The Role of the Fibronectin IGD Motif in Stimulating Fibroblast Migration
2007; Elsevier BV; Volume: 282; Issue: 49 Linguagem: Inglês
10.1074/jbc.m707532200
ISSN1083-351X
AutoresChristopher J. Millard, Ian Ellis, Andrew R. Pickford, Ana M. Schor, Seth L. Schor, Iain D. Campbell,
Tópico(s)Protease and Inhibitor Mechanisms
ResumoThe motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 106 times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN. The motogenic activity of migration-stimulating factor, a truncated isoform of fibronectin (FN), has been attributed to the IGD motifs present in its FN type 1 modules. The structure-function relationship of various recombinant IGD-containing FN fragments is now investigated. Their structure is assessed by solution state NMR and their motogenic ability tested on fibroblasts. Even conservative mutations in the IGD motif are inactive or have severely reduced potency, while the structure remains essentially the same. A fragment with two IGD motifs is 100 times more active than a fragment with one and up to 106 times more than synthetic tetrapeptides. The wide range of potency in different contexts is discussed in terms of cryptic FN sites and cooperativity. These results give new insight into the stimulation of fibroblast migration by IGD motifs in FN. Fibronectin (FN) 2The abbreviations used are: FN, fibronectin; MSF, migration-stimulating factor; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence; 7Fn1, 8Fn1, 8Fn1*, 8Fn1**, and 9Fn1, seventh, eighth, eighth (R503K), eighth (R503K, N497Q), and ninth type I fibronectin modules, respectively; ESI-MS, electrospray ionization mass spectrometry. 2The abbreviations used are: FN, fibronectin; MSF, migration-stimulating factor; NOE, nuclear Overhauser effect; HSQC, heteronuclear single quantum coherence; 7Fn1, 8Fn1, 8Fn1*, 8Fn1**, and 9Fn1, seventh, eighth, eighth (R503K), eighth (R503K, N497Q), and ninth type I fibronectin modules, respectively; ESI-MS, electrospray ionization mass spectrometry. is a multifunctional, multidomain adhesive glycoprotein that plays a prominent role in wound healing, embryogenesis, and hemostasis (1Hynes, R. (1990) in Fibronectins, (Rich, A., ed) pp. 1–175, Springer-Verlag, BerlinGoogle Scholar). It is found both in the extracellular matrix and in soluble form in blood plasma. The structure and diverse binding properties of FN have been much studied (2Potts J.R. Campbell I.D. Curr. Opin. Cell Biol. 1994; 6: 648-655Crossref PubMed Scopus (184) Google Scholar, 3Mao Y. Schwarzbauer J.E. Matrix Biol. 2005; 24: 389-399Crossref PubMed Scopus (601) Google Scholar). We have previously described a novel truncated form of FN (70 kDa) that was able to act as a potent migration-stimulating factor (MSF) (4Schor S.L. Schor A.M. Grey A.M. Rushton G. J. Cell Sci. 1988; 90: 391-399Crossref PubMed Google Scholar, 5Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar). MSF was originally identified in fetal and cancer patient fibroblasts (4Schor S.L. Schor A.M. Grey A.M. Rushton G. J. Cell Sci. 1988; 90: 391-399Crossref PubMed Google Scholar). In vitro, exposure of human dermal fibroblasts to MSF provokes a change in phenotype, causing cells to migrate into three-dimensional gels of native type I collagen (5Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar). Fulllength FN is, however, devoid of MSF-like activity, suggesting that cryptic sites are exposed in MSF (5Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar) as well as in certain N-terminal FN fragments (6Schor S.L. Ellis I. Dolman C. Banyard J. Humphries M.J. Mosher D.F. Grey A.M. Mould A.P. Sottile J. Schor A.M. J. Cell Sci. 1996; 109: 2581-2590Crossref PubMed Google Scholar).The N-terminal fragment of FN is composed of independently folded modules, the majority of which are classified as type I (Fn1), each consisting of ∼45 amino acids. Fn1 modules have five short β-strands that form two sheets and a small hydrophobic core; they are further stabilized by two disulfide