Angelo M. Scanu

Solubility in aqueous solutions of ethanol of the small molecular weight peptides of the serum very low density and high density lipoproteins: Relevance to the recovery problem during delipidation of serum lipoproteins

Abstract Extraction of human serum very low density (VLDL) and high density lipoproteins (HDL) by ethanol/ethyl ether at low temperature is associated with only partial recovery of delipidated apoproteins, apo VLDL and apo HDL. In an attempt to explain this phenomenon, many of the parameters involved in delipidation were investigated systematically. In the case of apo VLDL, it was found that 3:1 ethanol/ether mixtures commonly adopted to extract VLDL may solubilize up to 20% by weight of the VLDL protein, involving specifically the fraction containing the low molecular weight peptides. Such a fraction was increasingly soluble in aqueous solutions of ethanol (ethanol/H2O, 95:5 to 50:50 v/v), less soluble in ethanol/ethyl ether mixtures, and totally insoluble in ethyl ether, chloroform, methanol, or mixtures thereof. Extraction of HDL2 (d 1.063 – 1.125 gm/ml) or HDL3 (d 1.125 – 1.21 gm/ml) by 3:2 ethanol/ethyl ether was associated with the solubilization of a small portion of apo HDL2 (about 1% by weight of the original HDL2 protein) into the organic phase. Such a soluble fraction was made up of the low molecular weight peptides which, as shown in a previous study, are separable from the other apo HDL components by gel filtration (fraction V). The solubility pattern of such a fraction in an organic solvent was the same as that of the low molecular weight peptides from apo VLDL. From the quantitative standpoint, however, V was much less soluble in ethanol than the products from apo VLDL. The current studies provided a better insight into the chemical basis for the chemical basis for the delipidation of VLDL and HDL and also allowed for the design of procedures insuring total recoveries of delipidated apo VLDL and apo HDL (apo HDL2 and apo HDL3).

Lipoprotein(a) and atherosclerosis.

Lipoprotein(a) [Lp(a)], a lipoprotein variant, was relegated for almost 25 years to the study of a few specialists. During the past 3 to 4 years, however, there has been a tremendous upsurge of interest in Lp(a), primarily because of multidisciplinary efforts in structural and molecular biology. Findings emerging from these efforts include the following: Lp(a) represents a cholesteryl-ester, low-density-lipoprotein (LDL)-like particle with apolipoprotein (apo) B-100 linked to apo(a); apo(a) is a glycoprotein coded by a single gene locus on the long arm of chromosome 6, which has several alleles, accounting for its remarkable size polymorphism (300 to 800 kD); apo(a) size polymorphism relates to plasma levels and density distribution of Lp(a); apo(a) is strikingly similar to plasminogen; and in vitro, Lp(a), in appropriate levels, competes for some physiologic functions of plasminogen in the coagulation and fibrinolytic cascade and may thus be thrombogenic. The LDL-like properties of Lp(a) may also confer atherogenic potential, but the mechanisms underlying this atherogenicity remain to be defined. In epidemiologic studies, high plasma Lp(a) levels have been associated with an increased incidence of atherosclerotic cardiovascular disease, especially in patients less than 60 years of age. Moreover, Lp(a) has been found as an intact particle in the arterial intima, particularly in association with atherosclerotic plaque. This finding suggests that Lp(a) can transverse the endothelium, possibly by a non-receptor-mediated process, and, at the intimal level, acquire thrombogenic and atherogenic potentials. Current information justifies the need to determine plasma Lp(a) levels in patients with a history of atherosclerotic cardiovascular disease. Unfortunately, the available techniques need to be standardized. Apolipoprotein(a) exists in isoforms of different sizes, and the importance of determining apo(a) phenotypes in clinical practice remains to be established.

