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Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

Ogawa R, Nagao K, Taniuchi K, Tsuchiya M, Kato U, Hara Y, Inaba T, Kobayashi T, Sasaki Y, Akiyoshi K, Watanabe-Takahashi M, Nishikawa K, Umeda M - PLoS ONE (2015)

Bottom Line: Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA.Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane.The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

View Article: PubMed Central - PubMed

Affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.

ABSTRACT
We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

No MeSH data available.


Related in: MedlinePlus

Analysis of PAB-TP by fluorescence measurement of tryptophan residues.(A) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPA/DOPC/cholesterol (10:60:30), (5:65:30), (2:68:30), (1:69:30), and (0:70:30) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and the wavelength of maximum fluorescence (λmax) was plotted against PA content (mol%). (mean ± SE, n = 3). (B) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPC, cholesterol, and indicated lipids (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was calculated. (mean ± SE, n = 3). (C) PAB-TP was incubated with LUVs (0–160 μM total lipids) composed of DOPC, cholesterol, and indicated phospholipid (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was plotted against the molar ratio of lipid to peptide. (mean ± SE, n = 3).
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pone.0131668.g007: Analysis of PAB-TP by fluorescence measurement of tryptophan residues.(A) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPA/DOPC/cholesterol (10:60:30), (5:65:30), (2:68:30), (1:69:30), and (0:70:30) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and the wavelength of maximum fluorescence (λmax) was plotted against PA content (mol%). (mean ± SE, n = 3). (B) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPC, cholesterol, and indicated lipids (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was calculated. (mean ± SE, n = 3). (C) PAB-TP was incubated with LUVs (0–160 μM total lipids) composed of DOPC, cholesterol, and indicated phospholipid (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was plotted against the molar ratio of lipid to peptide. (mean ± SE, n = 3).

Mentions: Fluorescence of Trp is highly sensitive to the local environment; in particular, the emission maximum (λmax) of a Trp residue is dependent on its interaction with the membrane bilayer [33, 36]. When PAB-TP was incubated with LUVs composed of DOPA, DOPC, and cholesterol at the molar ratio of 10:60:30, an extensive blue shift in the Trp emission spectra of PAB-TP was observed, while no significant shift was observed upon incubation with the vesicles without PA (Fig 6A). A progressive shift of λmax to shorter wavelength was observed in a dose-dependent manner of the PA-containing vesicles, while no significant shift was seen even with the highest concentration of the vesicles without PA (Fig 6B). Together with the results obtained by the inhibition analysis of SPVB assay (Fig 4A) and the amino acid replacement analysis (Fig 5A), these results indicate that the interaction with the hydrophobic interior of the lipid bilayer is responsible for the stable association of PAB-TP to the membrane. We next reevaluated the interaction between the soluble form of PAB-TP and PA-containing vesicles by measuring the shift in the Trp emission spectra. As shown in Fig 7A, the shift of λmax was highly dependent on the contents of PA in the membrane, which was consistent with the results obtained with the SPVB assay (Fig 2A). Analysis of the phospholipid specificity of PAB-TP in the fluorescence shift assay demonstrated that the peptide showed a weak cross-reaction with PS, ceramide-1-phosphate (C1P), PG, and GM3, but a significant cross-reactivity with PI(4,5)P2 was observed (Fig 7B). Quantitative analysis showed that PAB-TP cross-reacted with PI(5)P, PI(4)P and PI(4,5)P2 to the same extent as with PA, suggesting that the phosphomonoester head group of phospholipids is required for the high affinity interaction with PAB-TP (Fig 7C). It is noteworthy that PAB-TP exhibited differential reactivity to the phosphomonoester-containing phospholipids, i.e. PA and C1P, implying that the structural or physicochemical properties of C1P might affect the interaction with PAB-TP [37].


Development of a Novel Tetravalent Synthetic Peptide That Binds to Phosphatidic Acid.

Ogawa R, Nagao K, Taniuchi K, Tsuchiya M, Kato U, Hara Y, Inaba T, Kobayashi T, Sasaki Y, Akiyoshi K, Watanabe-Takahashi M, Nishikawa K, Umeda M - PLoS ONE (2015)

