Leila Kotite

Triglyceride-rich lipoproteins isolated by selected-affinity anti-apolipoprotein B immunosorption from human atherosclerotic plaque.

We isolated and characterized immunoreactive apolipoprotein B (apoB)-containing lipoproteins from human atherosclerotic plaque and plasma to determine whether very-low-density lipoprotein (VLDL) can enter and become incorporated into the atherosclerotic lesion and how plaque apoB-containing lipoproteins differ from apoB-containing lipoproteins isolated from plasma. Atherosclerotic plaques were obtained during aortic surgery and processed immediately. Lipoproteins were extracted from minced plaque in a buffered saline solution (extract A). In selected cases a second extraction was done after plaque was incubated with collagenase (extract B). Lipoproteins were then isolated from the extracts by anti-apoB immunosorption and separated into VLDL + intermediate-density lipoprotein (IDL) (d < 1.019 g/mL) and low-density lipoprotein (LDL) (1.019 < d < 1.070 g/mL) fractions by ultracentrifugation. The VLDL + IDL fractions from plaque contained more than one third of the total apoB-associated lipoprotein cholesterol in both extracts A and B. The lipid composition of VLDL + IDL in both extracts was related to that of plasma VLDL + IDL. By electron microscopy mean particle diameters of VLDL + IDL from extracts A and B were 9% and 23%, respectively, greater than VLDL + IDL diameters from plasma. Mean diameters of LDL from extracts A and B were 11% and 31% greater than LDL diameters from plasma. The apoE-apoB ratio of extract A VLDL + IDL was nearly twice that of plasma VLDL + IDL and severalfold higher than that of extract A LDL. Immunoblots of both VLDL + IDL and LDL from extract A demonstrated minimal fragmentation of apoB.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentration and composition of lipoproteins in blood plasma of the WHHL rabbit. An animal model of human familial hypercholesterolemia.

Lipoproteins in blood plasma have been quantified and characterized in homozygous Watanabe-heritable hyperlipidemic (WHHL) rabbits, an animal model of human familial hypercholesterolemia. Like homozygous human hypercholesterolemics, WHHL rabbits have a severe deficiency of low density lipoprotein (LDL) receptors, a prolonged residence time for LDL, and an increased absolute rate of LDL catabolism. Although lipoproteins containing apolipoprotein B in WHHL rabbits are enriched in cholesteryl esters, their LDL as well as intermediate density lipoproteins (IDL) and very low density lipoproteins (VLDL) also contain a substantial amount of triglycerides and they consistently exhibit hypertrigiyceridemia as well as hypercholesterolemia. The cholesteryl esters accumulating in lipoproteins of WHHL rabbits are rich in cholesteryl linoleate and appear to be produced almost exclusively by lecithin-cholesterol acyltransferase. Levels of apolipoprotein B-100 are elevated in VLDL and IDL as well as in LDL of WHHL rabbits and only trace amounts of apolipoprotein B-48 are present. Plasma levels of apolipoprotein E are also substantially increased, and VLDL and IDL are enriched in this protein. The accumulation of lipoproteins with the expected characteristics of remnants of hepatogenous triglyceride-rich lipoproteins contrasts with the efficient hepatic clearance of chylomicron remnants in WHHL rabbits.

Isoelectric heterogeneity of the cofactor protein for lipoprotein lipase in human blood plasma

Abstract Several of the protein componets of lipoproteins in mammalian blood plasma exhibit charge polymorphism (1–7). In some cases, polymorphism in gel electrophoretograms is clearly evident only upon isoelectric focusing (2,3,6,7). In the case of the human apolipoprotein R-ala (apo C-III), variable sialylation accounts for the polymorphism (4,5), but in other cases the basis for polymorphism is unknown. Deficiency of one of the polymorphic components of the arginine-rich apolipoprotein (apo E) is associated with one genetically determined human hyperlipoproteinemia, familial dysbetalipoproteinemia (6,7). We have isolated a new component of the “C” group of apoproteins of human VLDL and have found it to be an isoelectric variant of the cofactor protein for lipoprotein lipase (R-glu; apo C-II).

Simultaneous quantification of apolipoproteins B-100, B-48, and E separated by SDS-PAGE.

Publisher Summary The two species of apolipoprotein (apo) B in humans, apoB-100, and apoB-48, define the hepatogenous and intestinal contributions to the secretion of triglyceride-rich lipoproteins (TRL) into the blood. A number of methods have been proposed to quantify apoB-100 and apoB-48. Several procedures based on separation by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) have given inconsistent results. With electrophoresis in 3.3% polyacrylamide tube gels, followed by Coomassie blue staining, Poapst et al. reported a lower chromogenicity for apoB-48 than for apoB-100, as well as a nonlinear response, between the intensity of dye uptake and apolipoprotein mass. This method has been used to quantify apolipoproteins in human triglyceride-rich lipoproteins (TRL) and has yielded apoB-100 and apoB-48 levels that are considerably higher than those reported, by others, using electrophoresis in slab gels. Zilversmit and Shea, using slab gels, found equal chromogenicities for rat apoB-48 and rat and human apoB-100 and a linear relationship between dye uptake and apolipoprotein mass. These observations are consistent with the findings here and those of others who have carried out the electrophoresis of apoB and other proteins in slab gels and they support the conclusion that apoB data obtained by tube gel electrophoresis, in which only a rim of dye penetrates into the gel, should be viewed with caution. A major advantage to the SDS-polyacrylamide slab gel electrophoretic method that has been developed is the ability to quantify apoB-100 and apoB-48 together with apoE in various lipoprotein fractions in humans and other species.

