A three-dimensional molecular model of lipid-free apolipoprotein A-I determined by cross-linking/mass spectrometry and sequence threading

Apolipoprotein (apo) A-I, a 243-residue, 28.1-kDa protein is a major mediator of the reverse cholesterol transport (RCT) pathway, a process that may reduce the risk of cardiovascular disease in humans. In plasma, a small fraction of lipid-free or lipid-poor apoA-I is likely a key player in the first step of RCT. Therefore, a basic understanding of the structural details of lipid-free apoA-I will be useful for elucidating the molecular details of the pathway. To address this issue, we applied the combined approach of cross-linking chemistry and high-resolution mass spectrometry (MS) to obtain distance constraints within the protein structure. The 21 lysine residues within apoA-I were treated with homo bifunctional chemical cross-linkers capable of covalently bridging two lysine residues residing within a defined spacer arm length. After trypsin digestion of the sample, individual peptide masses were identified by MS just after liquid chromatographic separation. With respect to the linear amino acid sequence, we identified 5 short-range and 12 long-range cross-links within the monomeric form of lipid-free apoA-I. Using the cross-linker spacer arm length as a constraint for identified Lys pairs, a molecular model was built for the lipid-free apoA-I monomer based on homology with proteins of similar sequence and known three-dimensional structures. The result is the first detailed model of lipid-free apoA-I. It depicts a helical bundle structure in which the N- and C-termini are in close proximity. Furthermore, our data suggest that the self-association of lipid-free apoA-I occurs via C- and N-termini of the protein based on the locations of six cross-links that are unique to the cross-linked dimeric form of apoA-I.     

Cytotoxicity of lipid-free apolipoprotein B

To investigate the effect of apolipoprotein B (apoB) on cell viability, we used lipid-free apoB as a model for denatured apoB. Lipid-free apoB had cytotoxicity to J774 macrophages, CHO cells and HepG2 cells, whereas apoB bound to low density lipoprotein (LDL) and lipid-free apolipoprotein A-I had no effect on cell viability. Lipid-free apoB induced apoptosis in J774 macrophages assessed by caspase-3 activation and annexin V binding. LDL receptor, heparan sulfate proteoglycans, and class A scavenger receptor were involved in the binding/uptake of lipid-free apoB, but lipid-free apoB binding/uptake by the cells did not correlate with cytotoxicity. Lipid-free apoB disrupted the lipid bilayer of large unilamellar vesicles containing calcein. We evaluated the interaction between apoB and cellular membrane by monitoring the change in intracellular Ca²⁺ concentration using Fura-2, and found that lipid-free apoB rapidly disrupted the cellular membrane in the absence or presence of the inhibitors for cellular binding/uptake mediated by the receptors. Therefore, it is suggested that lipid-free apoB induces cell death by disturbance of the plasma membrane. In addition to other lipid component in modified LDL, apoB itself has an ability to induce apoptosis and plays a crucial role in the development of atherosclerotic lesions.     

Identification of protein-protein interactions using in vivo cross-linking and mass spectrometry

The purification of protein complexes can be accomplished by different types of affinity chromatography. In a typical immunoaffinity experiment, protein complexes are captured from a cell lysate by an immobilized antibody that recognizes an epitope on one of the known components of the complex. After extensive washing to remove unspecifically bound proteins, the complexes are eluted and analyzed by mass spectrometry (MS). Transient complexes, which are characterized by high dissociation constants, are typically lost by this approach. In the present study, we describe a novel method for identifying transient protein-protein interactions using in vivo cross-linking and MS-based protein identification. Live cells are treated with formaldehyde, which rapidly permeates the cell membrane and generates protein-protein cross-links. Proteins cross-linked to a Myc-tagged protein of interest are copurified by immunoaffinity chromatography and subjected to a procedure which dissociates the cross-linked complexes. After separation by SDS-PAGE, proteins are identified by tandem mass spectrometry. Application of this method enabled the identification of numerous proteins that copurified with a constitutively active form of M-Ras (M-Ras(Q71L)). Among these, we identified the RasGAP-related protein IQGAP1 to be a novel interaction partner of M-Ras(Q71L). This method is applicable to many proteins and will aid in the study of protein-protein interactions.     

