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• W1992666094 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 (“Ω-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 Ω-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 Ω-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. 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 (“Ω-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 Ω-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 Ω-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. Fibronectin, an adhesive glycoprotein of the blood plasma and extracellular matrix, contains two distinct sites with affinity for the glycosaminoglycan heparin (see Refs. 1Mosher, D. F. (ed) (1989) Fibronectin, Academic Press, New YorkGoogle Scholar and 2Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar for reviews). One of these sites, Hep2, near the carboxyl-terminal end of the protein comprises one or more disulfide-lacking type III fibronectin modules (3Barkalow F.J.B. Schwarzbauer J.E. J. Biol. Chem. 1991; 266: 7812-7818Abstract Full Text PDF PubMed Google Scholar, 4Ingham K.C. Brew S.A. Migliorini M.M. Busby T.F. Biochemistry. 1993; 32: 12548-12553Crossref PubMed Scopus (45) Google Scholar) and may mediate cell adhesion and neurite outgrowth (5Drake S.L. Varnum J. Mayo K.H. Letourneau P.C. Furcht L.T. McCarthy J.B. J. Biol. Chem. 1993; 268: 15859-15867Abstract Full Text PDF PubMed Google Scholar, 6Hines K.L. Kulkarni A.B. McCarthy J.B. Tian H. Ward J.M. Christ M. McCartney-Francis N.L. Furcht L.T. Karlsson S. Wahl S.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5187-5191Crossref PubMed Scopus (84) Google Scholar). The other heparin-binding site, Hep1, is coincident with fibronectin's compact, trypsin-resistant, 29-kDa amino-terminal domain. This domain, which consists of five disulfide-containing type I fibronectin modules (“fibronectin fingers”), apart from its heparin-binding capability, is an important site of binding to fibrin (7McDonagh R.P. McDonagh J. Peterson T.E. Thogersen H.C. Skørstengaard K. Søttrup-Jensen L. Magnusson S. Dell A. Morris H.R. FEBS Lett. 1981; 127: 174-178Crossref PubMed Scopus (76) Google Scholar, 8Sottile J. Schwarzbauer J. Selegue J. Mosher D.F. J. Biol. Chem. 1991; 266: 12840-12843Abstract Full Text PDF PubMed Google Scholar, 9Matsuka Y.V. Medved L.V. Brew S.A. Ingham K.C. J. Biol. Chem. 1994; 269: 9539-9546Abstract Full Text PDF PubMed Google Scholar, 10Rostagno A. Williams M.J. Baron M. Campbell I.D. Gold L.I. J. Biol. Chem. 1994; 269: 31938-31945Abstract Full Text PDF PubMed Google Scholar), thrombospondin (11Homandberg G.A. Kramer-Bjerke J. Thromb. Res. 1987; 48: 329-335Abstract Full Text PDF PubMed Scopus (14) Google Scholar), tumor necrosis factor-α (12Alon R. Cahalon L. Hershkoviz R. Elbaz D. Reizis B. Wallach D. Akiyama S.K. Yamada K.M. Lider O. J. Immunol. 1994; 152: 1304-1313PubMed Google Scholar), surface proteins ofStaphylococcus aureus (13Signas C. Raucci G. Jonsson K. Lindgren P.E. Anantharamaiah G.M. Höök M. Lindberg M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 699-703Crossref PubMed Scopus (309) Google Scholar, 14Huff S. Matsuka Y.V. McGavin M.J. Ingham K.C. J. Biol. Chem. 1994; 269: 15563-15570Abstract Full Text PDF PubMed Google Scholar), as well as an essential component of fibronectin's ability to assemble into an extracellular matrix (15Quade B.J. McDonald J.A. J. Biol. Chem. 1988; 263: 19602-19609Abstract Full Text PDF PubMed Google Scholar, 16Schwarzbauer J.E. J. Cell. Biol. 1991; 113: 1463-1473Crossref PubMed Scopus (179) Google Scholar). Previous studies have suggested that the heparin binding capability of the 29-kDa NH2-terminal domain of fibronectin (FnNTD) 1The abbreviations used are: 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. also helps mediate the cellular condensation process that occurs at sites of incipient chondrogenesis in the precartilage mesenchyme of the developing vertebrate limb (17Hall B.K. Miyake T. Anat. Embryol. 