iso-DGR sequences do not mediate binding of fibronectin N-terminal modules to adherent fibronectin-null fibroblasts

Abstract

Fibronectin (FN) without an RGD sequence (FN-RGE), and thus lacking the principal binding site for alpha5beta1 integrin, is deposited into the extracellular matrix of mouse embryos. Spontaneous conversion of (263)NGR and/or (501)NGR to iso-DGR possibly explains this enigma, i.e. ligation of iso-DGR by alphavbeta3 integrin may allow cells to assemble FN. Partial modification of (263)NGR to DGR or iso-DGR was detected in purified plasma FN by mass spectrometry. To test functions of the conversion, one or both NGR sequences were mutated to QGR in recombinant N-terminal 70-kDa construct of FN (70K), full-length FN, or FN-RGE. The mutations did not affect the binding of soluble 70K to already adherent fibroblasts or the ability of soluble 70K to compete with non-mutant FN or FN-RGE for binding to FN assembly sites. Non-mutant FN and FN-N263Q/N501Q with both NGRs mutated to QGRs were assembled equally well by adherent fibroblasts. FN-RGE and FN-RGE-N263Q/N501Q were also assembled equally well. Although substrate-bound 70K mediated cell adhesion in the presence of 1 mm Mn(2+) by a mechanism that was inhibited by cyclic RGD peptide, the peptide did not inhibit 70K binding to cell surface. Mutations of the NGR sequences had no effect on Mn(2+)-enhanced cell adhesion to adsorbed 70K but caused a decrease in cell adhesion to reduced and alkylated 70K. These results demonstrate that iso-DGR sequences spontaneously converted from NGR are cryptic and do not mediate the interaction of the 70K region of FN with the cell surface during FN assembly.

FIGURE 1.

Diagram of FN, proteolytic or recombinant FN fragments, and recombinant FN and 70K with different combinations of NGR or RGD mutations. Each subunit of FN dimer consists of 12 type 1 (thin rectangles), two type 2 (triangles), and 15 type 3 (thick rectangles) modules. The subunits are joined near the C termini by a pair of disulfide bridges. Sites of mutations are shown by white dots. Also included are the ranges of protein concentrations produced in multiple preparations as estimated by using purified plasma FN or proteolytic 70K as a standard in Western blotting.

FIGURE 2.

LTQ-MS/MS fragmentation sequencing (A) demonstrating both unmodified ²⁶³NGR (B) and modified ²⁶³iso-DGR or ²⁶³DGR (C) from proteolytic 70-kDa N terminus isolated from plasma FN. The b⁺¹ and y⁺¹ are labeled on B and C. The double-headed line indicates the b⁺¹ or y⁺¹ ion gap corresponding to residue 263.

FIGURE 3.

NGR sequences do not affect 70K binding to adherent FN-null fibroblasts. FN-null fibroblasts were allowed to adhere to coverslips coated with ¹FN3-C (4 μg/ml) (A) or LN (15 μg/ml) (B) for 1 (A) or 2 (B) h and then incubated with 70K, 70K-N263Q, 70K-N501Q, or 70K-N263Q/N501Q for 3 h. Cells were fixed and stained for 70K with 4D1.7 followed by FITC anti-mouse IgG. Bound protein was detected by fluorescence microscopy. The four proteins were added at a similar level (∼20 μg/ml) as determined by Western blotting. The result is representative of three sets of experiments. Bar = 20 μm.

FIGURE 4.

NGR sequences do not affect the ability of 70K to compete with FN or FN-RGE for binding to FN-null fibroblasts adherent to ¹FN3-C. FN-null fibroblasts adherent to ¹FN3-C were incubated with non-mutant FN (A) or FN-RGE (B) in the absence or presence of 1 μm of the following forms of recombinant 70K: wild type 70K, 70K-N263Q, 70K-N501Q, or 70K-N263Q/N501Q. All incubations were for 1 h and done in the presence of 100 mm NaBr, which was present in the 70K preparations to enhance solubility. Cells were fixed and stained with IST9 to ^AFN3, which was present in the recombinant FN or FN-RGE, but not in the substrate ¹FN3-C, and observed under the fluorescence microscope. The result is representative of two sets of experiments. Bar = 20 μm.

FIGURE 5.

NGR sequences do not affect FN binding to adherent FN-null fibroblasts. FN-null fibroblasts adherent to ¹FN3-C (A) or LN (B), as described in the legend for Fig. 4, were incubated for 3 h with recombinant non-mutant FN, FN-RGE, FN-N263Q/N501Q, or FN-N263Q/N501Q-RGE. Cells were fixed and stained for full-length FN with 4D1.7, detected by fluorescence microscopy. The four proteins were present in a similar level (∼1 μg/ml) as determined by Western blotting. The result is representative of three sets of experiments. Bar = 20 μm.

FIGURE 6.

Effects of RGD peptides in cell adhesion to 70K and in 70K binding to cell surface. A, cell adhesion assays of FN-null cells to VN, LN, 70K, or 70K-N263Q/N501Q were done with a high binding plate (CLS3590) as described under “Materials and Methods” with or without 1 mm Mn²⁺ and cycRGD peptide, linRGD peptide, or linRGE peptide during cell attachment. Adhesion is expressed as a fraction of the absorbance at 595 nm of wells coated with VN and without Mn²⁺ (1.0 corresponds to an absorbance at 595 nm of about 0.21 for FN-null cells). Error bars represent the S.E. (n = 2). B, FN-null fibroblasts adherent to ¹FN3-C were incubated with 70K or 70K-N263Q/N501Q in conditioned medium, or a non-infected conditioned medium control, in the absence or presence of 100 μm cycRGD peptide for 1 h. Cells were fixed and stained for 70K with 4D1.7 followed by FITC-anti-mouse IgG as described in the legend for Fig. 3. The result is representative of two sets of experiments. Bar = 20 μm.

FIGURE 7.

Adhesion of FN-null, GD25, or GD25-β1A cells to FN, VN, and different 70K constructs. A, adhesion to different 70K constructs. Cell adhesion assays of FN-null, GD25, or GD25-β1A cells were done with a high binding plate (CLS3590) as described under “Materials and Methods.” Adhesion is expressed as a fraction of the absorbance at 595 nm of wells coated with non-modified FN (1.0 corresponds to an absorbance at 595 nm of about 0.10 for GD25 cells, 0.10 for GD25-β1A cells, and 0.30 for FN-null cells). Error bars represent the S.E. of three separate experiments in which each condition was tested in triplicate. Parallel enzyme-linked immunosorbent assays demonstrated that similar amounts of 70K had adsorbed to all wells. *, p < 0.05 as compared with non-mutant 70K. B, adhesion of GD25 cells to VN, reduced and alkylated FITC-70K, or reduced and alkylated FITC-70K-N263Q/N501Q. Cell adhesion assays of GD25 cells were done with proteins coated in serial dilutions of 0.03, 0.1, 0.3, 1, 3, and 10 μg/ml. Adhesion is expressed as a fraction of the absorbance at 595 nm for VN at 10 μg/ml (corresponding to about 0.13). Error bars represent the S.E. (n = 3).