Photo-assisted peptide enrichment in protein complex cross-linking analysis of a model homodimeric protein using mass spectrometry

MS analysis of cross-linked peptides can be used to probe protein contact sites in macromolecular complexes. We have developed a photo-cleavable cross-linker that enhances peptide enrichment, improving the signal-to-noise ratio of the cross-linked peptides in mass spectrometry analysis. This cross-linker utilizes nitro-benzyl alcohol group that can be cleaved by UV irradiation and is stable during the multiple washing steps used for peptide enrichment. The enrichment method utilizes a cross-linker that aids in eliminating contamination resulting from protein-based retrieval systems, and thus, facilitates the identification of cross-linked peptides. Homodimeric pilM protein from Pseudomonas aeruginosa 2192 (pilM) was investigated to test the specificity and experimental conditions. As predicted, the known pair of lysine side chains within 14?? was cross-linked. An unexpected cross-link involving the protein's amino terminus was also detected. This is consistent with the predicted mobility of the amino terminus that may bring the amino groups within 19?? of one another in solution. These technical improvements allow this method to be used for investigating protein-protein interactions in complex biological samples.     

The use of chemical cross-linking and mass spectrometry to elucidate the tertiary conformation of lipid-bound apolipoprotein A-I

PURPOSE OF REVIEW: The purpose of this review is to highlight recent advances in mass spectrometry and its use for identifying the lipid-bound conformation of apolipoprotein A-I. Given the current interest in understanding the structure of HDL apolipoprotein A-I, this approach seems ideal in assessing its dual role as mediator of lipid efflux and modulator of cellular inflammation. RECENT FINDINGS: A large number of different technical approaches have been employed over the past 25 years in attempts to solve the lipid-bound conformation of apolipoprotein A-I. Since the X-ray crystal structure of lipid-free Delta43 apolipoprotein A-I was reported in 1997, a 'double belt' model describing lipid-bound apolipoprotein A-I conformation for recombinant HDL has prevailed. Recent studies have focused on determining the exact helix-helix registry and salt-bridging partners found on a two apolipoprotein A-I molecule disc as well as on spherical HDL particles. Investigations are all aimed at defining the conformation of lipid-bound apolipoprotein A-I which may provide an explanation for how specific domains of apolipoprotein A-I interact with important HDL-modifying proteins that ultimately determine the apolipoprotein's fate in circulation. SUMMARY: Recent advances in mass spectrometric sequencing of cross-linked peptides provide an excellent tool to help define protein tertiary structure. This approach has provided refined structural information on apolipoprotein A-I folding which had eluded all previous approaches.     

Radioimmunoassay of rat apolipoprotein A-IV

We have developed a specific and sensitive radioimmunoassay for rat apolipoprotein A-IV (apoA-IV). The protocol includes treatment of the samples for 1 h at 60 degrees C with 0.7% Tween 20. Under these conditions, linear logit-log plots have been obtained for apoA-IV in lymph and plasma lipoprotein fractions as well as for purified apoA-IV. The sensitivity of the assay is to 20 ng. Absolute mass values obtained with the assay were validated by comparison with values obtained with an independent method of colorimetric reading of apoA-IV separated by polyacrylamide gel electrophoresis from plasma high density lipoproteins. The concentration of apoA-IV in fasting plasma averaged 10.2 mg/dl and in the mesenteric duct lymph 15.8 and 12.6 mg/dl during the fasting and the fat absorption states, respectively.     

