Chem Biol Drug Des 2014; 84: 443–449 Research Letter
Novel Fluorescently Labeled Peptide Compounds for Detection of Oxidized Low-Density Lipoprotein at High Specificity Akira Sato*, Hikaru Yamanaka, Keitaro Oe, Yoji Yamazaki and Keiichi Ebina Faculty of Pharmacy, Iwaki Meisei University, 5-5-1, Chuodai-Iino, Iwaki, Fukushima 970-8551, Japan *Corresponding author: Akira Sato, [email protected] The probes for specific detection of oxidized lowdensity lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the e-amino group of K at the N-terminus to FITC in the presence/ absence of 6-amino-n-caproic acid (AC) linker to FITC —(FITC-AC)KP6 and (FITC)KP6—both bound with high specificity to ox-LDL in a dose-dependent manner. In contrast, a tetrapeptide (YKDG) labeled with FITC at the N-terminus and a pentapeptide (YKDGK) coupled through the e-amino group of K at the C-terminus to FITC did not bind selectively to ox-LDL. Furthermore, (FITC)KP6 and (FITC-AC)KP6 bound with high specificity to the protein in mouse plasma (probably ox-LDL fraction). These findings strongly suggest that (FITC) KP6 and (FITC-AC)KP6 may be effective novel fluorescent probes for specific detection of ox-LDL. Key words: Asp-hemolysin, atherosclerosis, fluorescent probe, oxidized LDL, synthetic peptide Received 15 January 2014, revised 17 March 2014 and accepted for publication 3 April 2014
capable of functioning as a macrophage scavenger receptor ligand, and is also involved in macrophage proliferation. Many studies have reported that ox-LDL is abundantly present in atherosclerotic plaques and is associated with plaque instability (7–9). Moreover, several clinical studies have demonstrated that elevated ox-LDL levels were present in the plasma of patients with cardiovascular diseases. Therefore, the probes for highly specific detection of oxLDL in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. We have recently reported that Asp-hemolysin, a hemolytic protein from Aspergillus fumigatus, binds to ox-LDL with high affinity (dissociation constant, Kd = 0.63 lg/mL) (10– 13) and that its binding specificity is distinct from that of any ox-LDL receptor (14). Moreover, Asp-hemolysin-related synthetic peptides (P4-P29) comprise of 4–29 amino acid residues, all of which contain a YKDG sequence that binds to ox-LDL with high specificity (15,16). A YKDG sequence in these peptides is essential for binding to ox-LDL, especially its major lipid component lysophosphatidylcholine [1-acyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC)] (17– 19). In this study, to develop a fluorescent probe for the specific detection of ox-LDL, we designed various types of fluorescently-labeled peptides, and the binding analysis of these peptides to ox-LDL was performed. The results indicated that two heptapeptides (KWYKDGD and KP6) coupled through the e-amino group of N-terminal Lys (K), in the presence/absence of 6-amino-n-caproic acid (AC) linker, to fluorescein isothiocyanate (FITC)—(FITC)KP6 and (FITC-AC)KP6—are both useful as fluorescent probes for detecting ox-LDL.
Methods and Materials Atherosclerosis may be described as a chronic inflammatory process (1) characterized by increased cellular turnover concomitant with apoptotic and necrotic cell death within an atherosclerotic lesion (2,3). The oxidation of low-density lipoprotein (LDL) has been reported to play a primary role in the pathogenesis of atherosclerosis and is an essential step in chronic inflammation (4–6). Oxidized LDL (ox-LDL) is known to be a highly proinflammatory and proatherogenic factor found in abundance in atherosclerotic plaques, is ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12333
Materials Ultracentrifuge tubes (1PC tubes) were purchased from Hitachi Koki Co., Ltd (Tokyo, Japan). A VISKING dialysis tubing (MWCO 12000–14000) was purchased from Serva (Heidelberg, Germany). Bovine serum albumin (BSA, Fraction V RIA grade, A-7888) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethylenediamine-N,N, N0 ,N0 -tetraacetic acid (EDTA) disodium salt was purchased
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from Dojindo Laboratories, Inc (Kumamoto, Japan). 1-Palmitoyl-LPC was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), unless otherwise noted.
Synthetic peptides FITC-labeled peptides were all synthesized and purified (the purity more than 95% was assessed by high-performance liquid chromatography and mass spectroscopy) by GL Biochem Ltd. (Shanghai, China). The amino acid sequences and structures of FITC-labeled peptides used in this study are shown in Figure 1.
