Abstract
Determinants of the interaction of the 29-kDa NH2-terminal domain of fibronectin with heparin were explored by analysis of normal and mutant recombinant NH2-terminal fibronectin fragments produced in an insect cell Baculovirus host vector system. A genomic/cDNA clone was constructed that specified a secretable human fibronectin NH2 fragment. With the use of site-directed mutagenesis a set of 29 kDa fragments was obtained that contained glycine or glutamic acid residues in place of basic residues at various candidate sites for heparin binding in the five type I modules that make up the domain. The recombinant fragment containing the wild type sequence had a nearly normal circular dichroic spectra and a melting profile, as assayed by loss of ellipticity at 228 nm, that was indistinguishable from that of the native fragment obtained by trypsinization of plasma fibronectin. A substantial proportion of the wild type recombinant fragment bound to heparin-Sepharose, where it was eluted at the same NaCl concentration as the native fragment. The wild type fragment was capable of promoting matrix-driven translocation, a morphogenetic effect in artificial extracellular matrices that depends on the interaction of the fibronectin NH2 terminus with heparin-like molecules on the surfaces of particles. Mutant fragments in which arginines predicted to be most exposed in the folded fragment were converted to glycines retained the same affinity for heparin as the wild type fragment. In contrast, a mutant fragment in which the single basic residue (Arg99) in the minor loop ("Omega-loop") of the second type I module was converted to a glycine had an essentially normal melting profile but exhibited no binding to heparin and failed to promote matrix-driven translocation. A mutant fragment in which the single basic residue (Arg52) of the first type I module was converted to a glycine also completely lacked heparin binding activity, but one in which the single basic residue (Arg191) the fourth type I module was converted to a glycine retained the ability to bind heparin. A mutant fragment in which the single basic residue (Lys143) in the Omega-loop of the third type I module was converted to a glutamic acid lacked heparin binding activity but had a CD spectrum similar to the heparin-liganded native protein and was capable of promoting matrix-driven translocation. The results indicate that multiple residues in the Omega-loops of the fibronectin NH2-terminal domain participate in its interactions with heparin. In addition, the conformation of one of the nonbinding mutants may mimic the heparin-induced structural alteration in this fibronectin domain required for certain morphogenetic events.
Highlights
Determinants of the interaction of the 29-kDa NH2terminal domain of fibronectin with heparin were explored by analysis of normal and mutant recombinant NH2-terminal fibronectin fragments produced in an insect cell Baculovirus host vector system
The morphogenetic potential of the interaction between the FnNTD and heparin-like molecules was demonstrated in experiments that assayed for matrix-driven translocation (MDT), a morphological reorganization that occurs when an artificially constructed extracellular matrix containing heparin-coated polystyrene beads is placed adjacent to similar matrix containing fibronectin [23, 24]
MDT did not occur when dextran sulfate-coated beads were substituted for heparin-coated beads [23] or when tri- or tetrapeptides containing Gly-Arg-Gly were used as competitors of the bead-fibronectin interaction [20] and was greatly reduced when FnNTDs were used in which more than four arginines were blocked by the chemical agent 1,2-cyclohexanedione [20]
Summary
FnNTD, fibronectin 29-kDa NH2-terminal domain; Wt, wild type; MDT, matrix-driven translocation; kbp, kilobase pair; TPCK, tosylphenylalanyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis. MDT did not occur when dextran sulfate-coated beads were substituted for heparin-coated beads [23] or when tri- or tetrapeptides containing Gly-Arg-Gly were used as competitors of the bead-fibronectin interaction [20] and was greatly reduced when FnNTDs were used in which more than four arginines were blocked by the chemical agent 1,2-cyclohexanedione [20]. This degree of chemical modification essentially eliminated the heparin binding capacity of this fibronectin domain [20]. The results indicate that basic residues in the V-loops of the FnNTD are decisive in heparin binding and some morphogenetic effects mediated by this domain and suggest that loop-loop interactions may play a role in these processes
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