A proteolytic method for distinguishing between lipid-free and lipid-bound apolipoprotein A-I

Recent studies indicate that certain lipid-poor forms of apolipoprotein (apo)A-I may be particularly important in promoting cholesterol release from overburdened cells in the periphery. However, a detailed understanding of the physiological relevance of these species has been hampered by the difficulty in measuring them. As part of a search for a rapid assay for these forms of apoA-I, we have observed that the protease enteropeptidase can specifically cleave human lipid-free apoA-I but not its lipid-bound form. Enteropeptidase cleaved lipid-free apoA-I at a single site at amino acid 188, resulting in an N-terminal fragment of 22 kDa. However, apoA-I was not susceptible to enteropeptidase when present in reconstituted high-density lipoprotein (rHDL) particles as small as 6 nm in diameter or in human HDL(3) particles, even at extremely high enzyme-to-protein ratios and extended reaction times. We capitalized on this observation to develop an assay for the measurement of lipid-poor apoA-I in in vitro systems. Densitometry was used to generate a standard curve from sodium dodecyl sulfate polyacrylamide gels to determine the amounts of the N-terminal proteolytic fragment in unknown samples treated with enteropeptidase. This system could accurately quantify apoA-I that had been displaced from rHDL particles and human HDL(3) with purified apoA-II. On the basis of the results, a system of nomenclature is proposed for "lipid-free," "lipid-poor," and "lipid bound" apoA-I.The reported method distinguishes forms of apoA-I by a conformational parameter without previous separation of the species. This simple and inexpensive method will be useful for understanding the characteristics of plasma HDL that are favorable for the dissociation of apoA-I.     

A method for the determination of the three-dimensional structure of fluorescent proteins based on homology modeling and mass spectrometry

In the current work a method for the elucidation of the spatial structure of fluorescent GFP-like proteins is presented. The method is based on combining the results of homology modeling of the overall spatial protein structure and the chromophore structure determined experimentally by mass spectrometry. The approach developed can be used for the determination of the spatial structure of GFP homologues which are difficult to crystallize.     

A structural model of the erythrocyte spectrin heterodimer initiation site determined using homology modeling and chemical cross-linking

Spectrin assembles into an anti-parallel heterodimeric flexible rod-like molecule through a multistep process initiated by a high affinity interaction between discrete complementary homologous motifs or "repeats" near the actin binding domain. Attempts to determine crystallographic structures of this critical dimer initiation complex have so far been unsuccessful. Therefore, in this study we determined the subunit-subunit docking interface and a plausible medium resolution structure of the heterodimer initiation site using homology modeling coupled with structural refinement based on experimentally determined distance constraints. Intramolecular and intermolecular cross-links formed by the "zero length" cross-linking reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide were identified after trypsin digestion of cross-linked heterodimer complex using liquid chromatography-tandem mass spectrometry analysis. High confidence assignment of cross-linked peptides was facilitated by determination of cross-linked peptide masses with an uncertainty of a few parts per million using a high sensitivity linear ion trap mass spectrometer equipped with a Fourier-transform ion cyclotron resonance detector. Six interchain cross-links distinguished between alternative docking models, and these distance constraints, as well as three intrachain cross-links, were used to further refine an initial homology-based structure. The final model is consistent with all available physical data, including protease protection experiments, isothermal titration calorimetry analyses, and location of a common polymorphism that destabilizes dimerization. This model supports the hypothesis that initial docking of the correct alpha and beta repeats from among many very similar repeats in both subunits is driven primarily by long range electrostatic interactions.     

