the role of the fibronectin igd motif in stimulating fibroblast migration

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    The role of the fibronectin IGD motif in stimulating fibroblast

    migration

    Christopher J. Millard1, Ian R. Ellis2, Andrew R. Pickford3, Ana M. Schor2, Seth L. Schor2, Iain D. Campbell1*

    1Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK 2Unit of Cell and Molecular Biology, University of Dundee, Dundee DD1 4HR, Scotland, UK

    3School of Biological Sciences, University of Portsmouth, Portsmouth PO1 2DY, UK Running title: IGD motifs in fibronectin and cell migration

    *Corresponding author. Department of Biochemistry, University of Oxford, South Parks Road, Oxford, Oxfordshire OX1 3QU, UK. Tel.: +44 1865 275346; Fax: +44 1865 275253; E-mail:

    iain.campbell@bioch.ox.ac.uk Abstract: The motogenic activity of migration stimulating factor (MSF), 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 100x more active than a fragment with one and up to 106x more than synthetic tetra peptides. 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) is a multifunctional, multidomain adhesive glycoprotein that plays a prominent role in wound healing, embryogenesis and haemostasis (1). 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 (2,3). We have previously described a novel truncated form of FN (70kDa), which was able to act as a potent migration-stimulating factor (MSF) (4,5). MSF was originally identified in fetal and cancer patient fibroblasts (4). In vitro, exposure of human dermal fibroblasts to MSF provokes a change in phenotype, causing cells to migrate into 3-dimensional gels of native type-I collagen (5). Full length FN is, however, devoid of MSF-like activity, suggesting that cryptic sites are

    exposed in MSF (5) as well as in certain N-terminal FN fragments (6). 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 approximately 45 amino acids. Fn1 modules have five short -strands that form two sheets and a small hydrophobic core and further stabilised by two disulphide bonds in a 1-3, 2-4 configuration (7). These -strands form two anti-parallel -sheets which 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 (5). Furthermore, soluble synthetic tri- and tetra-peptides containing the IGD amino acid motif can mimic MSF properties (8). Like MSF, these peptides stimulate the migration of human dermal fibroblasts into 3-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 (8). The addition of Ser or Gln C-terminal to the IGD tri-peptide was shown to enhance MSF-like activity by a factor of 10 (8). The demonstration of bioactivity in a tri-peptide is reminiscent of several other activation motifs. The Arg-Gly-Asp (RGD) cell adhesion motif in the tenth Fn3 module was the first tri-peptide to be isolated and has been shown to interact with integrin receptors (9,10). Synthetic peptides based on Leu-Asp-Val (LDV) and Arg-Glu-Asp-Val (REDV), found in the IIICS alternatively

    http://www.jbc.org/cgi/doi/10.1074/jbc.M707532200The latest version is at JBC Papers in Press. Published on October 5, 2007 as Manuscript M707532200

    Copyright 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

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    spliced site in FN, can mimic the activity of intact FN at least partially (11,12). While 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 v3 (8), 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 paper 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 (13). The importance of the N-terminal 70kDa fragment was also emphasized recently when it was shown to initiate FN fibril formation in fibronectin-null mouse fibroblasts (14). These papers 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 paper 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 effect 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 procedures Protein 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 pPICZ. 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 pPICZA (Invitrogen). All constructs included the conservative mutation R503K in the 8Fn1 module, to avoid cleavage during secretion by the endogenous Pichia protease KEX2 (15). 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 which 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 (www.invitrogen.com) and expression of unlabelled and uniformly 15N-labelled protein was performed in a 1-litre fermenter (Electrolab Ltd., Tewksbury, U.K.) in an analogous fashion to that described previously for the 4F15F1 module pair (16). The protein was initially passed through a SP-Sepharose cation-exchange chromatography column at pH3.0 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 pH5.5. 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 was confirmed by electrospray ionisation mass spectroscopy (ESI-MS).

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    Cells: Collagen gel migration experiments were performed with human skin fibroblasts FSF44. Stock cultures were maintained in Eagles Minimal Essential Medium containing 15% donor calf serum, as previously described (17). Migration assays: The collagen gel assay was performed in 30mm plastic culture dishes containing preformed 2ml type I collagen gel, as previously described (5). 1ml serum-free MEM containing 4x the desired final concentration of effector molecule, and 1ml trypsinised fibroblasts (density 2x105 cells/ml) suspended in MEM containing 4% donor calf serum, were plated in duplicate onto the collagen gel to give a final volume of 4ml in 1% donor calf serum. After a 4 day incubation period at 37C, cell migration into the 3-dimensi