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:
[email protected] 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 β-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 β-strands form two anti-parallel β-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 αvβ3 (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 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 (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 Eagle’s 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 37°C, cell migration into the 3-dimensional gel in response to the effector was determined as previously described (17). 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 1-2mM protein in 150mM NaCl, 20mM sodium phosphate, 1mM dioxane, 95% H2O, 5% D2O at pH 6.6. Backbone NH and Hα assignments have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under the accession number 15447 for 8Fn1*-9Fn1 (8Fn1*-9Fn1 (R503K)). RESULTS The 8Fn1*-9Fn1 (R503K) protein is heterogeneously glycosylated Our earlier study concluded that the wild type 8Fn1-9Fn1 was proteolytically cleaved by the Pichia Pastoris endoprotease KEX2 between residues R503 and H504. The R503K mutation was therefore introduced to alleviate this proteolysis problem (15). 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 Asn511 was critical for optimal binding to gelatin whereas glycosylation of Asn497 had no significant effect on binding
activity (15). To increase the yield of homogeneous protein, and to simplify the purification procedure, Asn497 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 Asn497 glycosylation site. Approximately 80mg/l 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 to 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 weight was reduced from ~16kDa to ~11kDa 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 weight of 11075Da, indicating the presence of the complete module pair with a single GlcNAc sugar residue. The 8Fn1**-9Fn1 module pair was analysed 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 labelled by replacing the sole nitrogen source in the fermenter vessel with (15NH4)2SO4. Both module pairs were expressed, purified and characterised by ESI-MS, which gave an increased mass of 131Da, corresponding to label incorporation of 98.5%. The crosspeaks of the 8Fn1**-9Fn1-GlcNAc511 and 8Fn1*-9Fn1-GlcNAc511 [1H-15N]-HSQC spectra (a single N-linked GlcNAc sugar residue on Asn511), were shown to overlay as expected, with the exception of mutated residue N497 (data not shown). Direct comparison of the 8Fn1**-9Fn1 spectrum and the 8Fn1*-9Fn1 spectrum allowed assignment of the 8Fn1**-9Fn1 crosspeaks. 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.
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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 (18,19). 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 timescale, showing the module pair to be relatively rigid, especially in the short linker between modules and in the IGD loop of the 9Fn1 (Fig. 3). Those values lower than 0.65 indicate regions with fast internal mobility, the majority falling within the loop regions of the protein. Expression, purification and characterisation 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 labelled, expressed and purified; their fold was examined by comparing each HSQC spectrum with that of the unmodified 8Fn1**-9Fn1 module pair. All six 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 (Fig. S1 and S2). As expected, there were minor shifts of peaks from residues in the IGD and of residue S544 which immediately follows. Other observed localised shifts were in the cross-strand residue C530 (in strand A of 9F1) and C558 (in strand D of 9F1); these sidechains are predicted by structural homology models to point towards 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 behaviour 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 3-dimensional gels of native type I collagen fibres. In a similar manner to that of MSF and IGD peptides (8), 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 pmolar (Fig. 4A). As predicted from peptide studies (8), the module pairs with a mutation at I541 (VGD, AGD, RGD) did not affect cell migration (Fig. 4A). The DGI mutant was also unable to stimulate migration (Fig. 4B). In contrast, those module pairs carrying a mutation at D543 (IGE and IGA) showed some bioactivity but this was significantly reduced with respect to the intact IGD at the same protein concentrations (Fig. 4B). It has also been suggested that the fourth amino acid in the IGD loop could affect migration (8). 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. 4B). 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. 4B). 8Fn1**-9Fn1 (His) was also shown to exhibit the same bioactivity as the module pair without the His tag (Fig. 4B). 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 approximately 80mg/l and 40mg/l, respectively. The lower yield of the module triple is presumably due to some mis-folding, and therefore degradation, of this larger recombinant fragment. The identity of the proteins was confirmed by N-terminal sequencing and ESI-MS (expected molecular weights of 12061Da and 18393Da). The recombinant material was isotopically labelled with 15N and examined by NMR. The dispersion of crosspeaks 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 characterised by
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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 1D 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. 5). The 7Fn1-8Fn1**-9Fn1 with both IGD motifs stimulated fibroblast migration with a factor of 100 higher potency with maximal activity at 1 pmolar/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. 4B). DISCUSSION In this study we have been able to extend the earlier work on the bioactivity of soluble synthetic IGD peptides (8) 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 which promotes cell adhesion when in other regions of fibronectin e.g. 10Fn3 (9). 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 (Fig. 4A and 5) 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 (8), 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 favour the orientation of ligand binding which 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 (20). The insertion of RGD motif into mutant lysozyme significantly increased the cell adhesion activity (21), again implying that the introduction of conformational constraints can increase affinity to the integrin receptor, although a constrained RGD sequence in the IGD loop of Fn1 modules does not enhance cell migration. In the current study, the IGD motif as presented by the 9Fn module structure has markedly increased the bioactivity compared to free peptides. The heteronuclear {1H-}15N NOE data on 8Fn1*-9Fn1 here, as well as previous dynamics studies of 2Fn1-3Fn1 and 4Fn1-5Fn1 (18), show that the IGD motif is in a relatively immobile part of the module. This inflexibility in the IGD loop is quite different from the considerable flexibility of the RGD loop observed in 10F3 (22). We also explored possible cooperativity by studying the effect of two IGD motifs in one