bonds in a 1–3, 2–4 configuration (7Potts J.R. Campbell I.D. Matrix Biol. 1996; 15: 313-321Crossref PubMed Scopus (165) Google Scholar). These β-strands form two anti-parallel β-sheets that are linked together by a short loop of three amino acids. Four of the nine Fn1 modules contain a highly conserved Ile-Gly-Asp (IGD) sequence (see Fig. 1) within this loop. The motogenic activity of MSF on fibroblasts has been attributed to this loop since mutation of IGD to DGI in modules 7 and 9 abolishes MSF bioactivity (5Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar). Furthermore, soluble synthetic tri- and tetrapeptides containing the IGD amino acid motif can mimic MSF properties (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar). Like MSF, these peptides stimulate the migration of human dermal fibroblasts into three-dimensional type I collagen gels, although with much reduced potency; half maximal activity is observed with femtomolar and micromolar concentrations for MSF and IGD peptides, respectively (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar). The addition of Ser or Gln C-terminal to the IGD tripeptide was shown to enhance MSF-like activity by a factor of 10 (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar).The demonstration of bioactivity in a tripeptide is reminiscent of several other activation motifs. The Arg-Gly-Asp (RGD) cell adhesion motif in the tenth Fn3 module was the first tripeptide to be isolated and has been shown to interact with integrin receptors (9Ruoslahti E. Pierschbacher M.D. Science. 1987; 238: 491-497Crossref PubMed Scopus (3841) Google Scholar, 10Xiong J.P. Stehle T. Zhang R. Joachimiak A. Frech M. Goodman S.L. Arnaout M.A. Science. 2002; 296: 151-155Crossref PubMed Scopus (1382) Google Scholar). Synthetic peptides based on Leu-Asp-Val (LDV) and Arg-Glu-Asp-Val (REDV), found in the IIICS alternatively spliced site in FN, can mimic the activity of intact FN at least partially (11Mould A.P. Humphries M.J. EMBO J. 1991; 10: 4089-4095Crossref PubMed Scopus (165) Google Scholar, 12Humphries M.J. Akiyama S.K. Komoriya A. Olden K. Yamada K.M. J. Cell Biol. 1986; 103: 2637-2647Crossref PubMed Scopus (304) Google Scholar). Although the RGD sequence has been shown to bind integrins and promote cell adhesion and fibril formation, the exact binding partner for the IGD motif is unknown, but its ability to promote cell migration, which is abolished by antibody to αvβ3 (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar), suggests a possible interaction with integrins.Full-length fibronectin can form fibrils, and although much is known about promotion of fibrillogenesis by cells, little is known about the precise mechanism. Most models implicate integrin binding via the RGD recognition sequence. A recent report surprisingly showed that the absence of a functional RGD motif in FN did not compromise the assembly of FN fibrils in mutant embryos or cells. A possible explanation suggested was that FN contains a novel integrin binding motif in its N-terminal region (13Takahashi S. Leiss M. Moser M. Ohashi T. Kitao T. Heckmann D. Pfeifer A. Kessler H. Takagi J. Erickson H.P. Fassler R. J. Cell Biol. 2007; 178: 167-178Crossref PubMed Scopus (146) Google Scholar). The importance of the N-terminal 70-kDa fragment was also emphasized recently when it was shown to initiate FN fibril formation in fibronectin-null mouse fibroblasts (14Tomasini-Johansson B.R. Annis D.S. Mosher D.F. Matrix Biol. 2006; 25: 282-293Crossref PubMed Scopus (47) Google Scholar). These reports support the idea that fibrillogenesis might be initiated through integrin cell attachment at the N terminus via a binding site other than the RGD region.Here we set out to explore the structure-function relationships of the IGD sequence in the context of intact Fn1 domains. Of particular interest are the third and fourth IGD motifs in MSF, one in the seventh Fn1 (in this report these modules will be described using a superscript nomenclature: 