The alternatively spliced type III connecting segment of fibronectin is a zinc-binding module

Abstract

Fibronectin (FN) is a prototypic adhesive glycoprotein that is widely expressed in extracellular matrices and body fluids. The fibronectin molecule is dimeric, and composed of a series of repeating polypeptide modules. A recombinant fragment of FN incorporating type III repeats 12-15, and including the alternatively-spliced type three connecting segment (IIICS), was found to bind Ni(2+), Cu(2+) and Zn(2+) divalent cations, whereas a similar fragment lacking the IIICS did not. Mutation of two pairs of histidine residues in separate spliced regions of the IIICS reduced cation binding to near the level of the variant lacking the IIICS, suggesting a zinc finger-like mode of cation coordination. Analysis of native FNs purified from plasma or amniotic fluid revealed significant levels of zinc associated with those isoforms that contain the complete IIICS. Taken together, these data demonstrate that the IIICS region of FN is a novel zinc-binding module.

Figure 1

Diagram of FN structure showing the recombinant fragments of the HepII/IICS region (H variants) used in this study. The figure depicts the structure of a full-length human FN subunit, with its component type I, II and III repeats, together with the regions subject to alternative splicing. The numbers assigned to each recombinant H variant refer to the number of amino acids remaining in the spliced IIICS. The figure also shows the amino acid sequence of the IIICS-A and IIICS-C regions. The histidine residues mutated in this study are underlined, and the cell adhesion motifs LDV and REDV highlighted.

Figure 2

Binding of H/120 fragments to Ni-NTA-agarose. A. 50μg of H/120 without His-tag were mixed with 80μl of Ni-NTA resin for 30 minutes. The unbound material was retained, the resin washed, and bound protein eluted with 100mM imidazole. Starting material (lane 1), unbound (lanes 2,3,4) and bound (lanes 5,6,7) protein was separated by SDS-PAGE and the gel stained with Coomassie Blue. Note that the gel gives a qualitative rather than quantitative analysis of the different fractions. B. Bands from unbound and bound fractions were excised, subjected to trypsin digestion, and analysed by tandem mass spectrometry. Peptides consistently identified in unbound (dark grey) and bound grey) samples are highlighted.

Figure 2

Binding of H/120 fragments to Ni-NTA-agarose. A. 50μg of H/120 without His-tag were mixed with 80μl of Ni-NTA resin for 30 minutes. The unbound material was retained, the resin washed, and bound protein eluted with 100mM imidazole. Starting material (lane 1), unbound (lanes 2,3,4) and bound (lanes 5,6,7) protein was separated by SDS-PAGE and the gel stained with Coomassie Blue. Note that the gel gives a qualitative rather than quantitative analysis of the different fractions. B. Bands from unbound and bound fractions were excised, subjected to trypsin digestion, and analysed by tandem mass spectrometry. Peptides consistently identified in unbound (dark grey) and bound grey) samples are highlighted.

Figure 3

Binding of H variants to Ni-NTA-agarose. 50μg of each H variant was mixed with 80μl of Ni-NTA-agarose for 30 minutes. The unbound material was retained, the resin washed, and bound protein eluted with 100mM imidazole. The starting material, bound and unbound fractions were analysed by SDS-PAGE. Numbers refer to recombinant H variant.

Figure 4

Binding of radioactive Ni²⁺ to H variants immobilised on nitrocellulose. 500 pmol of each H variant or ovalbumin was bound to nitrocellulose using a slot blotter, and endogenous cations removed by washing with EDTA. The membrane was incubated with 0.5μCi/ml ⁶³NiCl₂ for 10 minutes, washed, and exposed to a phosphorimaging plate. Results are expressed as percent binding relative to Ni²⁺ bound by H/120.

Figure 5

Binding of cations to H/120. A. H/120 was immobilised on nitrocellulose and incubated with ⁶³NiCl₂ in the presence of 1mM of a series of monovalent and divalent cations as chloride salts. Results are expressed as percent labelled Ni²⁺ binding to H/120 with no added cation. B. Dose response of Cu²⁺, Ni²⁺ and Zn²⁺ inhibition of Ni²⁺ binding to H/120. H/120 was immobilised on nitrocellulose and incubated with ⁶³NiCl₂ in the presence of varying concentrations of CuCl₂(■), NiCl₂ (●) and ZnCl₂(▲). Results are expressed as percent labelled Ni²⁺ binding to H/120 with no added cation.

Figure 5

Binding of cations to H/120. A. H/120 was immobilised on nitrocellulose and incubated with ⁶³NiCl₂ in the presence of 1mM of a series of monovalent and divalent cations as chloride salts. Results are expressed as percent labelled Ni²⁺ binding to H/120 with no added cation. B. Dose response of Cu²⁺, Ni²⁺ and Zn²⁺ inhibition of Ni²⁺ binding to H/120. H/120 was immobilised on nitrocellulose and incubated with ⁶³NiCl₂ in the presence of varying concentrations of CuCl₂(■), NiCl₂ (●) and ZnCl₂(▲). Results are expressed as percent labelled Ni²⁺ binding to H/120 with no added cation.

Figure 6

Binding of ⁶³Ni²⁺ to wild type and mutated H/120. 500 pmol of wild-type and His-mutated H/120 were immobilised on nitrocellulose and incubated with ⁶³NiCl₂. Results are expressed as percent labelled Ni²⁺ bound compared to wild type H/120.

Figure 7

Cell adhesion to wild-type and mutated H/120. Attachment of Molt-4 cells (A) and spreading of B16-F10 cells (B) on H/120 (■) and H/120(1278) (●). Background adhesion to BSA alone was subtracted. Results are expressed as percent cells attached or spread.

Figure 7

Cell adhesion to wild-type and mutated H/120. Attachment of Molt-4 cells (A) and spreading of B16-F10 cells (B) on H/120 (■) and H/120(1278) (●). Background adhesion to BSA alone was subtracted. Results are expressed as percent cells attached or spread.