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Keywords:

  • Asp-hemolysin;
  • biotinylated peptide;
  • interaction;
  • lysophosphatidylcholine;
  • micelle;
  • synthetic peptide

Abstract

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References

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.

Methods and Materials

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References

Materials

Synthetic phospholipids, LPCs (LPC12:0, -14:0, -16:0, and -18:0), LPA16:0, LPS18:1, LPE18:1, LPI from bovine liver, PC molecular species (PCs; PC16:0/18:0 and -16:0/18:1), and phosphatidylserine (PS16:0/18:1) were all purchased from Avanti Polar Lipids (Alabaster, AL, USA). Phosphatidylethanolamine (PE, from sheep brain), phosphatidic acid (PA16:0/16:0), and sphingomyelin from bovine brain were purchased from Sigma-Aldrich (St. Louis, MO, USA). 8-Anilino-1-naphthalenesulfonic acid (1,8-ANS) was purchased from Tokyo Chemical Industries (Tokyo, Japan). Other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan), unless otherwise noted.

Synthetic peptides

P21 corresponding to 21–41 of Asp-hemolysin amino acid sequence (16) (IKNASLSWGKWYKDGDKDAEI), N-terminally biotinylated P21 (BP21), BP21 labeled with fluorescein isothiocyanate (FITC) at its C-terminus (FITC-labeled BP21), BGP21, in which the YKDG sequence in BP21 is modified to GGGG sequence, were synthesized by Bio Synthesis (Lewisville, TX, USA). For C-terminal labeling of FITC, a lysine residue and 6-aminohexanoic acid as the spacer were added to the C-terminus of BP21. All peptides were purified to >95% purity by reverse high-pressure liquid chromatography and subsequently analyzed by laser desorption mass spectrometry.

Intrinsic tryptophan fluorescence

Spectra were collected using a Hitachi F2500 fluorescence spectrophotometer (Tokyo, Japan) at room temperature (22–25 °C) in phosphate-buffered saline (PBS; 10 mm phosphate and 150 mm sodium chloride, pH 7.4) (23–25). Excitation and emission bandwidths were both adjusted to 5 nm, tryptophan excitation wavelength was set to 295 nm to minimize interference from tyrosine fluorescence, emission was monitored from 300 to 400 nm, and the values were read at 348 nm. Each peptide (P21, BP21, and BGP21), all containing two tryptophan residues, was used. Different concentrations of phospholipids (0–1 mm), in the absence or presence of each peptide (0.4 μm), were incubated for 30 min at 37 °C. Buffer containing phospholipids was measured as a blank.

Tryptophan-quenching experiment

Peptide–phospholipid interactions are accompanied by changes in the accessibility of the peptides to aqueous quenchers of tryptophan fluorescence. Acrylamide (25) and iodide (26) quenching experiments were carried out on 0.4 μm BP21 in PBS (pH 7.4) in the absence or presence of LPC16:0 (1 μm to 1 mm) by adding aliquots of an acrylamide solution or a potassium iodide (KI) solution containing sodium isothiosulfate (Na2S2O3) to prevent inline image formation. BP21–LPC16:0 mixtures were incubated for 30 min at 37 °C prior to the measurements, and the fluorescence spectrum was recorded with a Hitachi F2500 fluorescence spectrophotometer. The excitation wavelength was set to 295 nm to reduce the absorbance of acrylamide and iodide. Fluorescence intensities were read at 348 nm after the addition of quencher at 25 °C. Quenching studies were analyzed by the classical Stern–Volmer equations for collisional quenching (26), where F0 and F are the corrected emission intensities in the absence and presence of the quencher [Q], respectively. The quenching constants (Ksv) were obtained from the slopes of the Stern–Volmer plots of F0/F versus [Q]

  • image

Determination of the CMC

The CMCs of LPCs were measured using the fluorescent hydrophobic probe 1,8-ANS (27) with a Hitachi F2500 fluorescence spectrophotometer at room temperature (22–25 °C). The excitation and emission slits were both adjusted to 5.0 nm. Among the LPCs, LPC14:0 and LPC16:0 were used. Different concentrations of the LPCs (0–100 μm) in PBS (pH 7.4) were incubated with 1,8-ANS (10 μm) for 30 min at 37 °C. The fluorescence data were measured using excitation and emission wavelengths of 375 and 480 nm, respectively.

Native polyacrylamide gel electrophoresis (Native PAGE)

FITC-labeled BP21 (10 or 50 μm) was incubated with different concentrations (0–1 mm) of LPCs in PBS (pH 7.4) for 60 min at room temperature, and then, the reaction was terminated by the addition of native sample buffer (Bio-Rad Laboratories, Hercules, CA, USA) (28,29). The reaction mixture was subjected to 15% non-denaturing PAGE to separate LPC-bound and -free BP21. The gels were then visualized using the LAS-3000 luminescent image analyzer (Fujifilm, Tokyo, Japan). The images were analyzed using Image Gauge version 3.1 (Fujifilm).