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protein. Using a triple module construct (7Fn1-8Fn1**-9Fn1) containing an IGD motif in both 7Fn1 and 9Fn1. The maximal activity of two IGD motifs occurred at significantly lower concentration than a single IGD (the concentration dependence of the MSF activity of different peptides and proteins is summarised in Table 1). It is interesting to note that the gelatin binding domain, which contains the same two IGD motifs, is some 1000 fold more potent than the triple module (6). Migration stimulating activity was only lost from the triple module protein after mutation of both of the IGD sites, confirming that they are both able to stimulate migration independently as well as acting together. The profile of cell migration at different concentrations takes a bell shape suggesting that a threshold concentration is required to initiate cell migration and that a negative feedback mechanism occurs at higher concentrations. This is somewhat similar to a previous observation (23), where a 21kDa fragment of FN stimulated the migration of fibroblasts in a bell-shape dose-response pattern. We have previously shown (8) that fibroblast migration stimulated by IGD peptides is dependent on both αvβ3 functionality and tyrosine phosphorylation of FAK125. The reduced migration at high concentration may thus arise from effects on integrin clustering and/or subsequent downstream signalling. While integrin αvβ3 cannot be confirmed as the prime candidate receptor for the IGD motif, it is clear that the presence of αvβ3 and its downstream signalling components (P13 kinase and FAK125) are required for IGD motogenic activity (8). αvβ3 integrin also appears to cooperate with activated growth factor receptors, such as EGFR, in mediating their respective motogenic signalling in response to ligand binding (24). The RGD loop has been established as a key recognition sequence for αvβ3 (10), but changing IGD to RGD in Fn1 domains results in loss, not gain, of motogenic activity. This apparent discrepancy may be due to the inherent inflexibility of the RGD in this context A recent report suggested that Asn can spontaneously deamidate to isoD to create a DGR site (25) that can interact with αvβ3. There is a potential isoDGR in 7Fn1 that could constitute a binding ligand for the αvβ3. But
since the 8Fn1**-9Fn1 lacks the NGR that converts to isoDGA, and 7Fn1-8Fn1** and 8Fn1**-9Fn1 both exhibit the same potency in our assay, it is unlikely that this receptor-activation mechanism is part of the IGD response. Of course integrins could play a critical mechanistic role in mediating motogenic signalling by IGD ligation to some unknown cell surface receptor. The four IGD motifs at the N-terminus of FN are invariant in human, rat, and mouse (see Fig. 1). Three are also invariant in frogs, with a conservative mutation in the fourth. From the current work and knowledge of Fn1 module structure it seems likely that all these IGD motifs will display bioactivity as long as the Fn1 modules are exposed. Only two out of the four IGD motifs of MSF (7Fn1 and 9Fn1) appear to be active regarding stimulation of fibroblast migration (5). Consistent with this conclusion, a proteolytically-derived gelatin-binding domain of FN (GBD, Sigma), which contains both 7Fn1 and 9Fn1, displays motogenic activity, whereas the proteolytically-derived 1Fn1-5Fn1 (Sigma) was inactive (5). This suggests that 3Fn1 and 5Fn1 may not be exposed in MSF or 1Fn1-5Fn1. Even if all four IGD motifs were exposed and bioactive in MSF, a simple association between exposed IGD motifs and motogenic activity would not explain the huge differences in potency between MSF and IGD-containing peptides (Table 1). MSF (with four IGDs in 4 Fn1 modules) has been shown to stimulate fibroblast migration at 100 fold lower concentration than the gelatin-binding domain (GBD) (with two IGDs) (5,6). This suggests that IGD motifs in larger fragments can act cooperatively to give a much increased response. The enormous enhancements of IGD potency in different contexts is, however, not easy to explain. One possibility is that the increased bioactivity arises from a precise geometric presentation of the different binding motifs. Another more likely possibility is that several steps are involved; for example an initial binding to matrix, followed by receptor encounter and intracellular signalling. It is additionally possible that the IGD-containing Fn1 modules might partially denature the collagen triple helix, exposing RGD triplets, to which the αvβ3 integrin could then bind. This would explain the high potency of those IGD-containing Fn1 modules which have high collagen/gelatin binding activity, and distinguish them from the
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N-terminal domain, which has no motogenic activity. Of further interest is the observation that MSF displays proteolytic activity (26). Another intriguing observation is that full length FN does not show bioactivity (5). The usual explanation is that IGD motifs are hidden in assembled, intact FN but bioactive when exposed; for example, as shown here and previously (6), bioactivity appears in smaller fragments. Fibronectin fragments created by proteolysis exhibit many properties not seen in full length FN (27,28), and cryptic sites can be exposed simply by selective sequence deletion (29). Mechanical tension has also been shown to
unravel FN (30) and potential new sites can be revealed through conformational change and molecular rearrangement. The number of cryptic sites revealed at any one time could define the size and nature of the response. Previous studies have suggested that biological activities of fibronectin such as fibrillogenesis can be controlled by concealing active motifs within the tertiary structure (31). The 70kDa FN fragment has been shown to be critical in fibril assembly (32) and much work has gone into establishing the matrix assembly site. A possible explanation is that the IGD motifs studied here could also contribute to fibrillogenesis.