Regulation of intestinal and hypothalamic apolipoprotein A-IV

This review discusses the regulation of the intestinal and hypothalamic apolipoprotein A-IV (apo A-IV) gene and protein expression. Apo A-IV is a glycoprotein secreted together with triglyceride-rich lipoproteins by the small intestine. Intestinal apo A-IV synthesis is stimulated by fat absorption, probably mediated by chylomicron formation. This stimulation of intestinal apo A-IV synthesis is attenuated by intravenous leptin infusion. Chronic ingestion of a high-fat diet blunts the intestinal apo A-IV in response to dietary lipid. Intestinal apo A-IV synthesis is also stimulated by members of the pancreatic polypeptide family, including peptide YY (PYY), neuropeptide Y (NPY), and pancreatic polypeptide (PP). Recently, apo A-IV was demonstrated to be present in the hypothalamus as well. Hypothalamic apo A-IV level was reduced by food deprivation and restored by lipid feeding. Intracerebroventricular administration of apo A-IV antiserum stimulated feeding and decreased the hypothalamic apo A-IV mRNA level, implying that feeding is intimately regulated by endogenous hypothalamic apo A-IV. Central administration of NPY significantly increased hypothalamic apo A-IV mRNA levels in a dose-dependent manner.     

Metabolism of apolipoprotein A-IV in rat

The metabolism of apolipoprotein A-IV (apo-IV) has been investigated in the rat. In this animal species, apoA-IV is a major protein constituent of plasma HDL and lymph chylomicron. The apolipoprotein is also present in the lipoprotein-deficient fraction (LDF) of plasma and lymph. In vivo studies with the radioiodinated protein showed the apoA-IV does not exchange freely between HDL and LDF and that LDF apoA-IV had a faster catabolism than HDL apoA-IV. ApoA-IV in chylomicrons is a direct precursor of apoA-IV in plasma HDL but not of that in LDF. On the other hand lymph LDF apoA-IV is an important precursor of plasma LDF apoA-IV. Transfer of apoA-IV from plasma to lymph is negligible, and since most of apoA-IV in lymph is present in LDF, we speculate that LDF apoA-IV is the major apoA-IV secretory product of the intestine. Studies aimed at identifying the site of catabolism of apoA-IV utilizing either radioiodinated or [14C]sucrose labelled apoA-IV, gave results consistent with the view that the liver plays a major role. When tested, human apoA-IV behaved in vivo in rat as the autologous protein. These findings, together with others previously published (Ghiselli, G. et al. (1987) J. Lipid Res. 27, 813-827), support the conclusion that the plasma metabolism of apoA-IV is remarkably similar in rat and human. We speculate that in mammals the rapid plasma catabolism of apoA-IV is mediated by an efficient uptake by the liver.     

Structure and interfacial properties of chicken apolipoprotein A-IV

To gain insight into the evolution and function of apolipoprotein A-IV (apoA-IV) we compared structural and interfacial properties of chicken apoA-IV, human apoA-IV, and a recombinant human apoA-IV truncation mutant lacking the carboxyl terminus. Circular dichroism thermal denaturation studies revealed that the thermodynamic stability of the alpha-helical structure in chicken apoA-IV (DeltaH = 71.0 kcal/mol) was greater than that of human apoA-IV (63.6 kcal/mol), but similar to that of human apoA-I (73.1 kcal/mol). Fluorescence chemical denaturation studies revealed a multiphasic red shift with a 65% increase in relative quantum yield that preceded loss of alpha-helical structure, a phenomenon previously noted for human apoA-IV. The elastic modulus of chicken apoA-IV at the air/water interface was 13.7 mN/m, versus 21.7 mN/m for human apoA-IV and 7.6 mN/m for apoA-I. The interfacial exclusion pressure of chicken apoA-IV for phospholipid monolayers was 31.1 mN/m, versus 33.0 mN/m for human A-I and 28.5 mN/m for apoA-IV.We conclude that the secondary structural features of chicken apoA-IV more closely resemble those of human apoA-I, which may reflect the evolution of apoA-IV by intraexonic duplication of the apoA-I gene. However, the interfacial properties of chicken apoA-IV are intermediate between those of human apoA-I and apoA-IV, which suggests that chicken apoA-IV may represent an ancestral prototype of mammalian apoA-IV, which subsequently underwent further structural change as an evolutionary response to the requisites of mammalian lipoprotein metabolism.     

