Orientation of Heparin-binding Sites in Native Vitronectin: ANALYSES OF LIGAND BINDING TO THE PRIMARY GLYCOSAMINOGLYCAN-BINDING SITE INDICATE THAT PUTATIVE SECONDARY SITES ARE NOT FUNCTIONAL

Angelia D Gibson,John A Lamerdin,Ping Zhuang,Kunnumal Baburaj,Engin H Serpersu,Cynthia B Peterson

doi:10.1074/jbc.274.10.6432

Angelia D Gibson, John A Lamerdin + Show 4 more

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https://doi.org/10.1074/jbc.274.10.6432

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Abstract

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein.

Highlights

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein
The Somatomedin B domain has been expressed in Escherichia coli as a functional protein that mimics the ability of vitronectin to bind and stabilize the active conformation of PAI-1 [10, 11]. (ii) The amino acids 131–268 and 269 – 459 of vitronectin share homology with domains found in hemopexin and collagenase [12]. (iii) At the C terminus of vitronectin, a highly charged sequence interrupts the second hemopexin homology domain
Experiments were designed to evaluate ionic versus non-ionic interactions in this context, to identify potential residues that contribute to the interaction, and to assess further the extent to which the primary heparin-binding sequence is exposed on the surface of vitronectin

Summary

Introduction

A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. The label has been localized to arginine residues within the cyanogen bromide fragment-(341–380) that contains the primary heparin-binding site on vitronectin. (iii) At the C terminus of vitronectin, a highly charged sequence interrupts the second hemopexin homology domain This cationic sequence has been proposed to be the primary binding site for numerous ligands such as plasminogen, the complement C7, C8, and C9 components, PAI-1, and the widely administered anticoagulant drug, heparin [4, 5, 13]. A search for the glycosaminoglycan-binding site within vitronectin localized binding activity to a C-terminal vitronectin fragment produced by digestion with cyanogen bromide [4] Another group reported that a fragment corresponding to the C-terminal region of vitronectin bound heparin in ligand blotting assays and that synthetic peptides corresponding to the vitronectin residues 347–359 bound biotinylated heparin and diminished heparin activity [5, 16, 17]. The amino acid sequence of the region shown to bind heparin is highly basic and contains two short sequences that correspond to consensus heparin-binding motifs that are found in many structurally unrelated heparin-binding proteins, including antithrombin III, thrombin, low density lipoprotein, and endothelial growth factor [19, 20]

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