Celina Edelstein

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).

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.

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.

An improved detection system applied to the study of serum lipoproteins after single-step density gradient ultracentrifugation.

Abstract Starting from a single-spin ultracentrifugation procedure described previously (Foreman et al., J. Lipid Res. 18 , 759, 1977), we have improved the system for detection of the fractions eluted from the gradient by monitoring them continuously at 280 nm. A graphic display readily permits assessment of the distribution of the lipoproteins and their quantification with the aid of a computer program. By the use of appropriate factors, one can convert absorbance readings into actual lipoprotein values which correspond well (±7% for low-density lipoproteins and ±5% for high-density lipoproteins) to those obtained by means of chemical analyses. The examples provided indicate the versatility of the method and its sensitivity (down to 0.1 ml of serum).

Polymorphonuclear Cells Isolated from Human Peripheral Blood Cleave Lipoprotein(a) and Apolipoprotein(a) at Multiple Interkringle Sites via the Enzyme Elastase GENERATION OF mini-Lp(a) PARTICLES AND apo(a) FRAGMENTS

Incubation of polymorphonuclear cells (PMN), isolated from human peripheral blood, with either lipoprotein(a) (Lp(a)) or free apolipoprotein(a) (apo(a)), derived from the parent Lp(a), caused in both cases a multisite fragmentation of apo(a) inhibited by methoxysuccinyl-Ala-Ala-Pro-Val-CH2Cl, a specific elastase inhibitor. The major cut site was at the interkringle region between apo(a) kringles IV-4 and IV-5 (Ile3520-Leu3521). The other cleavages were between kringles IV-7 and IV-8 (Thr3846-Leu3847) and between kringles IV-10 and V (Ile4196-Gln4197). The elastase-induced fragmentation of apo(a) was the same whether free or as a member of Lp(a), indicating that the disulfide bond between apo(a) and the apoB100 component of Lp(a) did not hinder the elastase action. Lp(a) fragments containing kringle IV-9 retained the linkage to apoB100 via the disulfide bond, forming mini-Lp(a) particles in which the size of apo(a) varied according to the size of the fragments produced by the elastase digestion. The proteolytic fragmentation was unaffected by apo(a) size polymorphism within the range examined. PMN elastase also caused a partial proteolysis of apoB100 whether as a component of Lp(a), Lp(a) freed of apo(a), or authentic low density lipoprotein without an apparent destabilization of these lipoprotein particles. Proteolysis of Lp(a) by PMN was due to an elastase activity that was 3.5% of that observed when PMN were activated by N-formyl-Met-Leu-Phe. A portion of the released elastase was found to be associated in an active form with both Lp(a) and low density lipoprotein even in an ultracentrifugal field at high salt concentrations. Taken together, our results indicate that apo(a) undergoes important proteolytic modifications by PMN elastase, which exhibits specificity for peptide bonds located in the interkringle domains of apo(a). In the case of Lp(a), elastase cleavage causes the formation of mini-Lp(a) particles with a protein moiety containing a truncated apo(a). Elastase-mediated proteolytic events may occur in vivo under conditions associated with either an excessive leakage of elastase from PMN and/or deficiencies of natural inhibitors of this enzyme.

Molecular weight and subunit structure of human serum high density lipoprotein after chemical modification by succinic anhydride

Abstract The two human serum subclasses of high density lipoproteins, HDL2 (ρ1.063–1.125) and HDL3 (ρ 1.125–1.21) and their corresponding delipidated products, apo HDL2 and apo HDL3, were analyzed in dilute solutions by the technique of sedimentation equilibrium before and after treatment with succinic anhydride. Succinylation did not significantly affect the molecular weight of either HDL2 (3.86·105 ± 2.4·104) or HDL3 (1.86·105 ± 1.1·104). Delipidated and succinylated apo HDL2 and apo HDL3 behaved as a monodisperse system of a mol. wt. of about 27 500. The value was the same whether succinylation was carried out before or after delipidation or after reduction and alkylation. The figure for the molecular weight of the subunits was supported by chemical and enzymatic N- and C-terminal amino acid analysis and by the number of arginine-positive peptides in maps of tryptic digests of either apo HDL2 or apo HDL3. The results were taken to indicate that: (1) a large percentage of the free amino groups of lysine is readily accessible to chemical modification and is probably located at the surface of the molecule; (2) apo HDL2 and apo HDL3 are made of subunits of identical or very similar molecular weight; (3) the subunits are made of single chains having either threonine or glutamine as C-terminals and aspartic acid as N-terminal. Based on circular dichroism spectra and viscosity measurements, succinylation was found to affect the properties of either apo HDL2 or apo HDL3 only after removal of lipids, suggesting a role of the latter in the conformational stability of the various peptide chains.