7Fn1, etc.), and one in 9Fn1. Here we examine the role of the IGD motif by studying the properties of a series of fragments, namely the 8Fn1-9Fn1 and 7Fn1-8Fn1 module pairs, each containing one IGD motif, and the 7Fn1-8Fn1-9Fn1 fragment that contains two IGD motifs. The effects of site-specific mutations on migration and structure are examined using fibroblast migration assays and solution state NMR. Both IGD motifs are shown to have significant migration-stimulating activity while contained within fully folded fibronectin modules.EXPERIMENTAL PROCEDURESProtein Production—The human fibronectin modules 8Fn1-9Fn1 (residues 485–577), 7Fn1-8Fn1 (residues 437–528), and 7Fn1-8Fn1-9Fn1 (residues 437–577) were cloned into the Pichia pastoris expression vector pPICZλ. This vector was produced by transplanting the 54-bp EcoRI-XbaI fragment from the multiple cloning site of LITMUS 28i (New England Biolabs) into the corresponding restriction sites of pPICZαA (Invitrogen). All constructs included the conservative mutation R503K in the 8Fn1 module, to avoid cleavage during secretion by the endogenous Pichia protease KEX2 (15Millard C.J. Campbell I.D. Pickford A.R. FEBS Lett. 2005; 579: 4529-4534Crossref PubMed Scopus (17) Google Scholar). This single point mutant will be referred to as 8Fn1*-9Fn1. A second conservative point mutation (N497Q) was introduced into the 8Fn1 module to aid purification of the protein fragments, referred to as 8Fn1**-9Fn1, 7Fn1-8Fn1**, and 7Fn1-8Fn1**-9Fn1 (R503K,N497Q). Further point mutations were then introduced in these constructs to disrupt the IGD motifs. The following single mutations were introduced into 8Fn1**-9Fn1 to disrupt the IGD motif in 9Fn1: I541R, I541V, I541A, D543E, D543A, and S544Q. A single mutation, I449R, was introduced in 7Fn1-8Fn1** to disrupt the IGD motif in 7Fn1. The 9Fn1 IGD motif in 7Fn1-8Fn1**-9Fn1 was disrupted with mutation I541R, and both IGD motifs were disrupted using the I449R,I541R double mutant. The nomenclature 8Fn1**-9Fn1 (His) indicates a construct that contained a C-terminal His6 tag. A C-terminal His6 tag was present on all the 7Fn1-8Fn1** and 7Fn1-8Fn1**-9Fn1 constructs.Transformation by electroporation was performed according to standard Pichia protocols (Invitrogen) and expression of unlabeled and uniformly 15N-labeled protein was performed in a 1-liter fermenter (Electrolab Ltd., Tewksbury, UK) in an analogous fashion to that described previously for the 4F1-5F1 module pair (16Bright J.R. Pickford A.R. Potts J.R. Campbell I.D. Methods Mol. Biol. 2000; 139: 59-69PubMed Google Scholar). The protein was initially passed through a SP-Sepharose cation-exchange chromatography column at pH 3.0 to partially purify and reduce the volume of the secreted protein. High mannose sugars were trimmed back to single N-linked N-acetylglucosamine (GlcNAc) residues with Endo Hf at pH 5.5. High performance liquid chromatography (RP-HPLC) on a C4 column with a gradient of 24–38% acetonitrile and 1% trifluoroacetic acid was used to achieve homogeneity. The purity of the protein at each stage was assessed with SDS-PAGE, and the identity and final purity were confirmed by electrospray ionization mass spectroscopy (ESI-MS).Cells—Collagen gel migration experiments were performed with human skin fibroblasts FSF44. Stock cultures were maintained in Eagle's minimal essential medium (MEM) containing 15% donor calf serum, as previously described (17Schor S.L. J. Cell Sci. 1980; 41: 159-175Crossref PubMed Google Scholar).Migration Assays—The collagen gel assay was performed in 30-mm plastic culture dishes containing preformed 2 ml of type I collagen gel, as previously described (5Schor S.L. Ellis I.R. Jones S.J. Baillie R. Seneviratne K. Clausen J. Motegi K. Vojtesek B. Kankova K. Furrie E. Sales M.J. Schor A.M. Kay R.A. Cancer Res. 2003; 63: 8827-8836PubMed Google Scholar). 