Conformational properties of human and rat apolipoprotein A-IV

Apolipoprotein A-IV has been isolated from four sources: human and rat lymph and plasma. Conformational properties of the rat and human apoA-IV in solution and denaturation changes induced by guanidine hydrochloride (Gnd X HCl) were studied using circular dichroic and fluorescence spectroscopy, and analytical sedimentation equilibrium ultracentrifugation. We have shown that both rat and human apoA-IV have similar secondary structure with negative maxima in the circular dichroic spectra at 222 nm and 207 nm. Furthermore, we have found no significant difference in the alpha-helical content of the apoA-IV from rat plasma (33%), rat lymph (37%), human plasma (35%), or human lymph (35%). Our denaturation studies with Gnd X HCl demonstrated reversibility and the fact that each apoA-IV had a tendency to self-associate in solution and the self-association could be disrupted by low concentrations of Gnd X HCl (less than or equal to 0.4 M). Unfolding of the secondary structure of each apoA-IV occurred at higher concentrations of Gnd X HCl (midpoint less than or equal to 1.0 M). The apparent free energy of denaturation of the four apoA-IV proteins calculated from changes in the circular dichroic spectra upon addition of increasing concentrations of Gnd X HCl varied in a range from 3.0 to 4.2 kcal/mol. The fluorescence experiments revealed that apoA-IV from all sources had a maximum fluorescence emission at 342.5 nm, which shifted to the red region upon addition of increasing concentrations of Gnd X HCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Chemical cross-linking and mass spectrometry for protein structural modeling

The growth of gene and protein sequence information is currently so rapid that three-dimensional structural information is lacking for the overwhelming majority of known proteins. In this review, efforts towards rapid and sensitive methods for protein structural characterization are described, complementing existing technologies. Based on chemical cross-linking and offering the analytical speed and sensitivity of mass spectrometry these methodologies are thought to contribute valuable tools towards future high throughput protein structure elucidation.     

Quantitative interactome analysis with chemical cross-linking and mass spectrometry

Structural plasticity and dynamic protein–protein interactions are critical determinants of protein function within living systems. Quantitative chemical cross-linking with mass spectrometry (qXL-MS) is an emerging technology able to provide information on changes in protein conformations and interactions. Importantly, qXL-MS is applicable to complex biological systems, including living cells and tissues, thereby providing insights into proteins within their native environments. Here, we present an overview of recent technological developments and applications involving qXL-MS, including design and synthesis of isotope-labeled cross-linkers, development of new liquid chromatography–MS methodologies, and computational developments enabling interpretation of the data.     