Preparation and oxidation of LDL Preparation and oxidation of LDL was performed as previously described (20). Blood was collected from normolipidemic volunteers in their 20s and 30s, and plasma was isolated after centrifuging the blood samples at 1450 9 g for 10 min in the presence of 1 mg/mL EDTA. LDL (d = 1.019–1.063 g/mL) was isolated by sequential ultracentrifugation (604 000 9 g; 16 °C) using a S120-AT2 rotor (CS100GXL ultracentrifuge; Hitachi Koki Co., Ltd), and it was dialyzed at 4 °C against 0.3 mM EDTA-containing phosphate-buffered saline (PBS: 150 mM sodium chloride and 10 mM phosphate; pH 7.4). and then at against EDTA-free PBS. The isolated LDL was stored at 4 °C. The protein content of LDL was assayed using the Lowry method with BSA as the standard (21). LDL was oxidized by incubation with 5 lM copper sulfate in EDTA-free PBS at 37 °C for different time intervals (0–12 h) at a protein concentration of 0.2 mg/mL. Oxidation was arrested by the addition of 300 lM EDTA and 25 lM butylated
hydroxytoluene. Prepared ox-LDL was stored at 4 °C to prevent further natural oxidation.
Thiobarbituric acid-reactive substances assay The extent of oxidation of LDL was assessed using the thiobarbituric acid-reactive substances (TBARS) assay kit (Cayman Chemical, Ann Arbor, MI, USA), using the method described by the manufacturer for the fluorometric measurement of malondialdehyde (MDA). Briefly, 100 lL of SDS solution was added to 100 lL of each LDL sample and standard curve solutions, followed by 4 mL of color reagent. The LDL samples and standard curve solutions were then boiled at 95 °C for 1 h, and the reaction was terminated by incubation on ice for 10 min followed by centrifugation at 1600 9 g for 10 min at 4 °C. After incubating for 30 min at room temperature, the fluorescence of the LDL samples and standard curve solutions at an excitation wavelength of 530 nm and an emission wavelength of 550 nm was determined using a Hitachi F2500 fluorescence spectrophotometer (Hitachi Koki Co., Ltd.) at 25 °C.
Animal experiments Animal care and experimental procedures used in this study were in accordance with the principles and guidelines of the Japanese Council on Animal Care, and they were also approved by the Animal Care and Use Committee for Iwaki Meisei University. Eight-week-old male ApoE-knockout (ApoE-KO) mice were obtained from Jackson Laboratory (West Grove, PA, USA). Eight-week-old male C57BL/6J wild-type mice (Charles River Laboratories Japan, Yokohama, Japan) were used as
Figure 1: Amino acid sequences and structures of fluorescein isothiocyanate (FITC)-labeled peptides derived from Asphemolysin. The sequences and structures of FITC-labeled peptides used are shown. Each amino acid is represented as a single letter. 6AC, 6-amino-n-caproic acid.
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Chem Biol Drug Des 2014; 84: 443–449
Fluorescent Compounds for Detection of Ox-LDL
the controls. The animals were housed individually under ordinary conditions with 12 h light/dark cycles. After a week of acclimation, the ApoE-KO mice were fed a highfat western diet, including 21% fat and 0.15% cholesterol (Oriental Yeast Co., Tokyo, Japan), while the C57BL/6J mice were fed a standard chow diet. All the animals were fed and provided water ad libitum. After 7 weeks, the mice were initially anesthetized with diethyl ether, and blood was collected and centrifuged at 1450 9 g for 10 min in the presence of 1 mg/mL EDTA for plasma isolation.
Native polyacrylamide gel electrophoresis FITC-labeled peptides were dissolved in sterilized saline containing 5% dimethyl sulfoxide (DMSO). Each peptide (10 lg) was incubated with ox-LDL (0–3.2 lg) and 1-palmitoyl-LPC (100 lM) in PBS (pH 7.4) for 90 min at 37 °C, or with plasma from the ApoE-KO mice and control mice (16 lL) for 5 min at room temperature. The reactions were terminated by the addition of native sample buffer (BioRad Laboratories, Hercules, CA, USA). The reaction mixture was subjected to 4% non-denaturing polyacrylamide gel electrophoresis (PAGE) to separate ox-LDL-bound and -free peptide. The gels were then visualized using an LAS3000 luminescent image analyzer (FujiFilm, Tokyo, Japan), and the images were analyzed using Image Gauge version 3.1 (FujiFilm).