1992; 186: 107-124Crossref PubMed Scopus (305) Google Scholar,18Newman S.A. Tomasek J.J. Structure and Function of Extracellular Matrices of Connective Tissues.in: Comper W.D. 2. Gordon & Breach, London1996Google Scholar). In particular, the formation of condensations in high density cultures of precartilage mesenchyme was inhibited by a monoclonal antibody directed against the FnNTD and by tri- or tetrapeptides containing Gly-Arg-Gly (19Frenz D.A. Jaikaria N.S. Newman S.A. Dev. Biol. 1989; 136: 97-103Crossref PubMed Scopus (129) Google Scholar), a conserved, repeated motif of the FnNTD (2Hynes R.O. Fibronectins. Springer-Verlag, New York1990Crossref Google Scholar) that represents a putative determinant of the heparin binding capacity of this domain (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar). Moreover, cell-sized polystyrene latex beads coated with heparin, when mixed with limb mesenchymal cells, accumulated at fibronectin-rich sites of cellular condensation during early chondrogenesis in vitro, but beads coated with dextran sulfate did not. This redistribution of heparin-coated beads was also inhibited by the anti-FnNTD antibody and by Gly-Arg-Gly (21Frenz D.A. Akiyama S.K. Paulsen D.F. Newman S.A. Dev. Biol. 1989; 136: 87-96Crossref PubMed Scopus (57) Google Scholar). In the condensing limb bud mesenchyme the presumed heparin-like ligand for the FnNTD is the cell surface heparan sulfate-containing proteoglycan, syndecan, which, like fibronectin, is abundant at sites of precartilage condensation (22Gould S.E. Upholt W.B. Kosher R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3271-3275Crossref PubMed Scopus (123) Google Scholar). The morphogenetic potential of the interaction between the FnNTD and heparin-like molecules was also 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 (23Newman S.A. Frenz D.A. Tomasek J.J. Rabuzzi D.D. Science. 1985; 228: 885-889Crossref PubMed Scopus (70) Google Scholar, 24Newman S.A. Frenz D.A. Hasegawa E. Akiyama S.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4791-4795Crossref PubMed Scopus (37) Google Scholar). Under the conditions of the assay the FnNTD was the only domain of fibronectin both necessary and sufficient for MDT to occur (24Newman S.A. Frenz D.A. Hasegawa E. Akiyama S.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4791-4795Crossref PubMed Scopus (37) Google Scholar). MDT did not occur when dextran sulfate-coated beads were substituted for heparin-coated beads (23Newman S.A. Frenz D.A. Tomasek J.J. Rabuzzi D.D. Science. 1985; 228: 885-889Crossref PubMed Scopus (70) Google Scholar) or when tri- or tetrapeptides containing Gly-Arg-Gly were used as competitors of the bead-fibronectin interaction (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar) and was greatly reduced when FnNTDs were used in which more than four arginines were blocked by the chemical agent 1,2-cyclohexanedione (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar). This degree of chemical modification also essentially eliminated the heparin binding capacity of this fibronectin domain (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar). It was concluded from these studies that the arginines (and possibly a lysine) in the highly flexible minor loops of the FnNTD type I modules (25Baron M. Norman D. Willis A. Campbell I.D. Nature. 1990; 345: 642-646Crossref PubMed Scopus (92) Google Scholar) were likely sites of interaction with heparin. Because none of the individual minor loops contains a canonical heparin binding site (26Cardin A.D. Weintraub H.J. Arteriosclerosis. 1989; 9: 21-32Crossref PubMed Google Scholar) and limited internal cleavage of the major loops by cyanogen bromide destroyed the MDT promoting activity of the FnNTD (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar), it was further suggested that cooperation among multiple minor loops is required for the interaction of this fibronectin domain with heparin (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar). (Following Leszczynski and Rose (27Leszczynski J.F. Rose G.D. Science. 