- Top of page
- Methods and Materials
- Conclusions and Future Directions
- Conflict of interest
Lysophosphatidylcholine, a major phospholipid component of oxidized low-density lipoprotein, is implicated in many inflammatory diseases, including atherosclerosis. We previously reported that Asp-hemolysin-related synthetic peptide (P21) composed of 21 amino acid residues markedly inhibits the bioactivities of oxidized low-density lipoprotein and lysophosphatidylcholine, by directly binding to oxidized low-density lipoprotein and lysophosphatidylcholine. Here, to clarify whether P21 specifically binds to lysophosphatidylcholine and what forms of lysophosphatidylcholine with which P21 interact, we investigated the interaction between P21 containing two tryptophan residues and lysophosphatidylcholine by using fluorescence spectroscopy, polyacrylamide gel electrophoresis, and surface plasmon resonance. From tryptophan fluorescence measurements, N-terminally biotinylated P21 specifically interacted with lysophosphatidylcholine, at concentrations exceeding the critical micelle concentration. From tryptophan fluorescence quenching, the tryptophan residues in biotinylated P21 in the presence of lysophosphatidylcholine were mostly exposed on the outer side of the peptide. From polyacrylamide gel electrophoresis and surface plasmon resonance, bound to 1-palmitoyl-lysophosphatidylcholine at concentrations higher than 100 μm, ensuring stable micelles. These results indicate that biotinylated P21 specifically recognizes lysophosphatidylcholine micelles. Further study of the interaction between biotinylated P21 and lysophosphatidylcholine micelles may provide important information for the prevention and treatment for many inflammatory diseases caused by lysophosphatidylcholine micelles.
Phospholipids are important components of cell membranes. The hydrolysis of phospholipids by phospholipases generates fatty acids and lysophospholipids, including lysophosphatidylcholine (LPC), lysophosphatidic acid (LPA), lysophosphatidylethanolamine (LPE), lysophosphatidylinositol (LPI), and lysophosphatidylserine (LPS). Lysophospholipids play important roles in cellular signal transduction (1–3) and are implicated in numerous biological processes, including tumorigenesis, angiogenesis, immunity, atherosclerosis, arteriosclerosis, and neuronal survival (4). In particular, LPC, the most abundant lysophospholipid in plasma and tissues, is present at high concentrations in the circulation where it predominantly associates with albumin and lipoprotein (5). The production of LPC by phospholipase A2 (PLA2) from phosphatidylcholine (PC) pools in cells and oxidized low-density lipoprotein (ox-LDL) (6–8) plays an important role in the regulation of cell activity, and especially, in conditioning the immune arrangement and the immune stabilization of the organism (5,9,10). Furthermore, LPC is a major phospholipid component of ox-LDL and is implicated in numerous inflammatory diseases, including atherosclerosis (11,12).
The LPC molecule consists of one hydrophobic fatty acid chain and one hydrophilic polar choline group, attached to a glycerol backbone. The amphipathic nature of LPC gives it surfactant- and detergent-like properties. At low concentrations, LPC exists as a single molecule, a monomer. However, at concentrations exceeding the critical micelle concentration (CMC), LPC forms small micelles. For instance, 1-palmitoyl-LPC (LPC16:0) forms small micelles with a Stokes radius of 34 Å between 7 and 50 μm (13). At approximately 50 μm, LPC16:0 transitions to larger micelles with a Stokes radius of 72 Å (13). The average aggregation number of an LPC16:0 is 139 (14). Different LPC molecular species (LPCs), including 1-lauroyl-LPC (LPC12:0), 1-myristoyl-LPC (LPC14:0), LPC16:0, and 1-stearoyl-LPC (LPC18:0), all form micelles at concentrations exceeding their CMCs. LPC itself, with a relatively large hydrophilic moiety, virtually organizes in a micellar structure, and the hydrophobic core is remarkably larger than that of other surfactants.
Asp-hemolysin is a hemolytic and toxic protein derived from Aspergillus fumigatus (15). The gene for Asp-hemolysin has been cloned, and the gene sequence has been reported (16). The primary Asp-hemolysin gene product consists of 131 amino acid residues and has a molecular mass of 14 275 Da. We previously reported that ox-LDL inhibits the hemolytic activity of Asp-hemolysin, and that Asp-hemolysin binds to ox-LDL in a concentration-dependent manner (17,18). We have shown that Asp-hemolysin binds specifically to ox-LDL with a high affinity (the dissociation constant, Kd = 0.63 μg/mL). Its binding specificity is distinct from any receptor for ox-LDL (18). In addition, we have shown that LPC inhibits the binding of Asp-hemolysin to ox-LDL, and that Asp-hemolysin is a binding protein for LPC (19,20). Moreover, we have also reported that an Asp-hemolysin-related synthetic peptide (P21) composed of 21 amino acid residues (16), which corresponds to the amino acids 21–41 of Asp-hemolysin – a sequence rich in positive charges – inhibits ox-LDL-induced macrophage proliferation and LPC-induced apoptosis in human umbilical vein endothelial cells (HUVECs) by directly binding to ox-LDL and LPC (21,22). Finally, we have also shown that the Tyr-Lys-Asp-Gly (YKDG) sequence in P21 is important for binding to ox-LDL (16), and that the binding of ox-LDL to P21 is attributable to LPC (20). However, it remains unknown whether P21 specifically binds to LPC and what LPC forms P21 interacts with, for example monomeric or micellar LPC.
