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Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide.

Leali D, Bianchi R, Bugatti A, Nicoli S, Mitola S, Ragona L, Tomaselli S, Gallo G, Catello S, Rivieccio V, Zetta L, Presta M - J. Cell. Mol. Med. (2010)

Bottom Line: In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective.In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding.These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

View Article: PubMed Central - PubMed

Affiliation: Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Brescia, Italy.

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STD NMR analysis of Ac-ARPCA-NH2/FGF2 interaction. (A) STD spectra of Ac-ARPCA-NH2 peptide and its mutants acquired at 280 K on a 500 MHz Bruker spectrometer. (a) 1D NMR reference spectrum of 1.9 μM Ac-ARPCA-NH2 peptide alone; (b)–(e) STD spectra of Ac-ARPCA-NH2 and the three Ac-GRPCA-NH2, H-ARPCA-NH2 and Ac-ARPCG-NH2 peptides recorded in the presence of 50 μM FGF2 in 30 μM buffer phosphate (95% D2O, 5% H2O), 8 μM DTT, 40 μM NaCl, pH 6.8. A saturation time of 3 sec. was used. The assignment of the STD signals is reported. (B) Plot of STD factors (ASDT) of the Ac-ARPCA-NH2/FGF2 system versus saturation time. A 1:40 FGF2:peptide ratio was used. T1 relaxation time (sec) of each signal is shown on the right side of the curves. (C) ASTD× (ligand excess) versus different concentrations of Ac-ARPCA-NH2 peptide.
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fig04: STD NMR analysis of Ac-ARPCA-NH2/FGF2 interaction. (A) STD spectra of Ac-ARPCA-NH2 peptide and its mutants acquired at 280 K on a 500 MHz Bruker spectrometer. (a) 1D NMR reference spectrum of 1.9 μM Ac-ARPCA-NH2 peptide alone; (b)–(e) STD spectra of Ac-ARPCA-NH2 and the three Ac-GRPCA-NH2, H-ARPCA-NH2 and Ac-ARPCG-NH2 peptides recorded in the presence of 50 μM FGF2 in 30 μM buffer phosphate (95% D2O, 5% H2O), 8 μM DTT, 40 μM NaCl, pH 6.8. A saturation time of 3 sec. was used. The assignment of the STD signals is reported. (B) Plot of STD factors (ASDT) of the Ac-ARPCA-NH2/FGF2 system versus saturation time. A 1:40 FGF2:peptide ratio was used. T1 relaxation time (sec) of each signal is shown on the right side of the curves. (C) ASTD× (ligand excess) versus different concentrations of Ac-ARPCA-NH2 peptide.

Mentions: In order to map the peptide residues making direct contacts with FGF2, STD NMR methods were applied [46]. STD NMR experiments were performed in the presence of DDT in order to avoid the formation of disulphide bridges between free cysteines. The STD spectrum of ARPCA peptide in the presence of FGF2 proves that methyl protons of Ala1, Ala5 and of the N-terminal blocking acetyl group receive saturation transfer, giving rise to STD NMR signals (Fig. 4A). The hydrophobic patch defined by the three methyl groups is therefore involved in binding to the protein surface. In an attempt to compare the relative contribution of each methyl group to the contact area, a build-up curve of the saturation degree (STD factor, ASTD) against the saturation time was performed for each NMR signal (Fig. 4B). It has been previously reported that both the build–up rate and the height of STD factor plateau are strongly affected by T1 relaxation of the single protons [49]. Indeed at long saturation times higher STD factors are measured for protons with higher T1 values while at short saturation times, if all sites of the protein are sufficiently saturated, ASDT reflects the average proximity of the ligand protons to the protein surface. Due to the relatively low molecular weight of FGF2, saturation times lower than 1 sec. were not sufficient to saturate the entire protein and to monitor any STD effect. However, from the behaviour of the ASTD curves obtained for Ala1 and Ala5 methyl signals, showing comparable T1 values, a similar contribution to contact area of Ala1 and Ala5 methyls can be inferred. The proximity of the two methyls to FGF2 protein was also evaluated by performing a series of STD titration experiments and calculating the STD factors as a function of ARPCA excess. STD factors obtained for the different chemical groups (Fig. 4C) suggest that the proximity to the protein of the methyl groups of the two Ala residues is comparable. A higher STD amplification factor is observed for the N-terminus acetyl group but the result could be biased by the longer relaxation time T1 of this group.


Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide.