1986; 234: 849-855Crossref PubMed Scopus (452) Google Scholar), we refer to these small, compact loops as “Ω-loops”). In the present study we have tested these inferences directly by producing recombinant FnNTDs with site-directed modifications that eliminated one or more basic side chains at sites predicted to be the most highly exposed in the human FnNTD and at the somewhat less exposed sites in the Ω-loops of modules I-1, I-2, I-3, and I-4. We found, as predicted from the earlier chemical modification studies, that elimination of arginines outside the Ω-loops had no effect on heparin binding of the FnNTD, whereas modifications of the single basic residues in the Ω-loops of I-1, I-2, or I-3 eliminated all of the fragment's heparin binding activity. The mutant fragments containing either or both of the alterations in the Ω-loops of I-2 and I-3 were tested as well for ability to promote the MDT in vitromorphogenetic effect and, by CD, for retention of native conformation in the absence and the presence of heparin. Two of these mutant fragments were incapable of promoting MDT, as expected. However, the fragment mutated in the Ω-loop of I-3, in which a positively charged side chain was replaced by a negatively charged one, retained the ability to promote MDT, despite the virtual elimination of its heparin binding activity. The CD measurements suggested that in this case the mutation may have thrown the fragment into its “morphogenetically active” conformation. The results indicate that basic residues in the Ω-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. Human plasma fibronectin was obtained from the New York Blood Center and exchanged into phosphate-buffered saline (0.02 m sodium phosphate buffer, pH 7.2, 0.15 mNaCl) and used directly or after being digested with tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Worthington) as described (28Hayashi M. Yamada K.M. J. Biol. Chem. 1983; 258: 3332-3340Abstract Full Text PDF PubMed Google Scholar). Purified beef lung heparin was generously provided by the late Dr. Isidore Danishefsky. Rat monoclonal antibody 304, reactive against the FnNTD (24Newman S.A. Frenz D.A. Hasegawa E. Akiyama S.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4791-4795Crossref PubMed Scopus (37) Google Scholar), was a gift from Dr. Steven Akiyama (Laboratory of Developmental Biology, NIDR). cDNA clone pFH6, encoding the human FnNTD and a portion of the adjacent collagen-binding domain (29Kornblihtt A.R. Vibe-Pedersen K. Baralle F.E. EMBO J. 1984; 3: 221-226Crossref PubMed Scopus (143) Google Scholar), was a gift from Dr. Francisco Baralle (International Center for Genetic Engineering and Biotechnology, Trieste, Italy). Genomic DNA clone pgHF3.7, encoding the human fibronectin promoter, secretory signal sequence, and the initial portion of the FnNTD (30Dean D.C. Bowlus C.L. Bourgeois S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84 (2nd Ed.): 1876-1880Crossref PubMed Scopus (76) Google Scholar), was a gift from Dr. Douglas Dean (Washington University, St. Louis, MO). Oligonucleotides for sequencing and site-directed mutagenesis were synthesized in the Department of Biochemistry and Molecular Biology (New York Medical College) or purchased from Gene Link, Inc. (Thornwood, NY). Sf21 insect cells were obtained from Invitrogen (San Diego, CA) and maintained in shaker flasks in serum-free insect cell culture medium (Life Technologies, Inc.). Type I collagen was obtained by acetic acid extraction of tail tendons of young adult rats and kept frozen at −20 °C as described (23Newman S.A. Frenz D.A. Tomasek J.J. Rabuzzi D.D. Science. 1985; 228: 885-889Crossref PubMed Scopus (70) Google Scholar). The collagen was dialyzed into acidified 1:10 strength Ham's F-12 medium without bicarbonate (Flow Laboratories, McLean, VA) before being used in the MDT assay (23Newman S.A. Frenz D.A. Tomasek J.J. Rabuzzi D.D. Science. 