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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

Effect of PE and cholesterol on the binding of PAB-TP to PA-containing vesicles.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/DOPE (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE (10:88:2) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).
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pone.0131668.g003: Effect of PE and cholesterol on the binding of PAB-TP to PA-containing vesicles.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/DOPE (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE (10:88:2) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).

Mentions: Since Kooijman et al. have shown that the ionization of PA is enhanced by coexistence of PE and cholesterol [7], we next analyzed the effect of PE and cholesterol on the binding of PAB-TP to PA. As shown in Fig 3A, the PAB-TP binding to PA in the PC membranes was significantly enhanced by the presence of PE. The coexistence of cholesterol also drastically increased the binding of PAB-TP to the PA-containing vesicles (Fig 3B). To examine the effect of PE and cholesterol on the electrostatic properties of the membranes, we measured the zeta potential of the PA-containing vesicles with various amounts of PE and cholesterol. A significant increase in the negative zeta potential was observed with the presence of PE in the PA-containing vesicles (Table 1), reflecting the formation of a hydrogen bond between PE and phosphomonoester of PA that causes further deprotonation and ionization of PA [7]. The negative zeta potential of the PA-containing vesicles was also remarkably increased in a dose-dependent manner by cholesterol (Table 1). Addition of cholesterol into the vesicles in the absence of PA did not significantly affect the zeta potential (-0.4±0.4 and -1.7±0.4 mV for vesicles composed of only DOPC and DOPC/cholesterol (50:50), respectively), indicating that the effect of cholesterol reflects the increased ionization of PA. Furthermore, the size of the PA-containing vesicle was not significantly affected by the presence of PE or cholesterol (Table 1). These results suggest that the presence of PE or cholesterol increases the negative surface charge of the PA-containing vesicles, causing the enhanced binding of PAB-TP to the membrane. Although PE and cholesterol caused the comparable increase in the negative zeta potential, cholesterol has a more striking effect on the binding of PAB-TP to the PA-containing vesicles. As proposed by Kooijman et al. it is likely that cholesterol has additional effects such as induction of negative curvature stress in the planar lipid bilayers that enhance the insertion of hydrophobic residues into the hydrophobic interior of lipid bilayer [7]. Taken together, these results suggest that PAB-TP can distinguish the cholesterol and PE-mediated changes in the ionization properties of PA molecules as well as the microenvironment of PA molecules such as curvature stress of the lipid bilayer.


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)

Effect of PE and cholesterol on the binding of PAB-TP to PA-containing vesicles.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/DOPE (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE (10:88:2) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131668.g003: Effect of PE and cholesterol on the binding of PAB-TP to PA-containing vesicles.(A) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/DOPE (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (B) Binding of PAB-TP to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:88:2:0), (10:78:2:10), (10:58:2:30), and (10:38:2:50) was examined by the SPVB assay (mean ± SE, n = 3). (A, B) The binding of LUVs composed of DOPA/DOPC/biotin-DOPE (10:88:2) at the concentration of 125 μM to PAB-TP was represented as 100 (arbitrary units).
Mentions: Since Kooijman et al. have shown that the ionization of PA is enhanced by coexistence of PE and cholesterol [7], we next analyzed the effect of PE and cholesterol on the binding of PAB-TP to PA. As shown in Fig 3A, the PAB-TP binding to PA in the PC membranes was significantly enhanced by the presence of PE. The coexistence of cholesterol also drastically increased the binding of PAB-TP to the PA-containing vesicles (Fig 3B). To examine the effect of PE and cholesterol on the electrostatic properties of the membranes, we measured the zeta potential of the PA-containing vesicles with various amounts of PE and cholesterol. A significant increase in the negative zeta potential was observed with the presence of PE in the PA-containing vesicles (Table 1), reflecting the formation of a hydrogen bond between PE and phosphomonoester of PA that causes further deprotonation and ionization of PA [7]. The negative zeta potential of the PA-containing vesicles was also remarkably increased in a dose-dependent manner by cholesterol (Table 1). Addition of cholesterol into the vesicles in the absence of PA did not significantly affect the zeta potential (-0.4±0.4 and -1.7±0.4 mV for vesicles composed of only DOPC and DOPC/cholesterol (50:50), respectively), indicating that the effect of cholesterol reflects the increased ionization of PA. Furthermore, the size of the PA-containing vesicle was not significantly affected by the presence of PE or cholesterol (Table 1). These results suggest that the presence of PE or cholesterol increases the negative surface charge of the PA-containing vesicles, causing the enhanced binding of PAB-TP to the membrane. Although PE and cholesterol caused the comparable increase in the negative zeta potential, cholesterol has a more striking effect on the binding of PAB-TP to the PA-containing vesicles. As proposed by Kooijman et al. it is likely that cholesterol has additional effects such as induction of negative curvature stress in the planar lipid bilayers that enhance the insertion of hydrophobic residues into the hydrophobic interior of lipid bilayer [7]. Taken together, these results suggest that PAB-TP can distinguish the cholesterol and PE-mediated changes in the ionization properties of PA molecules as well as the microenvironment of PA molecules such as curvature stress of the lipid bilayer.

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