1 ml of serum-free MEM containing four times the desired final concentration of effector molecule and 1 ml of trypsinized fibroblasts (density 2 × 105 cells/ml) suspended in MEM containing 4% donor calf serum were plated in duplicate onto the collagen gel to give a final volume of 4 ml in 1% donor calf serum. After a 4-day incubation period at 37 °C, cell migration into the three-dimensional gel in response to the effector was determined as previously described (17Schor S.L. J. Cell Sci. 1980; 41: 159-175Crossref PubMed Google Scholar). In short, this involved using an inverted phase microscope to count the cells on the surface of the gel and then focusing down through the gel to count the cells within the gel. This operation was repeated with 15 randomly selected areas on the gel surface.NMR Spectroscopy—All NMR spectra were recorded at 25 °C using spectrometers built in-house, on samples of 1–2 mm protein in 150 mm NaCl, 20 mm sodium phosphate, 1 mm dioxane, 95% H2O, 5% D2O at pH 6.6. Backbone NH and Hα assignments have been deposited in the BioMagResBank under the accession number 15447 for 8Fn1*-9Fn1 (8Fn1*-9Fn1 (R503K)).RESULTSThe 8Fn1*-9Fn1 (R503K) Protein Is Heterogeneously Glycosylated—Our earlier study concluded that the wild type 8Fn1-9Fn1 was proteolytically cleaved by the P. pastoris endoprotease KEX2 between residues Arg-503 and His-504. The R503K mutation was therefore introduced to alleviate this proteolysis problem (15Millard C.J. Campbell I.D. Pickford A.R. FEBS Lett. 2005; 579: 4529-4534Crossref PubMed Scopus (17) Google Scholar). This 8Fn1*-9Fn1 module pair expressed in Pichia was found to be heterogeneously glycosylated at two sites; at least one of the sites was glycosylated and the different fractions could be separated by increasing the retention time on a C4 RP-HPLC column. We previously concluded that glycosylation of Asn-511 was critical for optimal binding to gelatin whereas glycosylation of Asn-497 had no significant effect on binding activity (15Millard C.J. Campbell I.D. Pickford A.R. FEBS Lett. 2005; 579: 4529-4534Crossref PubMed Scopus (17) Google Scholar). To increase the yield of homogeneous protein and to simplify the purification procedure, Asn-497 was mutated to remove the "non-essential" glycosylation site.The 7Fn1-8Fn1**, 8Fn1**-9Fn1 and 7Fn1-8Fn1**-9Fn1 Mutants (R503K,N497Q) Are Singly Glycosylated—The mutation N497Q was introduced into all constructs used in this study (with the exception of 8Fn1*-9Fn1, which was used as a control) as it was judged to be a suitable conservative mutation to remove the Asn-497 glycosylation site. Approximately 80 mg/liter of 8Fn1**-9Fn1 was expressed, as judged by SDS-PAGE of crude fermentation supernatant under non-reducing conditions (data not shown). This initial yield was comparable with that of the wild type and single point mutant (R503K) protein but the purification yield was greatly enhanced as the target protein was homogenously glycosylated. SDS-PAGE showed that the molecular mass was reduced from ∼16 to ∼11 kDa after treatment with Endo Hf, indicating the removal of a single high mannose sugar chain. N-terminal sequencing gave a single sequence DQCIVDDIY, and ESI-MS confirmed the expected molecular mass of 11,075 Da, indicating the presence of the complete module pair with a single GlcNAc sugar residue.The 8Fn1**-9Fn1 module pair was analyzed by NMR, both to compare the fold of this protein to that of the 8Fn1*-9Fn1 single point mutant and to study the sugar attachment site. The module pairs were isotopically labeled by replacing the sole nitrogen source in the fermenter vessel with (15NH4)2SO4. Both module pairs were expressed, purified, and characterized by ESI-MS, which gave an increased mass of 131 Da, corresponding to label incorporation of 98.5%. The cross-peaks of the 8Fn1**-9Fn1-GlcNAc511 and 8Fn1*-9Fn1-GlcNAc511 [1H-15N]-HSQC spectra (a single N-linked GlcNAc sugar residue on Asn-511) were shown to overlay as expected, with the exception of mutated residue Asn-497 (data not shown). Direct comparison of the 8Fn1**-9Fn1 spectrum and the 8Fn1*-9Fn1 spectrum allowed assignment of the 8Fn1**-9Fn1 cross-peaks. After this comparison, a C-terminal His tag was engineered onto the 8Fn1**-9Fn1 to aid identification of the module pair by Western blot. This addition did not significantly affect the NMR spectra other than by obvious addition of new peaks from mobile residues.The 8Fn1**-9Fn1 Module Pair Contains Typical Fn1 Folds and the IGD Motif Is Located in a Rigid Loop between Strand B and Strand C—The solution structures of several Fn1-type module pairs have already been calculated by NMR (18Potts J.R. Bright J.R. Bolton D. Pickford A.R. Campbell I.D. Biochemistry. 