1985; 228: 885-889Crossref PubMed Scopus (70) Google Scholar,24Newman S.A. Frenz D.A. Hasegawa E. Akiyama S.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4791-4795Crossref PubMed Scopus (37) Google Scholar). Clone pFNs54 was constructed from two pre-existing clones as follows. (i) Human fibronectin cDNA clone pFH6 (29Kornblihtt A.R. Vibe-Pedersen K. Baralle F.E. EMBO J. 1984; 3: 221-226Crossref PubMed Scopus (143) Google Scholar) was digested with EspI and BamHI, yielding a 1.2-kbp fragment encoding the initial 54 kDa of the mature protein. (ii) Human genomic clone pgHF3.7 (30Dean D.C. Bowlus C.L. Bourgeois S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84 (2nd Ed.): 1876-1880Crossref PubMed Scopus (76) Google Scholar) containing the 5′ end of the fibronectin gene, including a small region of overlap with pFH6, was digested with PstI andEspI, yielding a 291-base pair fragment containing the complete signal sequence and a 3′ terminus at the same position as the 5′ terminus of the fragment generated from pFH6. The two fragments were ligated at their common EspI site and cloned into thePstI-BamHI site of pUC18 (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), yielding the plasmid pFNs54, specifying a secretable form of the NH2-terminal 54 kDa of the mature fibronectin protein. The 1.5-kbp PstI-BamHI fibronectin cDNA fragment was excised from pFNs54 and used for single-stranded (Ref. 32Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4903) Google Scholar; Sculptor kit version 2.1, Amersham Corp.) or double-stranded (Ref. 33Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1079) Google Scholar; Chameleon kit, Stratagene, La Jolla, CA) site-directed mutagenesis. Mutagenic oligonucleotides were phosphorylated with T4 polynucleotide kinase and annealed with the target sequences. Seven different mutants were made; the corresponding mutagenic oligonucleotides used (with the mutated base underlined) and the predicted amino acid changes are as follows: M1, 5′-TCTCCCTCCCCCAGCCCC-3′ for Arg99 → Gly99; M2, 5′-ATTCTCCTTCTCCATTACC-3′ for Lys143 → Glu143; in M1+2, the second of the above substitutions was made in a clone in which the first substitution had already been made, yielding a clone that encoded both Gly99 and Glu143; M3, 5′-GGAGGAAGCGGAGGTTTTAACTGCG-3′ for Arg52 → Gly52; M4, 5′-GGTGACACCTGGGGGGGACCACATGAGACTGG-3′ for Arg124-Arg125 → Gly124-Gly125; M5, 5′-GGCAGCGGAGGCATCACTTGC-3′ for Arg191 → Gly191; M6, 5′-GCACTTCTGGAAATGGATGCAACGATCAGG-3′ for Arg197-Asn198-Arg199 → Gly197-Asn198-Gly199. The presence of the desired mutations was confirmed by the dideoxy sequencing method using the Sequenase sequencing kit (U. S. Biochemical Corp.) with appropriate primers. The 1.5-kbp PstI-BamHI wild type and mutant fibronectin cDNA fragments were ligated into the polylinker region of the Baculovirus transfer vector pVL1392 (Pharmingen, Inc., San Diego, CA) in the same orientation as the polyhedrin protein promoter (34O'Reilly D.R. Miller L.K. Luckow V.A. Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, Oxford1994Google Scholar). Co-transfection of Sf21 insect cells was carried out using linearized Baculovirus DNA (Baculogold, Pharmingen) and recombinant fibronectin/pVL1392 DNA in calcium chloride solution. The transfected cells were grown in serum-free insect cell culture medium at 27 °C for 4 days, at which time the recombinant viruses were amplified to obtain a high titer stock (1 × 108 viral particles/ml). Subsequent transfections were done in both T-flask and shaker flask culture at a multiplicity of infection of 5–10. The infected cells were grown for 3 or more days, after which the supernatant was harvested, centrifuged at 5,000 × g for 10 min, and cleared by an additional 10 min of centrifugation at 10,000 × g. Supernatants were analyzed on 12.5% SDS-PAGE gels (35Blattler D.P. Garner F. van Slyke K. Bradley A. J. Chromatogr. 