Interaction of Heparin with a Synthetic Pentadecapeptide from the C-Terminal Heparin-Binding Domain of Fibronectin

Abstract

The synthetic pentadecapeptide FN-C/H II (KNNQKSEPLIGRKKT-NH(2)) has the sequence of the carboxy-terminal heparin-binding domain of module III(14) of fibronectin. Interaction of FN-C/H II with bovine lung heparin has been studied by (1)H and (23)Na NMR spectroscopy and by heparin affinity chromatography. FN-C/H II binds to heparin from pD <2 up to pD approximately 10; at higher pD, the binding decreases as the lysine side-chain ammonium groups are titrated. Na(+) counterions are displaced from the counterion condensation volume that surrounds sodium heparinate by FN-C/H II, which provides direct evidence that the binding involves electrostatic interactions. The pK(A) values for each of the five ammonium groups of FN-C/H II increase upon binding to heparin which, together with chemical shift data, indicates that the binding involves both delocalized and direct electrostatic interactions between ammonium groups of FN-C/H II and carboxylate and/or sulfate groups of heparin. NMR data also provide evidence for the direct interaction of the guanidinium group of the arginine side chain with anionic sites on heparin. The affinity of heparin for FN-C/H II and for 13 analogue peptides in which lysine and arginine residues were systematically substituted with alanine increases as the number of basic residues increases. The relative contribution of each lysine and arginine to the affinity of heparin for FN-C/H II is R(12) > K(13) > K(14) > K(1) > K(5). Nuclear Overhauser enhancement (NOE) data indicate that, while FN-C/H II is largely unstructured in aqueous solution, the bound peptide interconverts among overlapping, turn-like conformations over the L(9) - T(15) segment of the peptide. NOE data for the interaction of FN-C/H II with a heparin-derived hexasaccharide, together with the number of Na(+) ions displaced from heparin by FN-C/H II as determined by (23)Na NMR, indicates that the peptide binds to a hexasaccharide segment of heparin. Identical NMR and heparin affinity chromatography results were obtained for the interaction of FN-C/H II and its D-amino acid analogue peptide with heparin, which is of interest for the potential use of peptides as therapeutic agents for diseases in which cell adhesion plays a critical role.

Interaction of Heparin with Two Synthetic Peptides that Neutralize the Anticoagulant Activity of Heparin

“…Experimental values of 0.58 and 0.63 have been reported (46,47). Counterions are released when heparin binds to peptide, as described by the following binding equilibrium for sodium heparinate (38,43,48): (5) The number of Na + ions displaced by peptide, n, is equal to Zψ, where Z represents the number of ionic interactions the peptide makes with heparin and ψ is the apparent fraction of a Na + condensed per heparin anionic charge. ψ includes both the fraction of a Na + ion condensed per heparin anionic charge (θ) and the screening effect of condensed Na + ions on the interactions of residual heparin anionic charges.…”

“…If so, the apparent pK A s of the lysine ammonium groups that interact with heparin will be increased (38). pK A values can be determined for each lysine ammonium group in a peptide from the pH dependence of the chemical shifts of the side chain C ε H 2 protons (39).…”

Structural and Energetic Characteristics of the Heparin-binding Site in Antithrombotic Protein C

“…Na ϩ ) can interact with heparin by delocalized, long range electrostatic interactions and by direct binding to ionic sites. Displacement of counter-ions upon heparin binding to protein has been experimentally demonstrated (31). In cases where a plot of log K d obs against log [salt] is linear, the magnitude of the slope corresponds to the effective number of heparin counter-ions released upon its binding to a protein.…”

The Cartilage-specific Fibronectin Isoform Has a High Affinity Binding Site for the Small Proteoglycan Decorin

“…This domain also supports cell adhesion and spreading through five peptides composed of basic residues that, in vitro, have been shown independently to bind heparin and/or chondroitin sulfate (33-36). Repeat III 14 contains three of the five basic peptides, one immediately N-terminal to the V region which may be inaccessible in the native molecule (37,38). X-ray crystallography studies by Sharma et al (16) confirmed the prediction that a cluster of six basic residues from one or more of these peptides come together to generate a positively charged "cradle" on one side of the molecule that forms the major heparin-binding site in III 13 .…”