Analysis of PAB-TP by fluorescence measurement of tryptophan residues.(A) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPA/DOPC/cholesterol (10:60:30), (5:65:30), (2:68:30), (1:69:30), and (0:70:30) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and the wavelength of maximum fluorescence (λmax) was plotted against PA content (mol%). (mean ± SE, n = 3). (B) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPC, cholesterol, and indicated lipids (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was calculated. (mean ± SE, n = 3). (C) PAB-TP was incubated with LUVs (0–160 μM total lipids) composed of DOPC, cholesterol, and indicated phospholipid (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was plotted against the molar ratio of lipid to peptide. (mean ± SE, n = 3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493020&req=5

pone.0131668.g007: Analysis of PAB-TP by fluorescence measurement of tryptophan residues.(A) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPA/DOPC/cholesterol (10:60:30), (5:65:30), (2:68:30), (1:69:30), and (0:70:30) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and the wavelength of maximum fluorescence (λmax) was plotted against PA content (mol%). (mean ± SE, n = 3). (B) PAB-TP was incubated with LUVs (160 μM total lipids) composed of DOPC, cholesterol, and indicated lipids (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was calculated. (mean ± SE, n = 3). (C) PAB-TP was incubated with LUVs (0–160 μM total lipids) composed of DOPC, cholesterol, and indicated phospholipid (60:30:10) for 1h at 25°C. Fluorescence spectra were obtained with the excitation wavelength at 280 nm and blue shift in the wavelength of maximum fluorescence (λmax) was plotted against the molar ratio of lipid to peptide. (mean ± SE, n = 3).
Mentions: Fluorescence of Trp is highly sensitive to the local environment; in particular, the emission maximum (λmax) of a Trp residue is dependent on its interaction with the membrane bilayer [33, 36]. When PAB-TP was incubated with LUVs composed of DOPA, DOPC, and cholesterol at the molar ratio of 10:60:30, an extensive blue shift in the Trp emission spectra of PAB-TP was observed, while no significant shift was observed upon incubation with the vesicles without PA (Fig 6A). A progressive shift of λmax to shorter wavelength was observed in a dose-dependent manner of the PA-containing vesicles, while no significant shift was seen even with the highest concentration of the vesicles without PA (Fig 6B). Together with the results obtained by the inhibition analysis of SPVB assay (Fig 4A) and the amino acid replacement analysis (Fig 5A), these results indicate that the interaction with the hydrophobic interior of the lipid bilayer is responsible for the stable association of PAB-TP to the membrane. We next reevaluated the interaction between the soluble form of PAB-TP and PA-containing vesicles by measuring the shift in the Trp emission spectra. As shown in Fig 7A, the shift of λmax was highly dependent on the contents of PA in the membrane, which was consistent with the results obtained with the SPVB assay (Fig 2A). Analysis of the phospholipid specificity of PAB-TP in the fluorescence shift assay demonstrated that the peptide showed a weak cross-reaction with PS, ceramide-1-phosphate (C1P), PG, and GM3, but a significant cross-reactivity with PI(4,5)P2 was observed (Fig 7B). Quantitative analysis showed that PAB-TP cross-reacted with PI(5)P, PI(4)P and PI(4,5)P2 to the same extent as with PA, suggesting that the phosphomonoester head group of phospholipids is required for the high affinity interaction with PAB-TP (Fig 7C). It is noteworthy that PAB-TP exhibited differential reactivity to the phosphomonoester-containing phospholipids, i.e. PA and C1P, implying that the structural or physicochemical properties of C1P might affect the interaction with PAB-TP [37].

Bottom Line: Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA.Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane.The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

View Article: PubMed Central - PubMed

Affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Kyoto, Japan.

ABSTRACT
We employed a multivalent peptide-library screening technique to identify a peptide motif that binds to phosphatidic acid (PA), but not to other phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS). A tetravalent peptide with the sequence motif of MARWHRHHH, designated as PAB-TP (phosphatidic acid-binding tetravalent peptide), was shown to bind as low as 1 mol% of PA in the bilayer membrane composed of PC and cholesterol. Kinetic analysis of the interaction between PAB-TP and the membranes containing 10 mol% of PA showed that PAB-TP associated with PA with a low dissociation constant of KD = 38 ± 5 nM. Coexistence of cholesterol or PE with PA in the membrane enhanced the PAB-TP binding to PA by increasing the ionization of the phosphomonoester head group as well as by changing the microenvironment of PA molecules in the membrane. Amino acid replacement analysis demonstrated that the tryptophan residue at position 4 of PAB-TP was involved in the interaction with PA. Furthermore, a series of amino acid substitutions at positions 5 to 9 of PAB-TP revealed the involvement of consecutive histidine and arginine residues in recognition of the phosphomonoester head group of PA. Our results demonstrate that the recognition of PA by PAB-TP is achieved by a combination of hydrophobic, electrostatic and hydrogen-bond interactions, and that the tetravalent structure of PAB-TP contributes to the high affinity binding to PA in the membrane. The novel PA-binding tetravalent peptide PAB-TP will provide insight into the molecular mechanism underlying the recognition of PA by PA-binding proteins that are involved in various cellular events.

No MeSH data available.


Related in: MedlinePlus