Leali D, Bianchi R, Bugatti A, Nicoli S, Mitola S, Ragona L, Tomaselli S, Gallo G, Catello S, Rivieccio V, Zetta L, Presta M - J. Cell. Mol. Med. (2010)

STD NMR analysis of Ac-ARPCA-NH2/FGF2 interaction. (A) STD spectra of Ac-ARPCA-NH2 peptide and its mutants acquired at 280 K on a 500 MHz Bruker spectrometer. (a) 1D NMR reference spectrum of 1.9 μM Ac-ARPCA-NH2 peptide alone; (b)–(e) STD spectra of Ac-ARPCA-NH2 and the three Ac-GRPCA-NH2, H-ARPCA-NH2 and Ac-ARPCG-NH2 peptides recorded in the presence of 50 μM FGF2 in 30 μM buffer phosphate (95% D2O, 5% H2O), 8 μM DTT, 40 μM NaCl, pH 6.8. A saturation time of 3 sec. was used. The assignment of the STD signals is reported. (B) Plot of STD factors (ASDT) of the Ac-ARPCA-NH2/FGF2 system versus saturation time. A 1:40 FGF2:peptide ratio was used. T1 relaxation time (sec) of each signal is shown on the right side of the curves. (C) ASTD× (ligand excess) versus different concentrations of Ac-ARPCA-NH2 peptide.
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Related In: Results  -  Collection

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fig04: STD NMR analysis of Ac-ARPCA-NH2/FGF2 interaction. (A) STD spectra of Ac-ARPCA-NH2 peptide and its mutants acquired at 280 K on a 500 MHz Bruker spectrometer. (a) 1D NMR reference spectrum of 1.9 μM Ac-ARPCA-NH2 peptide alone; (b)–(e) STD spectra of Ac-ARPCA-NH2 and the three Ac-GRPCA-NH2, H-ARPCA-NH2 and Ac-ARPCG-NH2 peptides recorded in the presence of 50 μM FGF2 in 30 μM buffer phosphate (95% D2O, 5% H2O), 8 μM DTT, 40 μM NaCl, pH 6.8. A saturation time of 3 sec. was used. The assignment of the STD signals is reported. (B) Plot of STD factors (ASDT) of the Ac-ARPCA-NH2/FGF2 system versus saturation time. A 1:40 FGF2:peptide ratio was used. T1 relaxation time (sec) of each signal is shown on the right side of the curves. (C) ASTD× (ligand excess) versus different concentrations of Ac-ARPCA-NH2 peptide.
Mentions: In order to map the peptide residues making direct contacts with FGF2, STD NMR methods were applied [46]. STD NMR experiments were performed in the presence of DDT in order to avoid the formation of disulphide bridges between free cysteines. The STD spectrum of ARPCA peptide in the presence of FGF2 proves that methyl protons of Ala1, Ala5 and of the N-terminal blocking acetyl group receive saturation transfer, giving rise to STD NMR signals (Fig. 4A). The hydrophobic patch defined by the three methyl groups is therefore involved in binding to the protein surface. In an attempt to compare the relative contribution of each methyl group to the contact area, a build-up curve of the saturation degree (STD factor, ASTD) against the saturation time was performed for each NMR signal (Fig. 4B). It has been previously reported that both the build–up rate and the height of STD factor plateau are strongly affected by T1 relaxation of the single protons [49]. Indeed at long saturation times higher STD factors are measured for protons with higher T1 values while at short saturation times, if all sites of the protein are sufficiently saturated, ASDT reflects the average proximity of the ligand protons to the protein surface. Due to the relatively low molecular weight of FGF2, saturation times lower than 1 sec. were not sufficient to saturate the entire protein and to monitor any STD effect. However, from the behaviour of the ASTD curves obtained for Ala1 and Ala5 methyl signals, showing comparable T1 values, a similar contribution to contact area of Ala1 and Ala5 methyls can be inferred. The proximity of the two methyls to FGF2 protein was also evaluated by performing a series of STD titration experiments and calculating the STD factors as a function of ARPCA excess. STD factors obtained for the different chemical groups (Fig. 4C) suggest that the proximity to the protein of the methyl groups of the two Ala residues is comparable. A higher STD amplification factor is observed for the N-terminus acetyl group but the result could be biased by the longer relaxation time T1 of this group.

Bottom Line: In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective.In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding.These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

View Article: PubMed Central - PubMed

Affiliation: Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Brescia, Italy.

Show MeSH