Statistical analysis All results are expressed as mean SD (standard deviation). The data were analyzed statistically by means of a paired Student’s t-test. The difference was considered statistically significant when the p value was
Novel Fluorescently Labeled Peptide Compounds for Detection of Oxidized Low-Density Lipoprotein at High Specificity Akira Sato*, Hikaru Yamanaka, Keitaro Oe, Yoji Yamazaki and Keiichi Ebina Faculty of Pharmacy, Iwaki Meisei University, 5-5-1, Chuodai-Iino, Iwaki, Fukushima 970-8551, Japan *Corresponding author: Akira Sato, [email protected] The probes for specific detection of oxidized lowdensity lipoprotein (ox-LDL) in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. In this study, to develop a fluorescent peptide probe for specific detection of ox-LDL, we investigated the interaction of fluorescein isothiocyanate (FITC)-labeled peptides with ox-LDL using polyacrylamide gel electrophoresis. Two heptapeptides (KWYKDGD and KP6) coupled through the e-amino group of K at the N-terminus to FITC in the presence/ absence of 6-amino-n-caproic acid (AC) linker to FITC —(FITC-AC)KP6 and (FITC)KP6—both bound with high specificity to ox-LDL in a dose-dependent manner. In contrast, a tetrapeptide (YKDG) labeled with FITC at the N-terminus and a pentapeptide (YKDGK) coupled through the e-amino group of K at the C-terminus to FITC did not bind selectively to ox-LDL. Furthermore, (FITC)KP6 and (FITC-AC)KP6 bound with high specificity to the protein in mouse plasma (probably ox-LDL fraction). These findings strongly suggest that (FITC) KP6 and (FITC-AC)KP6 may be effective novel fluorescent probes for specific detection of ox-LDL. Key words: Asp-hemolysin, atherosclerosis, fluorescent probe, oxidized LDL, synthetic peptide Received 15 January 2014, revised 17 March 2014 and accepted for publication 3 April 2014
capable of functioning as a macrophage scavenger receptor ligand, and is also involved in macrophage proliferation. Many studies have reported that ox-LDL is abundantly present in atherosclerotic plaques and is associated with plaque instability (7–9). Moreover, several clinical studies have demonstrated that elevated ox-LDL levels were present in the plasma of patients with cardiovascular diseases. Therefore, the probes for highly specific detection of oxLDL in plasma and in atherosclerotic plaques are expected to be useful for the identification, diagnosis, prevention, and treatment for atherosclerosis. We have recently reported that Asp-hemolysin, a hemolytic protein from Aspergillus fumigatus, binds to ox-LDL with high affinity (dissociation constant, Kd = 0.63 lg/mL) (10– 13) and that its binding specificity is distinct from that of any ox-LDL receptor (14). Moreover, Asp-hemolysin-related synthetic peptides (P4-P29) comprise of 4–29 amino acid residues, all of which contain a YKDG sequence that binds to ox-LDL with high specificity (15,16). A YKDG sequence in these peptides is essential for binding to ox-LDL, especially its major lipid component lysophosphatidylcholine [1-acyl-2-hydroxy-sn-glycero-3-phosphocholine (LPC)] (17– 19). In this study, to develop a fluorescent probe for the specific detection of ox-LDL, we designed various types of fluorescently-labeled peptides, and the binding analysis of these peptides to ox-LDL was performed. The results indicated that two heptapeptides (KWYKDGD and KP6) coupled through the e-amino group of N-terminal Lys (K), in the presence/absence of 6-amino-n-caproic acid (AC) linker, to fluorescein isothiocyanate (FITC)—(FITC)KP6 and (FITC-AC)KP6—are both useful as fluorescent probes for detecting ox-LDL.
Methods and Materials Atherosclerosis may be described as a chronic inflammatory process (1) characterized by increased cellular turnover concomitant with apoptotic and necrotic cell death within an atherosclerotic lesion (2,3). The oxidation of low-density lipoprotein (LDL) has been reported to play a primary role in the pathogenesis of atherosclerosis and is an essential step in chronic inflammation (4–6). Oxidized LDL (ox-LDL) is known to be a highly proinflammatory and proatherogenic factor found in abundance in atherosclerotic plaques, is ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12333
Materials Ultracentrifuge tubes (1PC tubes) were purchased from Hitachi Koki Co., Ltd (Tokyo, Japan). A VISKING dialysis tubing (MWCO 12000–14000) was purchased from Serva (Heidelberg, Germany). Bovine serum albumin (BSA, Fraction V RIA grade, A-7888) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethylenediamine-N,N, N0 ,N0 -tetraacetic acid (EDTA) disodium salt was purchased
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Sato et al.
from Dojindo Laboratories, Inc (Kumamoto, Japan). 1-Palmitoyl-LPC was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). The other reagents were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan), unless otherwise noted.
Synthetic peptides FITC-labeled peptides were all synthesized and purified (the purity more than 95% was assessed by high-performance liquid chromatography and mass spectroscopy) by GL Biochem Ltd. (Shanghai, China). The amino acid sequences and structures of FITC-labeled peptides used in this study are shown in Figure 1.