SPR spectroscopy

Surface plasmon resonance measurements were taken with a Biacore 2000 instrument (GE Healthcare, Little Chalfont, UK) at 25 °C (30). BP21 was immobilized on the surface of a streptavidin-coated sensor chip (Sensor Chip SA; GE Healthcare) in PBS (pH 7.4), according to the manufacturer’s instructions. The mean amount of immobilized BP21 was approximately 800 resonance units (RU). LPC16:0 dissolved in PBS (pH 7.4) at various concentrations was passed over the chip surface at a flow rate of 10 μL/min. The results were analyzed by biaevaluation version 4.1 software (GE Healthcare). The corresponding association and dissociation parts of the sensorgrams were fitted to a Langmuir binding model, and the equilibrium dissociation constant, Kd, was calculated as the ratio of dissociation to association rate constants.

Results

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References

Analysis of interaction between synthetic peptides and phospholipids using the intrinsic tryptophan fluorescence

Because changes in the intrinsic tryptophan fluorescence of peptides and proteins often occur upon ligand binding or conformational changes, it is possible to use changes in tryptophan fluorescence intensity as a direct measure of ligand binding (23,24). To assess the interaction between P21 and LPC, intrinsic tryptophan fluorescence experiments were performed.

First, to examine the specificity of the interaction of BP21 with phospholipids, including LPC, the tryptophan fluorescence intensity of BP21 in the presence of different phospholipids (10 μm) was measured. As shown in Figure 1A, BP21 specifically interacted with PA, PE, and all lysophospholipids (LPC, LPA, LPS, LPE, and LPI), whereas BP21 did not interact with PCs, PS, and sphingomyelin. These results indicate that BP21 specifically interacts with phospholipids that form micellar structures (lysophospholipids) and hexagonal structures (PA and PE), but not with phospholipids that form lamellar structures (PCs, PS, and sphingomyelin).

image

Figure 1.  Effects of various phospholipids on the tryptophan fluorescence of P21. Each phospholipid in PBS (pH 7.4) was incubated in the absence or presence of each modified P21 (0.4 μm) for 30 min for 37 °C, and changes in tryptophan fluorescence intensity were measured at an excitation wavelength of 295 nm and an emission wavelength of 348 nm. Each figure shows the change in tryptophan fluorescence intensity of BP21 on the addition of (A) various phospholipids (10 μm), (B) lysophosphatidylcholine (LPC)16:0 or PC16:0/18:0 (0–10 μm), and (E) various LPCs (LPC12:0, -14:0, -16:0, and -18:0, each 0–100 μm) on the tryptophan fluorescence of BP21. Figure (C) shows the change in tryptophan fluorescence intensity of BP21, and BGP21 modified YKDG sequence to GGGG sequence, on the addition of LPC16:0 (0–10 μm). (D) The change in tryptophan fluorescence of BP21, P21, and P21-0.4 μm biotin mixture, on the addition of LPC16:0 (0–10 μm). Each value represents the change in fluorescence compared to the lipid-free sample and is presented as the mean ± SD (n = 3).

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To investigate differences in the interactions between BP21 and various PC molecular species (PCs or LPCs), we measured changes in the tryptophan fluorescence intensity of BP21 containing two tryptophan residues, in the presence of LPC16:0 or PC16:0/18:0 (each 0–10 μm) at the excitation and emission wavelengths of 295 and 348 nm, respectively. As shown in Figure 1B, the fluorescence intensity of BP21 markedly increased in the presence of LPCs, whereas it was unaffected in the presence of PCs.

To clarify the necessity of the YKDG sequence in BP21 for the interaction with LPC, we investigated differences in the interactions of BP21 and BGP21 with LPC16:0 (0–10 μm). As shown in Figure 1C, the change in intensity of BGP21 upon interaction with LPC16:0 was lower than that of BP21, indicating that the YKDG sequence in BP21 is necessary for BP21–LPC interaction.

To clarify the necessity of biotin bound to P21 for the interaction with LPC, we investigated differences in the interactions of BP21, P21, and P21-biotin mixture with LPC16:0 (0–10 μm). As shown in Figure 1D, BP21 interacted more strongly with LPC than P21 and P21-biotin mixture, indicating that biotin bound to N-terminus of P21 is necessary for the BP21–LPC interaction.