1999; 38: 8304-8312Crossref PubMed Scopus (37) Google Scholar, 19Rudino-Pinera E. Ravelli R.B. Sheldrick G.M. Nanao M.H. Korostelev V.V. Werner J.M. Schwarz-Linek U. Potts J.R. Garman E.F. J. Mol. Biol. 2007; 368: 833-844Crossref PubMed Scopus (30) Google Scholar). The secondary structure of the 8Fn1**-9Fn1 was mapped here using inter-strand NOEs. Both modules were found to have a typical type I module fold (Fig. 2A), and the observed hydrogen bonding pattern agrees well with existing structures. The position of the IGD motif in the 9Fn1 can be mapped to a tight turn between the B strand and the C strand, and a model based on the structure of the 2Fn1-3Fn1 pair confirms that the loop is accessible to the solvent (Fig. 2B). The heteronuclear {1H-}15N NOE was used to map the internal motion of residues on a subnanosecond time scale, showing the module pair to be relatively rigid, especially in the short linker between modules and in the IGD loop of the 9Fn1 (Fig. 2C). Those values lower than 0.65 indicate regions with fast internal mobility, the majority falling within the loop regions of the protein.FIGURE 2NMR structural analysis. A, secondary structure of 8Fn1**-9Fn1 module pair. Each module adopts a typical Fn1-type fold, with a two-stranded antiparallel β-sheet folded over a three-stranded β-sheet. Interstrand NOEs between protons are indicated by bold lines within the β-sheets. The three-dimensional NOESY strips (inset) show the interstrand NH-Hα and NH-NH NOEs between four of the residues in the 8F1 module. The position of the IGD loop is highlighted, and the mutated residues N497Q and R503K are starred. B, a three-dimensional model of the 8Fn1**-9Fn1 module pair based on the secondary structure data in Fig. 1 and the x-ray structure of the 2Fn1-3Fn1 module pair (19Rudino-Pinera E. Ravelli R.B. Sheldrick G.M. Nanao M.H. Korostelev V.V. Werner J.M. Schwarz-Linek U. Potts J.R. Garman E.F. J. Mol. Biol. 2007; 368: 833-844Crossref PubMed Scopus (30) Google Scholar). A short linker joins the two modules, and the IGD motif falls with a tightly constrained loop in the 9F1. The side chains of the tightly constrained IGD loop are highlighted (pyMOL). C, heteronuclear {1H-}15N NOE of 8Fn1*-9Fn1. The horizontal line indicates the module boundary, and the boxed area indicates the position of the IGD motif. The secondary structure is indicated in schematic form. The DE loop of the 9Fn1 remains unassigned due to intermediate exchange of the residues on the NMR time scale.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Expression, Purification, and Characterization of 8Fn1**-9Fn1 IGD Mutants—Conservative and non-conservative point mutations were introduced into 8Fn1**-9Fn1 to interrupt the IGD site in the 9Fn1 module. These constructs were labeled, expressed, and purified; their fold was examined by comparing each HSQC spectrum with that of the unmodified 8Fn1**-9Fn1 module pair. All seven mutants were equally soluble and were folded in the same way as shown by the limited 1HN and 15N chemical shift differences from the "wild-type" 8Fn1**-9Fn1 (supplemental Figs. S1 and S2). As expected, there were minor shifts of peaks from residues in the IGD and of residue Ser-544 which immediately follows. Other observed localized shifts were in the cross-strand residue Cys-530 (in strand A of 9F1) and Cys-558 (in strand D of 9F1); these side chains are predicted by structural homology models