Preparation and oxidation of LDL Preparation and oxidation of LDL was performed as previously described (20). Blood was collected from normolipidemic volunteers in their 20s and 30s, and plasma was isolated after centrifuging the blood samples at 1450 9 g for 10 min in the presence of 1 mg/mL EDTA. LDL (d = 1.019–1.063 g/mL) was isolated by sequential ultracentrifugation (604 000 9 g; 16 °C) using a S120-AT2 rotor (CS100GXL ultracentrifuge; Hitachi Koki Co., Ltd), and it was dialyzed at 4 °C against 0.3 mM EDTA-containing phosphate-buffered saline (PBS: 150 mM sodium chloride and 10 mM phosphate; pH 7.4). and then at against EDTA-free PBS. The isolated LDL was stored at 4 °C. The protein content of LDL was assayed using the Lowry method with BSA as the standard (21). LDL was oxidized by incubation with 5 lM copper sulfate in EDTA-free PBS at 37 °C for different time intervals (0–12 h) at a protein concentration of 0.2 mg/mL. Oxidation was arrested by the addition of 300 lM EDTA and 25 lM butylated
hydroxytoluene. Prepared ox-LDL was stored at 4 °C to prevent further natural oxidation.
Thiobarbituric acid-reactive substances assay The extent of oxidation of LDL was assessed using the thiobarbituric acid-reactive substances (TBARS) assay kit (Cayman Chemical, Ann Arbor, MI, USA), using the method described by the manufacturer for the fluorometric measurement of malondialdehyde (MDA). Briefly, 100 lL of SDS solution was added to 100 lL of each LDL sample and standard curve solutions, followed by 4 mL of color reagent. The LDL samples and standard curve solutions were then boiled at 95 °C for 1 h, and the reaction was terminated by incubation on ice for 10 min followed by centrifugation at 1600 9 g for 10 min at 4 °C. After incubating for 30 min at room temperature, the fluorescence of the LDL samples and standard curve solutions at an excitation wavelength of 530 nm and an emission wavelength of 550 nm was determined using a Hitachi F2500 fluorescence spectrophotometer (Hitachi Koki Co., Ltd.) at 25 °C.
Animal experiments Animal care and experimental procedures used in this study were in accordance with the principles and guidelines of the Japanese Council on Animal Care, and they were also approved by the Animal Care and Use Committee for Iwaki Meisei University. Eight-week-old male ApoE-knockout (ApoE-KO) mice were obtained from Jackson Laboratory (West Grove, PA, USA). Eight-week-old male C57BL/6J wild-type mice (Charles River Laboratories Japan, Yokohama, Japan) were used as
Figure 1: Amino acid sequences and structures of fluorescein isothiocyanate (FITC)-labeled peptides derived from Asphemolysin. The sequences and structures of FITC-labeled peptides used are shown. Each amino acid is represented as a single letter. 6AC, 6-amino-n-caproic acid.
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Chem Biol Drug Des 2014; 84: 443–449
Fluorescent Compounds for Detection of Ox-LDL
the controls. The animals were housed individually under ordinary conditions with 12 h light/dark cycles. After a week of acclimation, the ApoE-KO mice were fed a highfat western diet, including 21% fat and 0.15% cholesterol (Oriental Yeast Co., Tokyo, Japan), while the C57BL/6J mice were fed a standard chow diet. All the animals were fed and provided water ad libitum. After 7 weeks, the mice were initially anesthetized with diethyl ether, and blood was collected and centrifuged at 1450 9 g for 10 min in the presence of 1 mg/mL EDTA for plasma isolation.
Native polyacrylamide gel electrophoresis FITC-labeled peptides were dissolved in sterilized saline containing 5% dimethyl sulfoxide (DMSO). Each peptide (10 lg) was incubated with ox-LDL (0–3.2 lg) and 1-palmitoyl-LPC (100 lM) in PBS (pH 7.4) for 90 min at 37 °C, or with plasma from the ApoE-KO mice and control mice (16 lL) for 5 min at room temperature. The reactions were terminated by the addition of native sample buffer (BioRad Laboratories, Hercules, CA, USA). The reaction mixture was subjected to 4% non-denaturing polyacrylamide gel electrophoresis (PAGE) to separate ox-LDL-bound and -free peptide. The gels were then visualized using an LAS3000 luminescent image analyzer (FujiFilm, Tokyo, Japan), and the images were analyzed using Image Gauge version 3.1 (FujiFilm).
Statistical analysis All results are expressed as mean SD (standard deviation). The data were analyzed statistically by means of a paired Student’s t-test. The difference was considered statistically significant when the p value was
