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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

Identification of PA-binding peptide motif using tetravalent peptide libraries.(A) The tetravalent peptide library was comprised of compounds with 4 randomized peptides of sequence M1-A2-X3-X4-X5-R6-X7-X8-X9-AU (U; amino hexanoic acid), where X at positions 3–5 and 7–9 indicates any amino acids except cysteine. Screening of the library was performed to identify compounds that bound to vesicles containing PA but not to vesicles without PA. Sequences of PA binding peptides (peptide 1 to 4) were determined by comparing relative abundance of amino acids in each degenerate position of PA-, PC-, and PS-bound peptides. (B) Binding of the tetravalent peptides to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay (mean ± SE, n = 3). The binding of LUVs at the concentration of 125 μM to peptide 3 was represented as 100 (arbitrary units).
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pone.0131668.g001: Identification of PA-binding peptide motif using tetravalent peptide libraries.(A) The tetravalent peptide library was comprised of compounds with 4 randomized peptides of sequence M1-A2-X3-X4-X5-R6-X7-X8-X9-AU (U; amino hexanoic acid), where X at positions 3–5 and 7–9 indicates any amino acids except cysteine. Screening of the library was performed to identify compounds that bound to vesicles containing PA but not to vesicles without PA. Sequences of PA binding peptides (peptide 1 to 4) were determined by comparing relative abundance of amino acids in each degenerate position of PA-, PC-, and PS-bound peptides. (B) Binding of the tetravalent peptides to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay (mean ± SE, n = 3). The binding of LUVs at the concentration of 125 μM to peptide 3 was represented as 100 (arbitrary units).

Mentions: Screening of the tetravalent peptide libraries was performed as described previously [29]. In brief, the four peptide-chains present in a given tetravalent peptide (Fig 1A) were elongated at the same time from the amino groups of the core polylysine. The Met-Ala sequence at the amino terminus of the library peptides was included to verify that peptides from this mixture were being sequenced and to qualify the peptides, and the Arg residue at position 6 was fixed in the library peptides. The predicted degeneracy of a randomize peptide with 6 degenerate positions is 196 (47 million). LUVs composed of either DOPA/DMPC/biotin-DOPE/cholesterol (10:38:2:50), DMPC/biotin-DOPE/cholesterol (48:2:50) or DOPS/DMPC/biotin-DOPE/cholesterol (10:38:2:50) were incubated with Streptavidin-Agarose beads from Sigma Aldrich (St. Louis, MO) and washed with PBS. Vesicles containing DMPC were used for screening to avoid disruption of the membrane integrity during incubation with the library peptides and washing procedures. The LUVs bound to the beads were incubated with the library peptides for 16 h at 4°C. After washing with PBS, peptides bound to the LUVs were separated by SDS-PAGE and sequenced on an Applied Biosystems model 477A protein sequencer. To calculate the relative amino acid preference at each degenerate position, the corrected quantities of amino acids were compared with those obtained using LUVs of different phospholipid compositions. The relative abundance of individual amino acids at the degenerate positions reflects the relative abundance of high-affinity peptides containing these amino acids [29].


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)

Identification of PA-binding peptide motif using tetravalent peptide libraries.(A) The tetravalent peptide library was comprised of compounds with 4 randomized peptides of sequence M1-A2-X3-X4-X5-R6-X7-X8-X9-AU (U; amino hexanoic acid), where X at positions 3–5 and 7–9 indicates any amino acids except cysteine. Screening of the library was performed to identify compounds that bound to vesicles containing PA but not to vesicles without PA. Sequences of PA binding peptides (peptide 1 to 4) were determined by comparing relative abundance of amino acids in each degenerate position of PA-, PC-, and PS-bound peptides. (B) Binding of the tetravalent peptides to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay (mean ± SE, n = 3). The binding of LUVs at the concentration of 125 μM to peptide 3 was represented as 100 (arbitrary units).
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Related In: Results  -  Collection

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pone.0131668.g001: Identification of PA-binding peptide motif using tetravalent peptide libraries.(A) The tetravalent peptide library was comprised of compounds with 4 randomized peptides of sequence M1-A2-X3-X4-X5-R6-X7-X8-X9-AU (U; amino hexanoic acid), where X at positions 3–5 and 7–9 indicates any amino acids except cysteine. Screening of the library was performed to identify compounds that bound to vesicles containing PA but not to vesicles without PA. Sequences of PA binding peptides (peptide 1 to 4) were determined by comparing relative abundance of amino acids in each degenerate position of PA-, PC-, and PS-bound peptides. (B) Binding of the tetravalent peptides to LUVs composed of DOPA/DOPC/biotin-DOPE/cholesterol (10:58:2:30) was examined by the SPVB assay (mean ± SE, n = 3). The binding of LUVs at the concentration of 125 μM to peptide 3 was represented as 100 (arbitrary units).
Mentions: Screening of the tetravalent peptide libraries was performed as described previously [29]. In brief, the four peptide-chains present in a given tetravalent peptide (Fig 1A) were elongated at the same time from the amino groups of the core polylysine. The Met-Ala sequence at the amino terminus of the library peptides was included to verify that peptides from this mixture were being sequenced and to qualify the peptides, and the Arg residue at position 6 was fixed in the library peptides. The predicted degeneracy of a randomize peptide with 6 degenerate positions is 196 (47 million). LUVs composed of either DOPA/DMPC/biotin-DOPE/cholesterol (10:38:2:50), DMPC/biotin-DOPE/cholesterol (48:2:50) or DOPS/DMPC/biotin-DOPE/cholesterol (10:38:2:50) were incubated with Streptavidin-Agarose beads from Sigma Aldrich (St. Louis, MO) and washed with PBS. Vesicles containing DMPC were used for screening to avoid disruption of the membrane integrity during incubation with the library peptides and washing procedures. The LUVs bound to the beads were incubated with the library peptides for 16 h at 4°C. After washing with PBS, peptides bound to the LUVs were separated by SDS-PAGE and sequenced on an Applied Biosystems model 477A protein sequencer. To calculate the relative amino acid preference at each degenerate position, the corrected quantities of amino acids were compared with those obtained using LUVs of different phospholipid compositions. The relative abundance of individual amino acids at the degenerate positions reflects the relative abundance of high-affinity peptides containing these amino acids [29].

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