to point toward the IGD loop (data not shown). Apart from these minor local perturbations around the mutated residue, there was no evidence for a change in the overall fold.An Intact IGD Motif Is Required in 8Fn1**-9Fn1 for Stimulation of Fibroblast Migration—The effect of each module pair on the behavior of fibroblast cells was assessed in a collagen gel migration assay. Increasing concentrations of the module pairs were added to cells plated on the surface of three-dimensional gels of native type I collagen fibers. In a similar manner to that of MSF and IGD peptides (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar), 8Fn1**-9Fn1 with an intact IGD motif stimulated fibroblast migration into this matrix, in a bell-shaped dose-response fashion. Maximal bioactivity was expressed at concentrations of 100 pmol (Fig. 3A). As predicted from peptide studies (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar), the module pairs with a mutation at Ile-541 (VGD, AGD, RGD) did not affect cell migration (Fig. 3A). The DGI mutant was also unable to stimulate migration (Fig. 3B). In contrast, those module pairs carrying a mutation at Asp-543 (IGE and IGA) showed some bioactivity, but this was significantly reduced with respect to the intact IGD at the same protein concentrations (Fig. 3B). It has also been suggested that the fourth amino acid in the IGD loop could affect migration (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar). The amino acid immediately following the IGD was also mutated from Ser to Gln in 8Fn1**-9Fn1 but did not change the extent of cell migration (Fig. 3B).FIGURE 3The effects of 8Fn1**-9Fn1 module pairs on cell migration. Fibroblasts were plated on native collagen gels in the presence of intact IGD and mutant test proteins at the concentrations indicated. After a 4-day incubation period, the cells present on the surface and within the three-dimensional collagen matrix were counted. The percentage of cells that migrated into the gel is expressed relative to the control cultures. For clarity, the error bars are omitted for those results that fall within the error of the control, but are indicated by the shaded area. A, (R503K,N497) module pair; IGD (-♦-), VGD (-⋄-), AGD (-▴-), RGD (-□-). B, module pair; IGD (-▪-), IGE (-□-), IGA (-▴-), DGI (-⋄-), IGD (Q) {=8Fn1**-9Fn1 (S544Q)} (-X-), IGD′ {=8Fn1**-9Fn1 with C-terminal His tag} (-▵-), IGD (*) {=8Fn1*-9Fn1 (R503K)} (-○-).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The 8Fn1**-9Fn1 pair (R503K,N497Q) was shown to be as active as 8Fn1*-9Fn1 (R503K), indicating that the removal of the glycosylation site does not affect the bioactivity of the protein (Fig. 3B). 8Fn1**-9Fn1 (His) was also shown to exhibit the same bioactivity as the module pair without the His tag (Fig. 3B).The Relative Roles of IGD in 7Fn1-8Fn1** and 7Fn1-8Fn1**-9Fn1—The 7Fn1-8Fn1** and 7Fn1-8Fn1**-9Fn1 fragments were judged by SDS-PAGE to express with yields of ∼80 and 40 mg/liter, respectively. The lower yield of the module triple is presumably due to some misfolding, and therefore degradation, of this larger recombinant fragment. The identity of the proteins was confirmed by N-terminal sequencing and ESI-MS (expected molecular masses of 12,061 and 18,393 Da). The recombinant material was isotopically labeled with 15N and examined by NMR. The dispersion of cross-peaks in [1H-15N]-HSQC spectra was indicative of a fully folded protein.Following the results from the 8Fn1**-9Fn1 mutations the relative roles of the IGD motifs in 7F1 and 9F1 were explored by replacing IGD with RGD in 7Fn1-8Fn1** and 7Fn1-8Fn1**-9Fn1. The proteins were successfully expressed, purified to homogeneity, and characterized by Western blotting and ESI-MS. The two IGD motifs in 7Fn1-8Fn1**-9Fn1 were mutated in turn by introducing single point mutations I449R and I541R before introducing both mutations to create a double mutant. The structure of the mutants, before and after the Ile to Arg mutation, was judged not to have