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A., Monnot, C., and Corvol, P. (2005) Int J Cancer 116(3), 378-384 27. Homandberg, G. A. (1999) Front Biosci 4, D713-730 28. Ingham, K. C., Brew, S. A., and Erickson, H. P. (2004) J Biol Chem 279(27), 28132-28135 29. Sechler, J. L., Rao, H., Cumiskey, A. M., Vega-Colon, I., Smith, M. S., Murata, T., and
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FOOTNOTES
Acknowledgements: We thank Tony Willis for N-terminal sequencing, Jonathan Boyd and Nick Soffe for technical support with the NMR instrumentation. This work was supported by the Wellcome Trust, the BBSRC and the EPSRC. The abbreviations used are: FN, fibronectin; MSF, migration stimulating factor; GBD, gelatin binding domain; 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.
FIGURE LEGENDS
Fig. 1. The modular structure of fibronectin, MSF and Fn1 type module fragments.
Fig. 2a. 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 beta sheets. The 3D-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.
Fig. 2b. A 3-dimensional model of the 8Fn1**-9Fn1 module pair based on the secondary structure data in figure 1 and the X-ray structure of the 2Fn1-3Fn1 module pair (19). 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 (Isoleucine = blue, Glycine = grey, Aspartate = red). (http://www.pymol.org).
Fig. 3. 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 cartoon form. The DE loop of the 9Fn1 remains unassigned due to intermediate exchange of the residues on the NMR timescale.
Fig. 4. The 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 3D-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) 8Fn1**-9Fn1** (R503K, N497) module pair; IGD (▬♦▬), VGD (-◊-), AGD (-▲-), RGD (-□-). B) 8Fn1**-9Fn1** module pair; IGD (▬■▬), IGE (-□-), IGA (-▲-), DGI (-◊-), IGD (Q) (-X-) {= 8Fn1**-9Fn1 (S544Q)}, IGD’ (-Δ-) {= 8Fn1**-9Fn1 with C-terminal His tag}, IGD (*) (-○-) {= 8Fn1*-9Fn1 (R503K)}.
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Fig. 5. Fibroblast migration into collagen gels in response to different 7Fn1-8Fn1** and 7Fn1-
8Fn1**-9Fn1 constructs. The percentage of cells that migrated into the 3D-collagen matrix after 4 days is expressed relative to the control cultures. 7Fn1-8Fn1**-9Fn1 (R503K, N497); 2 x IGD (▬■▬), RGD 7Fn1 (-○-), RGD 9Fn1 (-x-), 2 x RGD (-□-). 7Fn1-8Fn1** module pair; IGD (▬▲▬), RGD (-Δ-).
Table 1. Relative potency of IGD motifs in different contexts The peptide/protein concentration (pmolar) showing maximal motogenic activity in the fibroblast migration assay was divided by the number of IGD motifs in that construct. GBD = gelatin-binding domain, MSF = migration stimulating factor. IGD peptides 8Fn1**-9Fn1 7Fn1-8Fn1**-9Fn1 GBD MSF
106 102 1 10-4 10-6
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SUPPLEMENTARY
Fig. S1. Changes in the [1H-15N]-HSQC spectrum due to mutation D543E in 8Fn1**-9Fn1. The mutated residue D543 disappears from the 8Fn1**-9Fn1 spectrum and the significant shifts of residues surrounding the IGD site are indicated. Chemical environments are unchanged for the other residues and the module fold is unperturbed. Spectra were taken at pH6.6 and 25°C on a 750MHz spectrometer.
Fig. S2. Overlay of the 8Fn1**-9Fn1 and I541R mutant [1H-15N]-HSQC spectra. The majority of the I541R spectrum can be assigned unambiguously from the 8Fn1**-9Fn1 spectrum, and those residues in close to the IGD loop are indicated. The majority of residues are unaffected by the mutation and the module fold is unperturbed. Spectra were taken at pH6.6 and 15°C on a 750MHz spectrometer.
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and Iain D. CampbellChristopher J. Millard, Ian R. Ellis, Andrew R. Pickford, Ana M. Schor, Seth L. Schor
The role of the fibronectin IGD motif in stimulating fibroblast migration
published online October 5, 2007J. Biol. Chem.
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