Isolation and Analysis of Plasma Lipoproteins by Ultracentrifugation.

Analysis of plasma lipoproteins and apolipoproteins is an essential part for the diagnosis of dyslipidemia and studies of lipid metabolism and atherosclerosis. Although there are several methods for analyzing plasma lipoproteins, ultracentrifugation is still one of the most popular and reliable methods. Because of its intact separation procedure, the lipoprotein fractions isolated by this method can be used for analysis of lipoproteins, apolipoproteins, proteomes, and functional study of lipoproteins with cultured cells in vitro. Here, we provide a detailed protocol to isolate seven lipoprotein fractions including VLDL (d<1.006 g/mL), IDL (d=1.02 g/mL), LDLs (d=1.04 and 1.06 g/mL), HDLs (d=1.08, 1.10, and 1.21 g/mL) from rabbit plasma using sequential floating ultracentrifugation. In addition, we introduce the readers how to analyze apolipoproteins such as apoA-I, apoB, and apoE by SDS-PAGE and Western blotting and show representative results of lipoprotein and apolipoprotein profiles using hyperlipidemic rabbit models. This method can become a standard protocol for both clinicians and basic scientists to analyze lipoprotein functions.

Proteomic Toolbox To Standardize the Separation of Extracellular Vesicles and Lipoprotein Particles.

Circulating in blood, extracellular vesicles (EVs) and lipoprotein particles (LPs) have diagnostic and prognostic value. To unambiguously define their functions, separation protocols need to be developed. However, because of their similar size and density, traditional approaches to separate EVs and LPs often fail to provide the required resolution. Further development and standardization of affinity-based protocols is necessary, and a quantitative method is needed to assess the efficiency of LP depletion from EV samples. In the present study, we propose the simultaneous quantification of three groups of proteins by mass spectrometry as a toolbox to evaluate prospective separation protocols. We generated 15N-labeled internal standards for quantification of (i) EV-specific proteins, (ii) all classes and subclasses of apolipoproteins constituting LPs, and (iii) several major serum proteins. These standards were then used in multiple reaction monitoring assay to evaluate the performance of size-exclusion chromatography, heparin-Sepharose, lipopolysaccharide-Sepharose, (2-hydroxypropyl)-ß-cyclodextrin-Sepharose, and concanavalin A-Sepharose in separating serum EVs and LPs. The efficiency of a resin to separate EVs from non-EV substances could be jeopardized by simultaneous EV aggregation. Therefore, dynamic light scattering analysis was used in this study in addition to the proteomic toolbox when making a recommendation to use particular resin for EV isolation. On the basis of our measurements, we concluded that none of the individual separation protocols used in this study resulted in LP-free EVs, and the combination of two protocols may be complex due to low EV yield. Overall, this further points to the importance of proposed proteomic toolbox for the future evaluation of EV separation protocols.

Serum Amyloid P Component (SAP) Interactome in Human Plasma Containing Physiological Calcium Levels.

The pentraxin serum amyloid P component (SAP) is secreted by the liver and found in plasma at a concentration of approximately 30 mg/L. SAP is a 25 kDa homopentamer known to bind both protein and nonprotein ligands, all in a calcium-dependent manner. The function of SAP is unclear but likely involves the humoral innate immune system spanning the complement system, inflammation, and coagulation. Also, SAP is known to bind to the generic structure of amyloid deposits and possibly to protect them against proteolysis. In this study, we have characterized the SAP interactome in human plasma containing the physiological Ca2+ concentration using SAP affinity pull-down and co-immunoprecipitation experiments followed by mass spectrometry analyses. The analyses resulted in the identification of 33 proteins, of which 24 were direct or indirect interaction partners not previously reported. The SAP interactome can be divided into categories that include apolipoproteins, the complement system, coagulation, and proteolytic regulation.

A Primate APOL1 Variant That Kills Trypanosoma brucei gambiense.

Humans are protected against infection from most African trypanosomes by lipoprotein complexes present in serum that contain the trypanolytic pore-forming protein, Apolipoprotein L1 (APOL1). The human-infective trypanosomes, Trypanosoma brucei rhodesiense in East Africa and T. b. gambiense in West Africa have separately evolved mechanisms that allow them to resist APOL1-mediated lysis and cause human African trypanosomiasis, or sleeping sickness, in man. Recently, APOL1 variants were identified from a subset of Old World monkeys, that are able to lyse East African T. b. rhodesiense, by virtue of C-terminal polymorphisms in the APOL1 protein that hinder that parasite's resistance mechanism. Such variants have been proposed as candidates for developing therapeutic alternatives to the unsatisfactory anti-trypanosomal drugs currently in use. Here we demonstrate the in vitro lytic ability of serum and purified recombinant protein of an APOL1 ortholog from the West African Guinea baboon (Papio papio), which is able to lyse examples of all sub-species of T. brucei including T. b. gambiense group 1 parasites, the most common agent of human African trypanosomiasis. The identification of a variant of APOL1 with trypanolytic ability for both human-infective T. brucei sub-species could be a candidate for universal APOL1-based therapeutic strategies, targeted against all pathogenic African trypanosomes.