In the present study, we assessed the interactions between P21 and LPC, by using fluorescence spectroscopy, polyacrylamide gel electrophoresis (PAGE), and surface plasmon resonance (SPR). The results presented in this manuscript indicate that N-terminally biotinylated P21 (BP21) specifically recognizes LPC in the micellar form, and that both the YKDG sequence in BP21 and biotin bound to the N-terminus of P21 are necessary for the development of the specific BP21–LPC micelle interaction.
- Top of page
- Methods and Materials
- Conclusions and Future Directions
- Conflict of interest
LPC, a major phospholipid component of ox-LDL, is believed to play an important role in many inflammatory diseases, including atherosclerosis (11,12). We have previously reported that Asp-hemolysin-related synthetic P21 inhibits ox-LDL-induced macrophage proliferation and LPC-induced apoptosis in HUVECs by direct binding to ox-LDL and LPC (21,22). In addition, we have suggested that the YKDG sequence contained in P21 is important for the interaction of P21 with ox-LDL and LPC (20). The main objective of the present study was to assess the specificity of the interaction between P21 and LPC, and then, to identify the LPC forms that interact with P21.
The fluorescent properties of aromatic amino acids (tryptophan, phenylalanine, and tyrosine) are useful for fluorometric assays (23,24). Tryptophan is the dominating intrinsic fluorophore in peptides and proteins, because it has the highest quantum yield of the aromatic amino acids (31). Its fluorescence is more sensitive to the local environment. Because changes in the intrinsic tryptophan fluorescence of peptides/proteins often occur when they bind ligands or undergo the conformational change, it is possible to use changes in tryptophan fluorescence intensity as a direct measurement of ligand binding to the peptides/proteins (23–25). In the present study, we have used this tool to gain insight into the interaction between BP21 and various phospholipids, including LPC. BP21 interacts much more strongly with phospholipids that form micellar structures (LPCs, LPS, LPA, LPE, and LPI) and with phospholipids that form hexagonal structures (PA and PE) than phospholipids that form lamellar structures (PCs and PS) (Figure 1A,B). Moreover, both the YKDG sequence in BP21 and biotin bound to N-terminus of P21 are essential for the specific interaction with LPC (Figure 1C,D). These results suggest that the addition of biotin to the N-terminus of P21 without a linker may significantly change the conformation of the parent P21 and therefore its binding properties.
The tryptophan fluorescence emission intensity of BP21 markedly increased in the presence of increasing amounts of LPCs (Figure 2). At lower concentrations, below the CMCs of the LPCs, interaction of BP21 with LPCs increased the fluorescence emission intensity of the tryptophan residues without any shift in the maximum emission wavelength. On the other hand, at higher concentrations of LPCs, above the CMCs of the LPCs (particularly 1 mm, but all high enough to ensure stable micellar structures), the maximum emission wavelength of the tryptophan residues in BP21 blue-shifted from approximately 351–346 nm. In general, the intensity of tryptophan fluorescence increases when tryptophan is exposed to a hydrophobic, ‘non-polar’ environment, whereas it decreases when it is exposed to a hydrophilic, ‘polar’ environment. A blueshift of the emission maximum wavelength (λmax) often accompanies quantum yield changes (32,33). Because the information on tryptophan fluorescence λmax indicates that a tryptophan is buried and in a non-polar environment, the degree of blueshift correlates with the depth of tryptophan insertion into the phospholipids. At the lower LPC concentrations, the absence of any significant blueshifts in the fluorescence emission maxima of tryptophan could be interpreted to mean that no polarity changes in the environment of the tryptophan residues occurred upon interaction of BP21 with LPCs, which is in agreement with the fact that the tryptophan residues in BP21 are not located at the LPC-binding site. Therefore, tryptophan residues in BP21 may not be involved directly in the interaction between BP21 and LPC at lower concentrations. On the other hand, at the higher concentrations of LPCs, above the CMCs, ensuring stable micellar structures, tryptophan residues in BP21 may strongly interact with LPC micelles, particularly with the sn-1 acyl chain methyl group of LPCs, yielding a blueshift of 5–8 nm, because tryptophan emission maximum strongly reflects the hydrophobicity surrounding tryptophan residues. Therefore, tryptophan residues in BP21 may be involved in the interaction of BP21 with LPC micelles.
