Gene A. Homandberg

Heparin-binding fragments of fibronectin are potent inhibitors of endothelial cell growth: structure-function correlations

Heparin-binding fragments derived from the amino- and carboxyl-terminal regions of human plasma fibronectin appear to be at least relatively specific potent inhibitors of the growth of bovine aortic endothelial cells in culture by as yet unknown mechanisms. In order to understand better the sites which subserve this activity, we have compared the relative potency of other major fragments of fibronectin, most of which have dissimilar properties and do not bind heparin. We have also proteolytically digested and chemically modified the most potent of these fragments, the amino-terminal 29-kDa fragment, in order to test whether structural alterations that affect heparin-binding also affect the inhibitory property. Not all chemical modifications that abolished heparin-binding also abolished endothelial cell growth. Neither an amino-terminal 20-kDa nor a carboxyl-terminal 8-kDa subfragment of the 29-kDa fragment bound heparin; however, both were as inhibitory as native 29-kDa fragment. Reduction of the disulfides of the 20-kDa and 8-kDa fragments did not abolish inhibitory activity. We therefore conclude that the activity is not strictly conformation-dependent and that although the inhibitory activity is distributed throughout the 29-kDa segment, it can be expressed by an 8-kDa carboxyl-terminal segment containing residues of the last Type I loop structure.

Thrombospondin binds to amino-terminal fragments of plasma fibronectin.

Thrombospondin is a 420-kD trimeric glycoprotein that can bind type V collagen, heparin, fibrinogen and certain cells and may be one of the lectins responsible for platelet aggregation. Thrombospondin binds another glycoprotein, fibronectin, that is also released during platelet aggregation and also binds similar ligands. This work shows that the amino-terminal 29-kD segment of fibronectin binds thrombospondin, the interaction occurs within minutes, and one 29-kD molecule binds per thrombospondin subunit. The interaction was not inhibited by fibrinogen, type V collagen, or heparin. Two subfragments of the 29-kD fragment, an amino-terminal 20-kD and a carboxyl-terminal 8-kD subfragment, the latter containing a single disulfide-rigidified type I loop of the five homologous loops in the 29-kD fragment, reacted with thrombospondin while the reduced counterparts did not.

Heparin modulates conformational states of plasma fibronectin: An electron spin resonance spin label approach

We have examined the interaction between heparin and human plasma fibronectin using electron spin resonance (ESR) spin label methods. The titratable sulfhydryl groups of plasma fibronectin were modified with a maleimide spin label [Lai and Tooney (1984) Arch. Biochem. Biophys. 228, 465-473]. Addition of heparin resulted in a decrease in the maximum splitting value of the ESR spectrum of spin-labeled fibronectin from 66.8 to 64.3 G, suggesting that heparin induces a conformational alteration of plasma fibronectin. This heparin effect was noticeable at a heparin-to-fibronectin ratio of 20 to 1 and reached a plateau at about 100 to 1. Other sulfated carbohydrates were tested; dextran sulfate was found to be as effective as heparin but chondroitin sulfates were ineffective. The results presented suggest that the binding of heparin changes the molecular conformation of plasma fibronectin to a more relaxed or flexible state.

Preparation of functionally intact monomers by limited disulfide reduction of human plasma fibronectin dimers

Most (90 to 95%) human plasma fibronectin (PFn) molecules exist as 450-kDa disulfide-rich dimers comprised of two major types of subunits (A, 220 kDa; B, 215 kDa) that are joined near the COOH terminus by two disulfide bonds. Smaller PFn species (Zone II; 190-235 kDa) consist mainly of monomers and/or a monomeric subunit joined covalently to a smaller peptide remnant presumably derived by proteolysis of a parent 450-kDa molecule. A relatively simple and selective method for preparing functionally active, partially reduced monomeric fibronectin subunits (PR-PFn) by limited and selective reduction of dimeric plasma fibronectin (PFn) has been developed. PR-PFn was prepared by incubating PFn in phosphate-buffered saline, pH 7.4, for 2 h at room temperature in the presence of 17 mM dithiothreitol (DTT). Following S-carboxymethylation or S-carboxyamidomethylation, the material was passed through a gelatin-Sepharose column and nonbinding material was discarded; gelatin-bound material was eluted using a 0 to 2 M KSCN gradient. Residual dimeric species (10-20%) could be separated from monomers in high yield by gel-sieving chromatography on a Sepharose 6B-Cl in the presence of a chaotropic salt, 0.3 M KSCN. Most new SH groups (74-81%) in that fraction of PR-PFn binding to gelatin were localized in proteolytic fragments containing the COOH terminus, thus suggesting that selective cleavage of the interchain disulfide bridges had taken place. The binding affinity of PR-PFn to gelatin- and fibrin-Sepharose was lower than that of dimeric PFn, but the same as that of Zone II PFn and other monomeric gelatin-binding proteolytic derivatives. PR-PFn also bound to heparin-Sepharose and promoted cell attachment and spreading. We conclude that PR-PFn monomers possess the same functional activities as those of the parent chains.

Near ultraviolet circular dichroism spectroscopy of plasma fibronectin and fibronectin fragments.

Abstract Comparison of the near uv CD spectrum of human plasma fibronectin with the spectra of the three major leukocyte elastase fragments 25 Kd, 60 Kd and 140 160 shows that the aromatic residues of these fragments are in different environments. In particular, the ellipticity bands associated with tryptophan in the 290–300 nm region differ for each fragment. The results also show that intrachain disulfide bridges are important in stabilizing regions of the fibronectin molecule against the structure-disrupting effects of 8M urea.

Interaction between fluorescence-labeled fibronectin fragments studied by gel high-performance liquid chromatography.

Fibronectin is a large, adhesive glycoprotein which self-associates on many cell surfaces. We have begun to study this reaction by determining the domains of fibronectin which interact with each other. To avoid possible solid-phase artifacts of affinity chromatography, we have devised a solution-phase assay in which the smallest fibronectin fragment is labeled with fluorescamine, mixed with unlabeled fibronectin, and complexation is observed by the appearance of a new higher-molecular-weight peak on gel high-performance liquid chromatography columns. The assay allowed use of excess unlabeled reactant, high-sensitivity, low background without removal of reagent, and fast analysis. Our results show that the amino- and carboxyl-terminal fibronectin fragments bind the native molecule in solution.

Inaccessibility to ligands of the amino-terminal region of plasma fibronectin

Fragments of plasma fibronectin can display properties not expressed by fibronectin. This work shows that the relative affinities of smaller fragments for gelatin or heparin increases with decreasing fragment size. When heparin or gelatin were added to fragments or to fibronectin, the affinities for the alternate ligand increased. The UV and CD spectra of a mixture of amino-terminal 29-kD and 50-kD fragments differed from that of the precursor, the intact 72-kD fragment, suggesting differences in secondary structure. Therefore, biologic activities of the 29-kD and 50-kD segments in fibronectin may be suppressed by structural constraints but may be expressed more fully by interaction with ligands.