The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III.

Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein-lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. (31)P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.

A Simple Method to Extract Whole Apolipoproteins for the Preparation of Discoidal Recombined High Density Lipoproteins as Bionic Nanocarriers for Drug Delivery.

PURPOSE: To develop a simple method to extract the whole apolipoproteins (apo) including apoA-I in native high density lipoproteins (HDLs) and prepare discoidal Tanshinone IIA-loaded reconstituted HDL (TA-rHDLs) as a dual functional drug delivery system with plaque-site target and therapeutic promises in atherosclerotic lesions. METHODS: A method based on isoelectric precipitation coupled with organic solvent precipitation was developed to isolate the whole apolipoproteins (apos). TA-rHDLs were prepared by incubating the resultant apos with liposomes and the incubation conditions were optimized using fluorescence quenching experiment. TA-rHDLs were characterized in terms of size, zeta potential, morphology, interaction between lipid and apos, safety, and bionic function. RESULTS: The extraction results showed that the yield of the HDL apos was 82.4%, with 59% being apoA-I type, similar ratio of apoA-I in the native apos. TA-rHDL prepared were disc-like with an average diameter of 157.6 ± 4.8 nm, zeta potential of -20.90 ± 0.15 mV, and entrapment efficiency of (90.13 ± 1.4) %. The interaction between the lipids and apos was electrostatic and hydrophobic force and was associated with amino acid sequence. Haemolysis and cytotoxicity assays showed good biocompatibility of TA-rHDL. Sterol efflux assay from macrophages mediated by TA-rHDLs and structure remodeling behavior from discs to spheres proved that TA-rHDL could resemble the biological activity of native nascent HDL irrespective of the size. CONCLUSIONS: The simple approach to isolate apos may provide a convenient and economical resource to support the development of rHDL as a potential targeting nanocarrier for lipophilic cardiovascular drugs.

Changes in the hemolymph protein profiles in Galleria mellonella infected with Bacillus thuringiensis involve apolipophorin III. The effect of heat shock.

This report concerns the effect of heat shock on host-pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis. We show enhanced activity against Gram-positive bacteria in the hemolymph of larvae pre-exposed to heat shock before infection with B. thuringiensis. Heat shock influenced the protein pattern in the hemolymph of infected larvae: more peptides with a molecular weight below 10 kDa were detected in comparison with nonshocked animals. Additionally, we noticed that the amount of apolipophorin III (apoLp-III) in the hemolymph decreased transiently following infection, which was considerably higher in larvae pre-exposed to heat shock. On the other hand, its expression in the fat body showed a consequent infection-induced decline, observed equally in shocked and nonshocked animals. This suggests that the amount of apoLp-III in the hemolymph of G. mellonella larvae is regulated at multiple levels. We also report that this protein is more resistant to degradation in the hemolymph of larvae pre-exposed to heat shock in comparison to nonshocked larvae. Two-dimensional analysis revealed the presence of three isoforms of apoLp-III, all susceptible to proteolytic degradation. However, one of them was the most abundant, both in the protease-treated and untreated hemolymph. Taking into consideration that, in general, apoLp-III has a stimulative effect on different immune-related hemolymph proteins and peptides, the reported findings bring us closer to understanding the effect of heat shock on the resistance of G. mellonella to infection.

Western blots.

Western analysis of apolipoproteins, lipoproteins, and proteins involved in lipoprotein metabolism can be challenging due to their size, hydrophobic nature, and, in some cases, low abundance. Here we describe a Western blotting method that has been used successfully for many proteins involved in lipoprotein metabolism, as well as intact LDL or HDL particles. Proteins or lipoprotein particles separated by gel electrophoresis are transferred to a PVDF membrane in a Hoefer TE22 transfer tank with Tris-Glycine-SDS-Methanol transfer buffer. The membrane is blocked with 3 % BSA/5 % milk to prevent nonspecific binding of antibody to the membrane and is then incubated with primary antibody that binds specifically to the protein of interest. After washing away unbound primary antibody, the membrane is then incubated with an HRP-labeled secondary antibody that binds primary antibody. After washing away unbound secondary antibody, the membrane is then incubated with a substrate for HRP, generating a chemiluminescent signal at the location of the protein of interest. The protein is visualized by exposing the membrane to an autoradiography film or an imaging device. Information on the use of several human antibodies, including apoA-I, A-II, apoB, apoC-II, apoC-III, apoD, apoL1, apoM, PON1, SAA, ABCA1, nitrotyrosine, and LCAT, is provided. This method can be used for Western blotting of virtually any protein as well as native lipoprotein particles.