Tryptophan appears to be uniquely sensitive to collisional quenching, apparently because indole rings tend to donate electrons when in the excitation state (34). Externally added quenchers such as acrylamide (25) and iodide (26) can quench tryptophan fluorescence. Given the molecules, quenching of intrinsic tryptophan fluorescence can be used to determine the location of tryptophan residues and to follow protein conformational changes (35). The emission intensity of a tryptophan residue located on the protein/peptide surface will strongly decrease in the presence of a charged water-soluble quencher such as iodide. On the other hand, emission of a buried tryptophan residue will be less affected by the presence of the quencher. In the absence of LPC, neither of the tryptophan residues in BP21 was accessible to the quenchers, whereas in the presence of LPC, the tryptophan residues were exposed on the outside of the peptide. The Ksv values obtained with iodide quenching were considerably lower than those obtained with acrylamide quenching (Table 1). The lower values are probably related to the inability of I− to penetrate past the surface of the protein/peptide because of LPC micelles (25,36,37), and BP21, therefore, may be surrounded by LPC micelles. Because the surface accessibility of tryptophan can be assessed by looking at Ksv, the increase in Ksv in the presence of LPC suggests that tryptophan residues in BP21 were exposed upon binding to LPC. Results from the intrinsic tryptophan fluorescence experiments (Figures 1 and 2) and the quenching experiments (Figure 4) suggest that BP21 undergoes a conformational change in the presence of various LPCs and that BP21 interacts with LPC micelles. Moreover, results from native PAGE and SPR spectroscopy (Figures 5 and 6) suggest that BP21 specifically interacts with the micellar LPCs, and that the interaction is strongly related to the length of the acyl chain at the sn-1 position of LPCs.
In general, pharmacological actions of lysophospholipids are mediated by specific G-protein-coupled receptors (38). High levels of lysophospholipids cause potent cytotoxicity and are associated with some disease states (39). In particular, LPC is the most abundant lysophospholipid in plasma and tissues, and its average concentration in the plasma derived from healthy subjects is approximately 200 μm (40). The most abundant LPCs in fresh plasma is LPC16:0, which comprises approximately 40% of the total LPC pool, followed by LPC18:0, 1-oleoyl-LPC (LPC18:1), and 1-lioneoyl-LPC (LPC18:2), which constitute 30–35% of the total LPC (40). LPCs, which have acyl chains longer than LPC16:0, such as LPC 18:0, LPC18:1, and LPC 18:2, have lower CMCs than LPC16:0. LPC is believed to play an important role in many inflammatory diseases, including arteriosclerosis, and LPC levels in the plasma increase in these diseases (41). Therefore, micelle-formed LPCs may play important roles in these diseases. Also, LPC is the major phospholipid component of ox-LDL, which plays an important role in the development and progression of atherosclerosis, and is present at a concentration of approximately 500 nmol/mg protein in ox-LDL. By contrast, in the native LDL derived from healthy subjects, the concentrations of LPC are only 25 nmol/mg protein (42,43). Although we previously reported that the binding of P21 to ox-LDL inhibits the induction of macrophage proliferation by ox-LDL, the levels of LPCs contained in ox-LDL that bound to the P21 were estimated to be 5–100 μm, whereas the levels of LPCs contained in native LDL were estimated to be 0.25–5 μm. Moreover, the levels of LPCs that P21 remarkably inhibit apoptosis in HUVECs were estimated to be 25–100 μm. LPC acts as detergents in the lipid membranes at concentrations above their CMCs and cause cell swelling, lysis, and eventually cell death (44). Moreover, ox-LDL (20 μg/mL) markedly increases the apoptosis of cultured human coronary arterial endothelial cells (45) and LPC induces hemolysis of rabbit erythrocytes at concentrations above the CMC (46). Therefore, our results in this study strongly suggest that ox-LDL contains micelle-rich LPCs, and that BP21 specifically recognizes the bioactive LPC micelles.
Recently, we have shown that synthetic peptides derived from Asp-hemolysin, especially their N-terminally biotinylated peptides including BP21, markedly inhibit the bioactivities induced by platelet-activating factor (PAF), which bears a marked resemblance to LPC in terms of structure and bioactivity, by direct binding to PAF and its metabolite/precursor lyso-PAF, both in vivo and in vitro (47). Therefore, the findings in this study suggest that BP21 can be useful for prevention and treatment for many inflammatory diseases caused by LPC, especially LPC micelles.