To determine whether BP21 interacts with particular forms of LPC, the change in the tryptophan fluorescence intensity of BP21 in the presence of different concentrations (0–100 μm) of LPCs (LPC12:0, -14:0, -16:0, and -18:0) was measured. As shown in Figure 1E, the fluorescence intensity of BP21 increased in the presence of each LPC in a concentration-dependent manner, indicating that the fluorescence intensity of BP21 strongly correlated with the length of the acyl chain at the sn-1 position of LPCs (LPC12:0 < -14:0 < -16:0 < -18:0).

The interaction of BP21 with LPCs was investigated using the wavelength of emission maxima in the absence and presence of LPCs at different concentrations. When BP21 interacted with LPCs (LPC14:0 and LPC16:0), the fluorescence emission intensity of the tryptophan residues in BP21 increased (Figure 2). There was no shift in the wavelength of the emission maxima of bound BP21 at lower LPC concentrations, below approximately 10 μm LPC16:0 and 100 μm LPC14:0, compared with the maximum emission peak of free BP21 (λmax = approximately 351 nm). On the other hand, at higher LPC concentrations, the emission maxima of the fluorescence emission spectra were significantly blue-shifted by 5–8 nm (λmax = 343–346 nm for LPC14:0 at 1 mm and for LPC 16:0 at 100 μm–1 mm, respectively), indicating that tryptophan residues in BP21 may interact with and then be buried in a hydrophobic region of the LPCs.

image

Figure 2.  Fluorescence emission spectra of tryptophan fluorescence of BP21 in the absence or presence of lysophosphatidylcholines (LPCs). BP21 (0.4 μm) in PBS (pH 7.4) was incubated in the absence or presence of LPC14:0 (A) or LPC16:0 (B) (1 μm–1 mm) for 30 min at 37 °C. Tryptophan excitation wavelength was set to 295 nm, and emission was monitored from 320 to 380 nm. All spectra data shown represent the change in fluorescence compared to the lipid-free sample.

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Determination of the CMCs of LPCs in PBS

The CMCs of LPCs in PBS were determined by a fluorescence method using 1,8-ANS (27). As shown in Figure 3, the fluorescence intensity began to increase remarkably as the concentrations of LPC14:0 and LPC16:0 increased beyond approximately 35.4 and 4.2 μm, respectively, indicating that the CMCs of LPC14:0 and LPC16:0 in PBS were 35.4 and 4.2 μm, respectively. In contrast, BP21 did not affect the increase in the 1,8-ANS fluorescence intensity induced by LPCs (data not shown).

image

Figure 3.  Determination of critical micelle concentrations (CMCs) of lysophosphatidylcholines (LPCs) by changes in the fluorescence intensity of the fluorescent hydrophobic probe 1,8-ANS. Different concentrations of LPCs (0–100 μm) in PBS (pH 7.4) were incubated with 1,8-ANS (10 μm) for 30 min at 37 °C. The fluorescence data were measured using excitation and emission wavelengths of 375 and 480 nm, respectively. The data shown are calculated as the fluorescence intensity changes compared to LPC alone.

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Acrylamide and iodide quenching of LPC-free and LPC-bound BP21

Quenching of tryptophan fluorescence in proteins/peptides has been critical to understanding protein dynamics and enzyme reactions by using tryptophan as a molecular optical probe. External added quenchers such as acrylamide (25) and iodide (26) can quench tryptophan fluorescence and then distinguish the location of tryptophan residues. The quenching of intrinsic fluorescence can also be used to follow the conformational changes of proteins/peptides. The surface accessibility of tryptophan residues can be assessed by looking at the quenching constant, Ksv. Fluorescence quenching by acrylamide and iodide was used to monitor the tryptophan environment of BP21 in the absence or presence of LPC. Stern–Volmer plots of acrylamide and iodide quenching are shown in Figure 4 for BP21 in buffer and in the presence of different concentrations of LPC16:0. The Ksv values, calculated from the slopes shown in Figure 4, are summarized in Table 1. Although a linear increase in F0/F of BP21 was observed in all cases, the Ksv values obtained for BP21 in the absence of LPC were lowest (Ksv = 1.38 m−1 for acrylamide and 0.90 m−1 for iodide), indicating that tryptophan residues in BP21 may be buried deeply in the molecule or may interact with other amino acid residues and cannot be accessed by either acrylamide or iodide. For acrylamide quenching (Figure 4A) in the presence of LPC16:0, a linear increase in F0/F of BP21 was observed in all cases and the Ksv values were about the same. On the other hand, for iodide quenching (Figure 4B), the Ksv values decreased in a LPC concentration-dependent manner, suggesting that the two tryptophan residues in BP21 are mostly exposed on the outside of the peptide in the presence of LPC and may be buried deeply in LPC micelles.

image

Figure 4.  Stern–Volmer plots for tryptophan fluorescence quenching of BP21 in the absence or presence of lysophosphatidylcholine (LPC)16:0. (A) Acrylamide and (B) iodide quenching experiments were carried out on 0.4 μm BP21 in PBS (pH 7.4) in the absence or presence of different concentrations of LPC16:0 (1 μm–1 mm), by the addition of a quencher. Tryptophan excitation wavelength was set to 295 nm, and F0 and F are the corrected fluorescence emission intensities at 348 nm in the absence or presence of a quencher, respectively.