Silkworm apolipophorin protein inhibits hemolysin gene expression of Staphylococcus aureus via binding to cell surface lipoteichoic acids.

We previously reported that a silkworm hemolymph protein, apolipophorin (ApoLp), binds to the cell surface of Staphylococcus aureus and inhibits expression of the saePQRS operon encoding a two-component system, SaeRS, and hemolysin genes. In this study, we investigated the inhibitory mechanism of ApoLp on S. aureus hemolysin gene expression. ApoLp bound to lipoteichoic acids (LTA), an S. aureus cell surface component. The addition of purified LTA to liquid medium abolished the inhibitory effect of ApoLp against S. aureus hemolysin production. In an S. aureus knockdown mutant of ltaS encoding LTA synthetase, the inhibitory effects of ApoLp on saeQ expression and hemolysin production were attenuated. Furthermore, the addition of anti-LTA monoclonal antibody to liquid medium decreased the expression of S. aureus saeQ and hemolysin genes. In S. aureus strains expressing SaeS mutant proteins with a shortened extracellular domain, ApoLp did not decrease saeQ expression. These findings suggest that ApoLp binds to LTA on the S. aureus cell surface and inhibits S. aureus hemolysin gene expression via a two-component regulatory system, SaeRS.

Quantitative analysis of apolipoproteins in human HDL by top-down differential mass spectrometry.

The field of quantitative, label-free proteomics has evolved significantly over time, with most experiments performed "bottom-up" using proteolyzed protein mixtures. In these experiments, statistically significant peptide abundance differences between two or more experimental conditions are determined, and their corresponding proteins later identified. Recently, the rationale for extending this experimental design to mixtures of intact proteins has become clear, as analysis at the protein level allows for the independent detection of each protein form present, including those modified posttranslationally. This provides a level of specificity lost in bottom-up experiments. As such, the application of label-free top-down differential mass spectrometry has provided a means for understanding the subtle protein changes that define a particular phenotype. Described here is an approach for the top-down label-free quantitative analysis of the proteins which constitute human high-density lipoprotein particles. The methodology is conceptually very straightforward; however, it does require a level of rigor and consistency typically not addressed by more conventional proteomics experiments.

Synergistic action of Galleria mellonella apolipophorin III and lysozyme against Gram-negative bacteria.

Insect immune response relies on the humoral and cellular mechanisms of innate immunity. The key factors are the antimicrobial polypeptides that act in concert against invading pathogens. Several such components, e.g. apolipophorin III (apoLp-III), lysozyme, and anionic peptide 2, are present constitutively in the hemolymph of non-challenged Galleria mellonella larvae. In the present study, we demonstrate an evidence for a synergistic action of G. mellonella lysozyme and apoLp-III against Gram-negative bacteria, providing novel insights into the mode of action of these proteins in insect antimicrobial defense. It was found that the muramidase activity of G. mellonella lysozyme considerably increased in the presence of apoLp-III. Moreover, apoLp-III enhanced the permeabilizing activity of lysozyme toward Escherichia coli cells. As shown using non-denaturing PAGE, the proteins did not form intermolecular complexes in vivo and in vitro, indicating that the effect observed was not connected with the intermolecular interactions between the proteins. Analysis of AFM images of E. coli cells exposed to G. mellonella lysozyme and/or apoLp-III revealed evident alterations in the bacterial surface structure accompanied by the changes in their biophysical properties. The bacterial cells demonstrated significant differences in elasticity, reflected by Young's modulus, as well as in adhesive forces and roughness values in comparison to the control ones. The constitutive presence of these two defense molecules in G. mellonella hemolymph and the fact that apoLp-III enhances lysozyme muramidase and perforating activities indicate that they can be regarded as important antibacterial factors acting at the early stage of infection against Gram-negative as well as Gram-positive bacteria.

Anti-Legionella dumoffii activity of Galleria mellonella defensin and apolipophorin III.