1972; 64: 147-155Crossref Scopus (188) Google Scholar) by Coomassie Blue staining or after electrotransfer to nitrocellulose by immunoblotting with monoclonal antibody 304 (24Newman S.A. Frenz D.A. Hasegawa E. Akiyama S.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4791-4795Crossref PubMed Scopus (37) Google Scholar) and an alkaline phosphatase-conjugated goat anti-mouse secondary antibody to confirm and estimate the level of expression of the FnNTDs. The cleared culture supernatants were dialyzed against 25 mm Tris-HCl, pH 7.6, 25 mm NaCl, 0.5 mm EDTA for 2 days, changing the buffer every 8 h. Protein concentrations were determined by the Bradford method (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) or, after purification of fibronectin fragments, by absorbance at 280 nm using a specific extinction coefficient of 12.8 (37Mosesson M.W. Umfleet R.A. J. Biol. Chem. 1970; 245: 5728-5736Abstract Full Text PDF PubMed Google Scholar). To generate the 29-kDa FnNTDs, the 54-kDa recombinant fibronectin NH2-terminal fragments were digested with TPCK-treated trypsin at a fibronectin:trypsin ratio of 500:1 at 25 °C for 15 min, followed by addition of phenymethylsulfonyl fluoride to a final concentration of 1 mm (28Hayashi M. Yamada K.M. J. Biol. Chem. 1983; 258: 3332-3340Abstract Full Text PDF PubMed Google Scholar). The digested protein was further dialyzed against 25 mm Tris-HCl, pH 7.0, 25 mm NaCl, 0.5 mm EDTA, 1 mm phenymethylsulfonyl fluoride. The dialyzed samples were passed through a DEAE-cellulose column equilibrated with the same buffer. The wild type recombinant FnNTD, like the native fragment obtained by digesting plasma fibronectin (28Hayashi M. Yamada K.M. J. Biol. Chem. 1983; 258: 3332-3340Abstract Full Text PDF PubMed Google Scholar), was not bound and was recovered from the prewash. For some experiments the wild type fragment was further purified by affinity chromatography on heparin-Sepharose (see below). Mutant fragment M4 was eluted from DEAE-cellulose with 100 mm NaCl, M2 and M5 were eluted with 150 mm NaCl, and M1, M1+2, M3, and M6 were eluted with 200 mm NaCl. The mutant proteins were purified away from contaminating species by gel filtration chromatography using Sephacryl-100 followed, where required, by fast protein liquid chromatography with a Superose-12 column (Pharmacia Biotech Inc.). Protein samples used in analytical experiments were >95% pure by the criterion of relative intensity of the 29-kDa band to other species on Coomassie-stained SDS-PAGE gels. CD measurements in the far UV region (200–250 nm) were carried out on a Jasco model 500C spectropolarimeter equipped with a microcell holder, ADLAB-PC adapter, and ADAPT program (Interactive Microware, State College, PA). Spectra were obtained in phosphate-buffered saline at room temperature with a time constant of 16 s, a sensitivity of 0.1 millidegree/cm and a scan rate of 5 nm/min in a cuvette with an optical path length of 0.1 cm. Protein concentrations were in the range of 0.32–0.48 mg/ml. When heparin was present, the weight ratio of heparin to protein was 0.005–0.025, and the signal for heparin alone in the same buffer, though minimal, was corrected for. The data were collected at 0.2 nm intervals, and the average of 10 scans was reported. Melting curves were obtained by monitoring ellipticity at 228 nm while heating a jacketed cell of the same path length, 0.1 cm, at 1 °C/min. A mean residue molecular weight of 112 was used in the calculations of ellipticity. Secondary structures were estimated from CD data using the fixed reference method (CDESTIMA program) of Yang et al. (38Yang J.T. Wu C.S. Martinez H.M. Methods Enzymol. 