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Table 1.   Stern–Volmer constant Ksv for the fluorescence emission-quenching tryptophan residues in BP21 on the addition of LPC16:0
LPC16:0Stern–Volmer constant Ksv (M−1)
AcrylamidePotassium Iodide (KI)
  1. LPC, lysophosphatidylcholine.

1.380.90
+1 μm3.343.21
+10 μm3.103.06
+100 μm3.482.67
+1 mm2.751.78

Analysis of direct binding between BP21 and LPCs

To analyze the direct binding between BP21 and LPC, we used another approach based upon the principle that tight binding to LPC could induce changes in the electrophoretic mobility of a peptide (gel shifts) under non-denaturing conditions (28,29). Interaction of BP21 with LPCs (LPC12:0, -14:0, -16:0, and -18:0) was evaluated by native PAGE after incubating FITC-labeled BP21 (50 μm) with various LPCs (1 mm to ensure stable micellar structures) for 60 min at room temperature. Figure 5A shows that the incubation of FITC-labeled BP21 with LPC micelles results in the formation of BP21-LPC micelle complexes, and that the interaction of BP21 with LPCs is strongly influenced by the length of the acyl chain at the sn-1 position of LPCs (LPC12:0 < -14:0 < -16:0 < -18:0). Figure 5B shows that FITC-labeled BP21 (10 μm) binds to both 100 μm and 1 mm of LPC16:0, indicating that BP21 strongly binds to LPC micelles because the CMC of LPC16:0 was calculated to be approximately 4.2 μm by a fluorescence method using 1,8-ANS (Figure 3).

image

Figure 5.  Analysis of binding between BP21 and lysophosphatidylcholines (LPCs) by native polyacrylamide gel electrophoresis (PAGE). Fluorescein isothiocyanate (FITC)-labeled BP21 (10 or 50 μm) in PBS (pH 7.4) was incubated in the absence or presence of (A) 1 mm LPCs (LPC12:0, -14:0, -16:0, and -18:0) or (B) different concentrations (1 μm–1 mm) of LPC16:0 for 60 min at room temperature. The reaction mixture was subjected to 15% non-denaturing PAGE for separating the free and LPC-bound BP21. The gels were then visualized using Fujifilm LAS-3000 luminescent image analyzer.

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To evaluate direct binding between BP21 and LPCs, we also utilized SPR. The Biacore instrument analyzes interactions in real time. Figure 6 shows a sensorgram of LPC16:0 at different concentrations (10 μm and 100 μm) binding to BP21 immobilized on the surface of a strepteavidin-coated sensor chip. LPC16:0 bound to BP21 at 100 μm, but not at 10 μm. We calculated the dissociation constant, Kd, at 100 μm of LPC16:0. Using Biacore software (biaevaluation version 4.1) and a Langmuir 1:1 (mol/mol) binding model, which describes a simple reversible interaction between BP21 and LPC16:0, we calculated the association rate constant (ka) to be 673/m/seconds and the dissociation rate constant (kd) to be 0.00213/seconds. The Kd obtained from these values was estimated to be approximately 3.1 μm.

image

Figure 6.  Surface plasmon resonance (SPR) measurement of the interaction between BP21 and lysophosphatidylcholine (LPC)16:0. Binding of LPC16:0 to the BP21 immobilized on the surface of Biacore sensor chip SA by biotin–streptavidin binding was shown as the sensorgram.

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Discussion

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References

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.

Conclusions and Future Directions

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References

In the present study, we have shown that an Asp-hemolysin-related synthetic biotinylated peptide, BP21, specifically recognizes LPC micelles, 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. Further study of the interaction between BP21 and LPC micelles may provide important information for the prevention and treatment for many inflammatory diseases caused by LPC micelles.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References

We thank Dr. Shota Endo (Department of Experimental Immunology, Institute of Development, Aging and Cancer, Tohoku University) and Ms. Hiromi Yoshida (Common Instrument Center, Institute of Development, Aging and Cancer, Tohoku University) for SPR measurements.

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  3. Methods and Materials
  4. Results
  5. Discussion
  6. Conclusions and Future Directions
  7. Acknowledgments
  8. Conflict of interest
  9. References
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