Evidence for a Proinflammatory and Proteolytic Environment in Plaques From Endarterectomy Segments of Human Carotid Arteries

ObjectivesBased on previous observations on apolipoprotein(a), apo(a), in human unstable carotid plaques, we explored whether in the inflammatory environment of human atheroma, proteolytic events affect other hepatic and topically generated proteins in relation to the issue of plaque stability. Methods and ResultsForty unstable and 24 stable plaques from endarterectomy segments of affected human carotid arteries were extracted with buffered saline (PBS) and then 6 mol/L guanidine-hydrochloride (GdHCl) to identify loosely and tightly bound products, respectively. The extracts were studied before and after ultracentrifugation at d 1.21 g/mL. In the extracts, the concentrations of interleukin (IL)-6, −8, and −18 were significantly higher in the unstable plaques and correlated to those of MMP-2 and MMP-9. By Western blots, both apoB and apo(a) were highly fragmented and mostly present in the d 1.21 bottom that also contained fragments of apoE (10 and 22 kDa), decorin, biglycan, and versican. Fragmentation was higher in the unstable plaques. In baseline plasmas, concentrations of lipids, lipoproteins, and ILs did not differ between patients with unstable and stable plaques. ConclusionsIn unstable and to a lesser extent in stable plaques, there is a proinflammatory and proteolytic microenvironment with the generation of fragments with potential pathobiological significance that requires investigation.

Apolipoprotein(a) binds via its C-terminal domain to the protein core of the proteoglycan decorin. Implications for the retention of lipoprotein(a) in atherosclerotic lesions.

Although it is known that lipoprotein(a) (Lp(a)) binds to proteoglycans, the mechanism for this binding has not been fully elucidated. In order to shed light on this subject, we examined the interactions of decorin, a proteoglycan with a well defined protein core and a single glycosaminoglycan (GAG) chain, with Lp(a) and derivatives, namely Lp(a) deprived of apo(a), or Lp(a−), free apo(a), and the two main proteolytic fragments, F1 and F2. By circular dichroism criteria, the decorin preparations used had the same secondary structure as that previously reported for native decorin. Authentic low density lipoprotein from the same human donor was used as a control. In a solid phase system, Lp(a−)and low density lipoprotein bound to decorin in a comparable manner. This binding required Ca2+/Mg2+ ions, was lysine-mediated, and was markedly decreased in the presence of GAG-depleted decorin, suggesting the ionic nature of the interaction likely involving apoB100 and the GAG component of decorin. Free apo(a) also bound to decorin; however, the binding was neither cation-dependent nor lysine-mediated, unaffected by sialic acid depletion of apo(a), and markedly decreased when either reduced and alkylated apo(a) or reduced and alkylated decorin was used in the assay. Of note, the binding of apo(a) was unaffected when it was incubated with a spectrally native decorin that had been renatured from either 4 m guanidine hydrochloride by extensive dialysis or cooled from 65 to 25 °C. On the other hand, the binding significantly increased when decorin was depleted of GAGs, which by themselves had no affinity for apo(a). The binding of apo(a) to the decorin protein core was also elicited by the C-terminal domain of apo(a), and it was favored by high NaCl concentrations, 1 to 2m. No binding was exhibited by the N-terminal domain accounting for the lack of effect of apo(a) size polymorphism on the binding. In the case of whole Lp(a), the binding to immobilized decorin was mostly GAG-dependent and ionic in nature. A minor contribution by apo(a) was detected when GAG-depleted decorin was used in the assay. Our results indicate that the binding of Lp(a) to decorin involves interactions both electrostatic (apoB100-GAG) and hydrophobic (apo(a)-decorin protein core), and that the binding of apo(a) requires decorin protein core to be in its native state.

Oleate stimulates the formation of triglyceride-rich particles containing APOB100-APO(a) in long-term primary cultures of human hepatocytes

Hepatocytes prepared from normal human liver donors were maintained in a synthetic, nutritionally defined, serum-free medium for up to 60 days with continuous secretion of the major plasma lipoproteins. By Western blot analysis, both cell lysates and culture media contained apo(a), both unbound and bound to apo B100, in varying proportions, dependent on the liver donor. In the medium of cells pulse-labeled for 16 h with [35S]methionine the apo(a)-apoB100 complex was predominantly found in the d < 1.006 g/ml triglyceride-rich particles (TRP). Incubation of the cells with a bovine serum albumin-potassium oleate complex in a molar ratio of 1:4 caused a twofold to threefold increase of the TRP-containing apoB100-apo(a). In the 48 h unlabeled conditioned medium apoB100-apo(a) was distributed among the d < 1.006, d < 1.063 and d < 1.21 g/ml lipoproteins, with a small amount in the d > 1.21 g/ml sedimenting fraction, suggesting that the newly secreted apoB100-apo(a)-containing TRP had undergone remodeling. The results indicate that primary human hepatocyte cultures produce both apo(a) and apoB100-apo(a) and that the latter affiliates preferentially with TRP, forming a lipoprotein complex that is affected by endogenous triglyceride synthesis, and can be metabolically modified in the culture medium of human primary hepatocytes.