1986; 130: 208-269Crossref PubMed Scopus (1740) Google Scholar). The ellipticity at each nm from 240 to 200 nm was used in the CDESTIMA analysis, as described (39Samuel M. Samuel E. Villanueva G.B. Thromb. Res. 1994; 75: 259-268Abstract Full Text PDF PubMed Scopus (6) Google Scholar). The reference spectra were pure component spectra extracted from CD analysis of proteins with structures previously determined by x-ray diffraction. Heparin-Sepharose mini-columns (Pharmacia) were equilibrated with 10 mm sodium phosphate, pH 7.2, 50 mm NaCl. Each purified FnNTD (0.2 ml; 0.11–0.22 mg/ml) was loaded onto a column, and a gradient of 50–750 mm NaCl in the same buffer was applied at room temperature at a flow rate of 0.5 ml/min. The absorbance of the column effluent at 280 nm was monitored with a flow cell. This assay was performed as described previously (23Newman S.A. Frenz D.A. Tomasek J.J. Rabuzzi D.D. Science. 1985; 228: 885-889Crossref PubMed Scopus (70) Google Scholar, 24Newman S.A. Frenz D.A. Hasegawa E. Akiyama S.K. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4791-4795Crossref PubMed Scopus (37) Google Scholar). Briefly, “primary gels” were constructed by suspending polystyrene latex beads (6 μm, Polysciences, Warrington, PA; final concentration, 5 × 106/ml) in a cooled, soluble collagen solution (1.7–3.5 mg/ml), which was simultaneously adjusted to physiological pH and ionic strength with concentrated Ham's F-12 medium and sodium bicarbonate. “Secondary gels” consisted of an identical collagen solution with or without 6 μm/ml FnNTD or one of its mutant variants. Drops of primary and secondary gel were placed contiguously on a plastic Petri dish, and the subsequent movement, or lack thereof, of beads and surrounding matrix across the original interface was recorded. Because certain commercially fabricated polystyrenes are well established mimics of heparin in MDT (20Jaikaria N.S. Rosenfeld L. Khan M.Y. Danishefsky I. Newman S.A. Biochemistry. 1991; 30: 1538-1544Crossref PubMed Scopus (20) Google Scholar) and other protein binding assays (40Jozefonvicz J. Jozefowicz M. J. Biomater. Sci. Polym. Ed. 1990; 1: 147-165Crossref PubMed Scopus (65) Google Scholar,41Boisson-Vidal C. Jozefonvicz J. Brash J.L. J. Biomed. Mater. Res. 1991; 25: 67-84Crossref PubMed Scopus (34) Google Scholar), uncoated beads having this property were used in most of these experiments. The modular organization of fibronectin is shown schematically in Fig.1, along with the relative positions of the fragments used in these studies. Sf21 insect cells infected with recombinantBaculovirus containing ∼1.5-kbp human fibronectin genomic/cDNA inserts derived from pFNs54 (Fig.2 A) were grown in serum-free medium where they secreted protein fragments corresponding to the first 54 kDa of the mature fibronectin molecule. These fragments were digested with trypsin and purified to yield wild type and mutant FnNTDs (Fig.2 B). Wild type (Wt) recombinant FnNTD was produced using the parental insert from pFNs54 and contained a nucleotide sequence that encoded the protein sequence of the first five type I modules (I-1–5) of human fibronectin (42Garcia-Pardo A. Pearlstein E. Frangione B. J. Biol. Chem. 1983; 258: 12670-12674Abstract Full Text PDF PubMed Google Scholar), plus I-6 and the protein's two type II modules (II-1 and II-2) (Fig. 1). Mutant 1 (M1) encoded an Arg → Gly substitution at the tip of the minor loop of module I-2 (amino acid 99 from the mature NH2 terminus). M2 encoded a Lys → Glu substitution at the corresponding position in module I-3 (amino acid 143), and M1+2 encoded both of these substitutions. M3 and M5 encoded Arg → Gly substitutions near the tips of the minor loops of modules I-1 (amino acid 52) and I-4 (amino acid 191), respectively. M4 encoded a Gly-Gly substitution for the Arg-Arg in the large loop of module I-3 (amino acids 124 and 125), and M6 encoded Arg → Gly substitutions for both of the arginine residues flanking an asparagine residue in the protein strand connecting the small loop of module I-4 with the large loop of module I-5 (amino acids 197 and 199) (Fig. 3). Sequencing of several hundred nucleotides to either side of the mutant sites disclosed no changes other than the desired mutations.Figure 2Production of" @default.
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