Human apolipoprotein C-I: Concentration in blood serum and lipoproteins

Abstract A radial immunodiffusion assay for apolipoprotein C-I in human serum and lipoproteins was developed and validated by comparison with analysis of these proteins in very-low-density and high-density lipoproteins by quantitative polyacrylamide gel electrophoresis. In normotriglyceridemic subjects, only about 10% of apoprotein C-I was in very-low-density lipoproteins; almost all of the remainder was in high-density lipoproteins. In subjects with widely varying serum triglyceride levels, apoprotein C-I comprised about 4% of the total apoprotein of the triglyceride-rich lipoproteins. In normotriglyceridemic subjects, apoprotein C-I comprised about 3% of the total apoprotein of high-density lipoproteins; this value was about twice as great in the high-density lipoproteins of density 1.063–1.125 g/ml as in those of density 1.125–1.21 g/ml. With increasing serum triglyceride level, the percentage of apoprotein C-I in triglyceride-rich lipoproteins increased and that in high-density lipoproteins fell progressively. Whereas apoprotein C-I comprised about 9% of the mass of the proteins of low molecular weight (

Human apoC-IV: isolation, characterization, and immunochemical quantification in plasma and plasma lipoproteins

Apolipoprotein C-IV (apoC-IV), the newest member of the low-molecular-weight apoC group, has been characterized in blood plasma of rabbits, in which it is a major proline-rich apoC component (Zhang, L-H., L. Kotite, and R. J. Havel. 1996. Identification, characterization, cloning, and expression of apoC-IV, a novel sialoglycoprotein of rabbit plasma lipoproteins. J. Biol. Chem. 271: 1776–1783). Although the decoded sequence of mouse and human apoC-IV is known, apoC-IV has not been identified in blood plasma from these or other species. Rabbit apoC-IV exists in several sialoforms, and the asialoform has an acidic isoelectric point. We show that apoC-IV is a basic protein in human, monkey, and mouse plasma, present as a minor apoC component of VLDL. Human apoC-IV, isolated from apo VLDL by DEAE-cellulose chromatography and two-dimensional electrophoresis, was identified by microsequencing four tryptic peptides. The protein exhibits two major isoforms; one is N-glycosylated, and both are variably sialylated. In normolipidemic plasma, greater than 80% of the protein is in VLDL (0.7% of total apo VLDL), with most of the remainder in HDL. The concentration of apoC-IV in the plasma and lipoproteins of ρ < 1.21 g/ml is closely related to plasma triglyceride concentration up to 1,770 mg/dl, varying from 0.1–1.9 mg/dl. Neither the human nor rabbit apoC-IV gene contains a typical TATA box in the 5′-flanking region, but the 5′-untranslated region of the rabbit gene contains a unique purine-rich sequence, GGGACAG(G/A), repeated nine times in tandem, with an additional two within the 5′-flanking sequence. This sequence, functioning as a GAGA box that has been implicated in the transcription of a number of genes, may explain the higher level of expression of apoC-IV in rabbits.

IDENTIFICATION, CHARACTERIZATION, CLONING, AND EXPRESSION OF APOLIPOPROTEIN C-IV, A NOVEL SIALOGLYCOPROTEIN OF RABBIT PLASMA LIPOPROTEINS

We have identified and characterized a novel proline- and arginine-rich protein component of lipoproteins, present in up to five sialylated isoforms, in rabbit blood plasma. The pI of the desialylated protein is 5.7. Based upon its N-terminal sequence, a complete cDNA sequence of 555 nucleotides was cloned from rabbit liver. The synthesized protein is predicted to contain 124 amino acids, including a typical signal peptide of 27 residues. The mature protein of 97 amino acids, designated apolipoprotein C-IV, is associated with the lipoproteins of blood plasma, primarily very low density and high density lipoproteins. It contains two potential amphipathic helices characteristic of plasma apolipoproteins and forms discoidal micelles with phosphatidylcholine. Northern analysis shows a single 0.6-kilobase apolipoprotein C-IV mRNA, detected only in the liver, and Southern analysis suggests a single copy gene. Sialylated apolipoprotein C-IV is secreted from transfected mammalian cells. Nucleotide sequence comparisons demonstrate a strong homology to portions of the upstream regions of the mouse and human apolipoprotein C2 genes, within each of which a distinct gene has recently been identified. The nucleotide sequences and the predicted amino acid sequences, as well as corresponding cDNA sequences in the rat and monkey, indicate that the apolipoprotein C4 gene has been highly conserved during mammalian evolution.