The gram-negative bacterium Legionella dumoffii is, beside Legionella pneumophila, an etiological agent of Legionnaires' disease, an atypical form of pneumonia. The aim of this study was to determine the antimicrobial activity of Galleria mellonella defense polypeptides against L. dumoffii. The extract of immune hemolymph, containing a mixture of defense peptides and proteins, exhibited a dose-dependent bactericidal effect on L. dumoffii. The bacterium appeared sensitive to a main component of the hemolymph extract, apolipophorin III, as well as to a defense peptide, Galleria defensin, used at the concentrations 0.4 mg/mL and 40 µg/mL, respectively. L. dumoffii cells cultured in the presence of choline were more susceptible to both defense factors analyzed. A transmission electron microscopy study of bacterial cells demonstrated that Galleria defensin and apolipophorin III induced irreversible cell wall damage and strong intracellular alterations, i.e., increased vacuolization, cytoplasm condensation and the appearance of electron-white spaces in electron micrographs. Our findings suggest that insects, such as G. mellonella, with their great diversity of antimicrobial factors, can serve as a rich source of compounds for the testing of Legionella susceptibility to defense-related peptides and proteins.

SERINE proteinase like activity in apolipophorin III from the hemolymph of desert locust, Schistocerca gregaria.

Apolipophorin III (apoLp-III) has been known as a lipid transport protein of insects. Recent studies indicated the involvement of apoLp-III in immune reactions and in the control of cell destruction, but no enzymatic activity has so far been detected. In the present study, a protease from the hemolymph of Schistocerca gregaria was purified to homogeneity and its enzymatic activity was examined. Identity as chymotrypsin-like proteinase was established by its high affinity toward bulky aromatic substrates and its catalytic specificity for amide or ester bonds on the synthetic substrates, Suc-Ala-Ala-Pro-Xaa-AMC (where Xaa was Phe, Tyr, Trp, and Lys, and AMC is 7-amino-4-methyl-coumarin) and thiolbenzyl ester substrate Suc-Ala-Ala-Pro-Phe-SBzl. The sensitivity for serine protease and chymotrypsin-specific covalent inhibitors, PMSF, TPCK, and noncovalent inhibitors SGCI, showed that it is a chymotrypsin-like proteinase. It showed its maximum activity at pH 8.0 and 55°C for the hydrolysis of Suc-Ala-Ala-Pro-Tyr-AMC. According to similarities in the amino terminal sequence, molar mass (19 kDa) and retention on reversed-phase analytical high-performance liquid chromatography (HPLC) column, this protein is S. gregaria homologue of Locusta migratoria apoLp-III. Our data suggest that apoLp-III also has an inherent proteolytic activity. Results indicated that S. gregaria apoLp-III is a good catalyst and could be used as a biotechnological tool in food processing and in agricultural biotechnology.

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi.

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal ß-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.

An apolipophorin III protein from the hemolymph of desert locust, Schistocerca gregaria.

Apolipophorin III (apoLp-III) from insects and apolipoprotein A-I from humans, are major component of the lipoprotein and share various properties. ApoLp-III is an abundant hemolymph protein. Besides its crucial role in lipid transport, apoLp-III is able to associate with fungal and bacterial membranes and stimulate cellular immune responses. ApoLp-III was isolated and purified from the hemolymph of desert locust Schistocerca gregaria by ion-exchange and reversed-phase chromatography. The purity and the molecular weight of apoLp-III were determined at ∼19,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. According to similarities in the amino terminal sequence, molar mass and retention on reversed-phase analytical HPLC column, this protein is a Schistocerca gregaria homologue of Locusta migratoria apoLp-III.

Atomic force microscopy of ex vivo amyloid fibrils.

Here, we report a study of ex vivo amyloid fibrils formed, respectively, by the Leu174Ser Apolipoprotein A-I (ApoA-I-LS) variant and by ß2-microglobulin (ß2-m) (Relini et al., J. Biol. Chem. 281:16521-16529, 2006; Relini et al., Biochim. Biophys. Acta 1690:33-41, 2004). In the work on ApoA-I-LS, the AFM has been used to characterize and compare the morphologies of amyloid fibrils isolated from two different patients, while in the study on ß2-m our investigation provided important information about the factors that can promote the aggregation in vivo.

An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria.

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. As revealed by LIVE/DEAD staining, incubation of Gram-negative and Gram-positive bacteria in the presence of Galleria mellonella apoLp-III led to growth inhibition of selected bacteria. An atomic force microscopy (AFM) study of bacterial cells after apoLp-III treatment showed considerable alterations in the cell surface of Bacillus circulans, Klebsiella pneumoniae and Salmonella typhimurium. Our results clearly demonstrate that apoLp-III disturbed the proper structure of the bacterial cell surface. The alterations were dissimilar to those caused by cationic antimicrobial peptide, cecropin B, suggesting a different mode of action against bacteria. The present results indicate that AFM provides a powerful tool for studying the interactions of apoLp-III with microbial cells.

Hydrophilic interaction chromatography of intact, soluble proteins.

The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae.

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.

Apolipoprotein M expression increases the size of nascent pre beta HDL formed by ATP binding cassette transporter A1.

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.