HDL: bridging past and present with a look at the future

Clinical and epidemiological studies have shown that HDLs, a class of plasma lipoproteins, heterogeneous in size and density, have an atheroprotective role attributed, for years, to their capacity to promote the efflux of cholesterol from activated cholesterol‐loaded arterial macrophages. Recent studies, however, have recognized that the physical heterogeneity of HDLs is associated with multiple functions that involve both the protein and the lipid components of these particles. ApoA‐I, quantitatively the major protein constituent, has an amphipathic structure suited for transport of lipids. It readily interacts with the ATP‐binding cassette transporter ABCA1, the SR‐B1 scavenger re‐ceptor;activates the enzyme lecithin‐cholesterol acyl transferase (LCAT), which is critical for HDL maturation. It also has antioxidant and antiinflammatory properties, along with the HDL‐associated enzymes paraoxonase, platelet activating factor acetylhydrolase (PAF), and glutathione peroxidase. Regarding the lipid moiety, an atheroprotective role has been recognized for lysosphingolipids, particularly sphingosine‐1‐phosphate (S1P). All of these atheroprotective functions are lost in the post‐translational dependent dysfunctional plasma HDLs of subjects with systemic inflammation, coronary heart disease, diabetes, and chronic renal disease. The emerging notion that particle quality has more predictive power than quantity has stimulated further exploration of the HDL proteome, already revealing unsuspected pro‐ or antiatherogenic proteins/peptides associated with HDL.— Scanu, A. M., Edelstein, C. HDL: bridging past and present with a look at the future. FASEB J. 22, 4044–4054 (2008)

[5] Precautionary measures for collecting blood destined for lipoprotein isolation

Publisher Summary Although the problem of artifacts originating after blood collection has been recognized, there is no general awareness of this problem among all of the workers in the field. In view of this, the chapter describes various precautionary measures for collecting blood destined for lipoprotein isolation. Studies have shown that proteolytic enzymes of different types can affect the cleavage of proapoA-I, apoA-II, apoB, and apoE; for example, the enzyme responsible for the cleavage of proapoA-I to apoA-I is present in circulation and is inhibited by ethylenediaminetetraacetic acid (EDTA). The chapter proposes to develop a cocktail that is added to the bottle before blood collection to prevent the multiple enzymatic degradations that can occur during and after the withdrawal of blood. However, proper mixing of the cocktail with the collected blood is essential, because it will ensure that all constituents have come in contact and that preventive measures have begun. The chapter concludes that the lipoprotein distribution varies from individual to individual and is a characteristic of each normolipemic subject independent of time.

Lysine-Phosphatidylcholine Adducts in Kringle V Impart Unique Immunological and Potential Pro-inflammatory Properties to Human Apolipoprotein(a)

Lipoprotein(a), Lp(a), an athero-thrombotic risk factor, reacts with EO6, a natural monoclonal autoantibody that recognizes the phophorylcholine (PC) group of oxidized phosphatidylcholine (oxPtdPC) either as a lipid or linked by a Schiff base to lysine residues of peptides/proteins. Here we show that EO6 reacts with free apolipoprotein(a) apo(a), its C-terminal domain, F2 (but not the N-terminal F1), kringle V-containing fragments obtained by the enzymatic digestion of apo(a) and also kringle V-containing apo(a) recombinants. The evidence that kringle V is critical for EO6 reactivity is supported by the finding that apo(a) of rhesus monkeys lacking kringle V did not react with EO6. Based on the previously established EO6 specificity requirements, we hypothesized that all or some of the six lysines in human kringle V are involved in Schiff base linkage with oxPtdPC. To test this hypothesis, we made use of a recombinant lysine-containing apo(a) fragment, rIII, containing kringle V but not the protease domain. EO6 reacted with rIII before and after reduction to stabilize the Schiff base and also after extensive ethanol/ether extraction that yielded no lipids. On the other hand, delipidation of the saponified product yielded an average of two mol of phospholipids/mol of protein consistent with direct analysis of inorganic phosphorous on the non-saponified rIII. Moreover, only two of the six theoretical free lysine amino groups per mol of rIII were unavailable to chemical modification by 2,4,6-trinitrobenzene sulfonic acid. Finally, rIII, like human apo(a), stimulated the production of interleukin 8 in THP-1 macrophages in culture. Together, our studies provide evidence that in human apo(a), kringle V is the site that reacts with EO6 via lysine-oxPtdPC adducts that may also be involved in the previously reported pro-inflammatory effect of apo(a) in cultured human macrophages.

A proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries

OBJECTIVES By using proteomics we isolated and identified proteins that were expressed/retained in stable and unstable human carotid artery atherosclerotic plaques. METHODS The criteria for plaque instability were the presence of a thin fibrous cap or fissured cap covering the foamy or necrotic core, and the presence of overt, hemorrhagic, ulcerated or thrombotic plaques. Proteins were extracted from finely minced endarterectomy specimens (19 stable and 29 unstable plaques) and separated by two-dimensional gel electrophoresis. Coomassie Blue-stained gels were analysed using PD-Quest software. RESULTS A total of 57 distinct spots corresponding to 33 different proteins were identified by matrix assisted laser desorption/ionization mass spectrometry using the NCBI database. Most of the spots were present in both types of extracts, although significantly (p<0.05) differing in abundance between them. Compared to stable plaque, unstable ones showed reduced abundance of: protective enzymes SOD3 and GST, small heat shock proteins HSP27 and HSP20, annexin A10, and Rho GDI. In unstable plaques the more abundant proteins were: ferritin light subunit, SOD 2 and fibrinogen fragment D. For fibrinogen fragment D, the increased levels in unstable versus stable plaques was confirmed by Western blot analysis. CONCLUSIONS Since many of the differentially expressed proteins are known to have a functional role in inflammation and oxidative stress, we speculate that they may be involved in events relating to plaque stability.

Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells.

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Uptake of endogenous cholesterol by a synthetic lipoprotein

The addition of cholesterol-poor phospholipid liposomes to canine plasma in vivo and in vitro substantially alters the distribution of phospholipids, apoproteins, and, especially, cholesterol. In vivo, intravenously injected phospholipid liposomes remain discrete particles, which are readily distinguished from the normally occurring lipoproteins by their buoyant density and electrophoretic mobility. They acquire unesterified cholesterol from endogenous sources, thereby producing an acute rise in the concentration of this sterol in plasma. The liposomes also accumulate endogenous proteins, one of which is identified as apolipoprotein A-I. In vitro, phospholipid liposomes incubated with plasma acquire unesterified cholesterol and apolipoprotein A-I at the expense of high-density lipoproteins (HDL), the major carrier of cholesterol in normal canine plasma. In exchange, the HDL particles are enriched in phospholipids and become larger. At sufficiently high concentrations, the liposomes nearly completely deplete HDL of its unesterified cholesterol. Thus, there are generated two types of particles, both rich in apolipoprotein A-I and phospholipid, but one (modified HDL) containing mainly esterified cholesterol in its core and the other (modified liposomes) containing mainly unesterified cholesterol at its surface. It is concluded that phospholipid liposomes produce important changes in the distribution of lipids and protein in canine plasma, particularly at the expense of HDL. These changes appear to favor the mobilization of tissue cholesterol into the plasma, and may have application to atherosclerosis.

Isoelectric fractionation and characterization of polypeptides from human serum very low density lipoproteins.

Abstract Tris-soluble polypeptides obtained from totally delipidated human serum very low density lipoprotein were isolated and characterized. The very low density apolipoproteins were separated by a newly developed method of isoelectric focusing in a narrow pH gradient. Four polypeptides were isolated that differed from the major proteins of the high density or low density lipoproteins. Three of these proteins had indistinguishable amino acid compositions, but different isoelectric points, COOH-terminal alanine, no isoleucine, cysteine or cystine. Two of these three polypeptides had NH 2 -terminal serine. The polymorphism of apolipoprotein-Ala, so designated from the COOH-terminal residue, was related to sialic acid content; one form contained 2 moles of sialic acid per mole of protein, the second, 1 mole per mole of protein, and the third, no sialic acid. The fourth polypeptide had an amino acid composition different from the first three polypeptides and from other polypeptides obtained from very low density lipoprotein. This polypeptide had NH 2 -terminal threonine, COOH-terminal resistant to carboxypeptidase A, no histidine, cysteine, cystine or sialic acid. These four polypeptides constituted approx. 40% of the total protein in very low density lipoprotein.

FURTHER CHARACTERIZATION OF THE HUMAN SERUM D 1.063-1.21, α1-LIPOPROTEIN

The high density lipoprotein class floating at a solvent density between 1.063 and 1.21 g per ml can be fractionated by flotation in solvents of intermediate density into two major subfractions (1, 2), which according to Shore (3) have a protein moiety with identical amino acid composition and C- and N-terminal amino acids. Homogeneity of the D 1.063-1.21 lipoprotein in terms of protein moiety has also been indicated by immunochemical studies (4). It would appear, therefore, that the human serum high density lipoprotein of D 1.0631.21 constitutes a group of molecules identical as to protein moiety and possibly differing only in lipid complement. The experiments reported below were designed to test this hypothesis. The serum D 1.063-1.21 lipoprotein was arbitrarily fractionated into three fractions floating respectively at solvent densities ofD 1.0631.125, 1.125-1.168, and 1.168-1.21. The results dealing with some of the physicochemical and biological properties of these lipoprotein subfractions form the object of this report.