Distribution of apolipoprotein A-IV in human plasma

Human apoA-IV was purified from delipidated urinary chylomicrons. Monospecific antibodies were raised in rabbits and used to develop a double antibody radioimmunoassay (RIA). Displacement of 125I-labeled apoA-IV by plasma or purified chylomicron apoA-IV resulted in parallel displacement curves, indicating that apoA-IV from both sources share common antigenic determinants. The apoA-IV level in plasma from normal healthy fasting male subjects (n = 5) was 37.4 +/- 4.0 mg/dl, while fat-feeding increased the level to 49.1 +/- 7.9 mg/dl (P less than 0.05) at 4 hr. The apoA-IV level in plasma from abetalipoproteinemic fasting subjects was 13.7 +/- 3.1 mg/dl (n = 5). Plasma from a single fasting Tangier subject showed a reduced apoA-IV level of 21.1 mg/dl. The distribution of apoA-IV in fasting and postprandial plasma was determined by 6% agarose gel chromatography. Fifteen to 25% of plasma apoA-IV eluted in the region of plasma high density lipoprotein (HDL), with the remainder eluting in subsequent column fractions. In abetalipoproteinemic plasma this HDL fraction is reduced and lacks apoA-IV, suggesting that at least some of the apoA-IV on these particles is normally derived from triglyceride-rich lipoproteins. Lipemic plasma from a fat-fed subject showed a small rise (3%) in chylomicron-associated apoA-IV. Gel-filtered HDL and subsequent apoA-IV-containing fractions were subjected to 4-30% polyacrylamide gradient gel electrophoresis (4/30 GGE), and apoA-IV was identified by immunolocalization following transfer of proteins to nitrocellulose paper. In normal plasma apoA-IV was localized throughout all HDL fractions. In addition, normal plasma contained apoA-IV localized in a small particle (diameter 7.8-8.0 nm). This particle also contained apoA-I and lipid. A markedly elevated saturated to unsaturated cholesteryl ester ratio was present in gel-filtered plasma fractions containing small HDL, suggesting an intracellular origin of these particles. In abetalipoproteinemic plasma apoA-IV was absent from all HDL fractions except for the small HDL particles, suggesting that they are not derived from the surface of triglyceride-rich particles. All plasmas contained free apoA-IV. In contrast to gel-filtered plasma, lipoprotein subfractions of fasted normal plasma prepared in the ultracentrifuge primarily contained apoA-IV in the d greater than 1.26 g/ml fraction, suggesting an artifactual redistribution of the apolipoprotein during centrifugation. Overall, these data suggest that apoA-IV secretion into plasma is increased with fat feeding, and that apoA-IV normally exists as both a free apolipoprotein and in association with HDL particles.     

The primary structure of human apolipoprotein A-IV

Human apolipoprotein (apo) A-IV was purified from chylous ascites fluid. Proteolytic peptides produced by trypsin and Staphylococcus aureus V8 proteinase digestions were purified by high-performance liquid chromatography and sequenced. Human apoA-IV contains 376 amino acid residues. The peptide-derived sequence generally matches two previously reported DNA-derived amino acid sequences except for discrepancies in five positions. In order to examine these discrepancies further, one complete apoA-IV cDNA clone and another partial clone were sequenced. Comparison of all the available information indicates that the peptide-derived sequence reported here is accurate. Sequencing errors probably account for some of the discrepancies between the two primary sequences predicted by earlier nucleotide analyses. In certain positions, however, bona fide sequence heterogeneity or cloning artifact cannot be excluded.     

Characterization of apolipoprotein A-IV complexes and A-IV isoforms in human lymph and plasma lipoproteins

We have isolated and characterised A-IV apolipoprotein (apo-A-IV) from human lymph and plasma by immunoabsorbance chromatography and two-dimensional electrophoresis. Two different apo-A-IV-containing lipoproteins were isolated from four different sources, human lymph triglyceride-rich fraction (TRL), lymph lipoprotein-deficient fraction (LDF), plasma high-density lipoprotein (HDL), and plasma lipoprotein-deficient fraction (LDF). The lipoprotein complexes obtained from lymph TRL and plasma HDL were similar and contained apo-A-IV, apo-A-I, and small molecular weight peptides (apo-C or -A-II). The second lipoprotein complex was isolated from lymph LDF and plasma LDF, and contained apo-A-IV, apo-A-I, and a peptide of Mr = 59,000. The lipid composition of the lipoprotein complexes varied according to the source: triglyceride predominating in lymph TRL and phospholipid and cholesteryl ester from the other sources. Free cholesterol was conspicuously present in very small amounts. Using two-dimensional electrophoresis and immunoblotting techniques, eleven isoproteins of apo-A-IV were identified (pI-4.98, 5.06, 5.10, 5.15, 5.20, 5.22, 5.25, 5.30, 5.34, 5.42, and 5.48). The isoprotein pattern of lymph TRL and plasma HDL was similar, but that of lymph and plasma LDF were different patterns. These results suggest that apo-A-IV associated with d less than 1.21 lipoproteins and apo-A-IV present in LDF may be in metabolically separate lipoproteins and may have different physiological roles.