoC‐III sequence derived from the cDNA sequence and differs at 4 positions from the previously reported sequence of apoC‐III derived from a patient with type V hyperlipoproteinemia.

Purification and characterization of apolipoprotein C-II from human plasma by high-pressure liquid chromatography

Apolipoprotein C-II, which activates lipoprotein lipase, was isolated from normal subjects and purified to homogeneity by reverse-phase high-pressure liquid chromatography (HPLC). The partially purified product from DEAE-cellulose chromatography was eluted from a Radial Pak C18 cartridge in a radial compression module using a linear gradient of 0.01 M ammonium bicarbonate and acetonitrile. The final product was homogeneous by polyacrylamide gel electrophoresis (pH 8.9), isoelectric focusing (pH 2.5-6.5), Ouchterlony double immunodiffusion, analytical HPLC and amino acid analysis. The purification of apolipoprotein C-II from normal subjects will permit the elucidation of its amino acid sequence and subsequent comparison with the known sequence of apolipoprotein C-II isolated from patients with hyperlipoproteinemia.

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.