changed significantly by comparing resolved up-field methyl peaks in one-dimensional NMR spectra (data not shown).The IGD Motifs in 7F1 and 9F1 Can Stimulate Fibroblast Migration Independently—The 7Fn1-8Fn1** with the intact IGD stimulated fibroblast migration at the same concentration as the 8Fn1**-9Fn1 (Fig. 4). The 7Fn1-8Fn1**-9Fn1 with both IGD motifs stimulated fibroblast migration with a factor of 100 higher potency with maximal activity at 1 pmol/IGD. Mutation of the 7Fn1 IGD motif in 7Fn1-8Fn1** caused significant loss of bioactivity. Independent mutation of the seventh and the ninth IGD motifs in 7Fn1-8Fn1**-9Fn1 reduced the maximal bioactivity to the level of the 7Fn1-8Fn1**, but the activity was absent in the double mutant (Fig. 3B).FIGURE 4Fibroblast migration into collagen gels. The response to different constructs is shown, 7Fn1-8Fn1** and 7Fn1-8Fn1**-9Fn1. The percentage of cells that migrated into the three-dimensional collagen matrix after 4 days is expressed relative to the control cultures. 7Fn1-8Fn1**-9Fn1 (R503K,N497); 2 × IGD (-▪-), RGD 7Fn1 (-○-), RGD 9Fn1 (-x-), 2 × RGD (-□-). 7Fn1-8Fn1** module pair, IGD (-▴-), RGD (-▵-).View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONIn this study we have been able to extend the earlier work on the bioactivity of soluble synthetic IGD peptides (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar) to that of IGD motifs in the context of folded Fn1-type modules. Various Fn1 module fragments, including several IGD mutants, were produced in P. pastoris, and their ability to stimulate fibroblast migration was assessed in a collagen gel migration assay. All of the proteins produced had a similar fold, as judged by NMR. A 10,000-fold increase in potency was observed in moving from peptides to recombinant module pairs.The conservative I541V mutation was designed to mimic the VGD of the first Fn1-type module; it was unable to promote cell migration in the context of 8Fn1**-9Fn1. The I541R mutation was designed to introduce an RGD sequence, a motif that promotes cell adhesion when in other regions of fibronectin, e.g. 10Fn3 (9Ruoslahti E. Pierschbacher M.D. Science. 1987; 238: 491-497Crossref PubMed Scopus (3841) Google Scholar). This mutation did not, however, promote cell migration here; neither did I541A. These results suggest that the isoleucine is critical for the observed bioactivity.The bioactivity of 7Fn1-8Fn1** is very similar to that of 8Fn1**-9Fn1 (Figs. 3A and 4). Substitution of I449R in the 7Fn1-8Fn1** module pair also resulted in a protein devoid of migration-stimulating activity.About half the bioactivity remained in the protein with the conservative D543E mutation. Some activity also remained for the D543A mutation but it was significantly lower than the wild type 8Fn1**-9Fn1. These results suggest that the aspartate residue in the IGD is required for optimal bioactivity but is less critical than the isoleucine. The "reverse" mutant DGI was also devoid of motogenic activity.It has been suggested that the IGDS of 9Fn1 is more active than the IGDQ in 7Fn1 (8Schor S.L. Ellis I. Banyard J. Schor A.M. J. Cell Sci. 1999; 112: 3879-3888Crossref PubMed Google Scholar), but the mutation S544Q did not change the bioactivity of the protein. This finding suggests that the fourth amino acid is less important in the context of the 8Fn1**-9Fn1 module pair than in the unconstrained peptide form.The increased potency of the protein in comparison to the peptide is remarkable. Constraining the ends of the IGD motif in the 9Fn1 module may favor the orientation of ligand binding that accounts for migration stimulation at much lower concentrations. A cyclic IGD peptide containing 20 amino acids from around the IGD site is also more potent than a similar length unconstrained IGD peptide (data not shown). Cyclic RGD peptides can inhibit cell attachment at a 20-fold lower concentration than linear peptides (20Kumagai H. Tajima M. Ueno
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