Definition of the native and denatured type II collagen binding site for fibronectin using a recombinant collagen system

Abstract

Interaction of collagen with fibronectin is important for extracellular matrix assembly and regulation of cellular processes. A fibronectin-binding region in collagen was identified using unfolded fragments, but it is not clear if the native protein binds fibronectin with the same primary sequence. A recombinant bacterial collagen is utilized to characterize the sequence requirement for fibronectin binding. Chimeric collagens were generated by inserting the putative fibronectin-binding region from human collagen into the bacterial collagen sequence. Insertion of a sufficient length of human sequence conferred fibronectin affinity. The minimum sequence requirement was identified as a 6-triplet sequence near the unique collagenase cleavage site and was the same in both triple-helix and denatured states. Denaturation of the chimeric collagen increased its affinity for fibronectin, as seen for mammalian collagens. The fibronectin binding recombinant collagen did not contain hydroxyproline, indicating hydroxyproline is not essential for binding. However, its absence may account, in part, for the higher affinity of the native chimeric protein and the lower affinity of the denatured protein compared with type II collagen. Megakaryocytes cultured on chimeric collagen with fibronectin affinity showed improved adhesion and differentiation, suggesting a strategy for generating bioactive materials in biomedical applications.

Keywords: Binding; Collagen; Extracellular Matrix Proteins; Fibronectin; Gelatin; Protein Chimeras; Recombinant Protein Expression; Triple Helix.

FIGURE 1.

Constructs of bacterial collagen-human collagen chimeras (SCl-Fn). Schematic diagram of type II collagen highlighting the putative fibronectin-binding region based on sequence homology with type I collagen (top panel), together with a diagram of the bacterial collagen constructs designed and produced (bottom panel). The constructs are named streptococcal collagen-like protein with Fn-binding site (SCl-Fn), supplemented with the starting and ending residue numbers of the inserted sequence in type II collagen. For example, the GLPGQRGER sequence is located in type II collagen triple-helix region from residue Gly⁷⁸⁴ to Arg⁷⁹², thus it is denoted SCl-Fn_G784-V792. The MMP cleavage site in the sequence is marked by ∧.

FIGURE 2.

A, SDS-PAGE of SCl-Fn recombinant collagen constructs. Top panel from left to right: marker, SCl-Fn_G784-R792, SCl-Fn_G781-R792, SCl-Fn_G778-R792, and SCl-Fn_G775-R792. Bottom panel from left to right: SCl-Fn_G772-V783, marker, SCl-Fn_G769-P786, SCl-Fn_G769-R789, marker, and SCl-Fn_G769-R792. Lower bands in SCl-Fn_G769-R789 and SCl-Fn_G769-R792 represent truncated products with a single CL unit. Triple-helical proteins migrate slower than normal in SDS-PAGE, thus the M_r prediction from the protein standard is higher than the actual M_w. B, examples of MALDI-TOF mass spectra showing M_r of SCl-Fn. SCl-Fn_G775-R792, top panel; and SCl-Fn_G769-R792, bottom panel.

FIGURE 3.

Structural and thermal analysis of SCl-Fn recombinant collagens. A, circular dichroism temperature scans showing the T_m values of SCl-Fn_G784-R792 (solid black), SCl-Fn_G781-R792 (solid red), SCl-Fn_G778-R792 (solid green), and SCl-Fn_G775-R792 (solid cyan) in the left panel and SCl-Fn_G772-V783 (dash black), SCl-Fn_G769-P786 (dash red), SCl-Fn_G769-R789 (dash green), and SCl-Fn_G769-R792 (dash cyan) in the right panel. The CD wavelength spectra of these proteins are shown in the inset. Insets share the same y axis label with the parent figures. B, differential scanning calorimetry of SCl-Fn_G775-R792 (green), SCl-Fn_G769-R792 (blue), and SCl-Fn_G784-R792 (red) as well as SCl control without insertion (black) showing heat capacity as a function of temperature. Areas under the curve indicate the calorimetric enthalpy. C, trypsin digestion of SCl-Fn_G775-R792, SCl-Fn_G778-R792, and SCl-Fn_G784-R792 on a time course of 0.5, 1, and 3 h and overnight (>16 h).

FIGURE 4.

Solid-phase binding assay of SCl-Fn recombinant collagen to soluble human plasma fibronectin. A, Fn binding affinity of all SCl-Fn constructs in both triple-helical (solid) and denatured (hatched) states. Two concentrations of fibronectin were used, 10 (blue) and 100 μg/ml (red). The binding signals of native SCl-Fn_G769-R792 and SCl-Fn_G775-R792 are statistically different from their respective signals in the denatured state, as evaluated using a paired t test; an asterisk (*) is used to indicate pairs where the means are statistically different (p < 0.05). B, binding of SCl-Fn_G769-R792 and SCl-Fn_G775-R792 to Fn prevented the recognition of Fn by the anti-Fn GBD antibody (black). A non-competitive anti-Fn C-terminal antibody (blue) was used to show the presence of Fn in ELISA plates with no competition to the gelatin-binding domain. Both native (solid) and denatured states (hatched) were tested. The beginning and ending residues of the inserted sequence are used to represent each SCl-Fn construct. Bovine collagen type II and recombinant collagen SCl with no insertion were used as control. C, soluble gelatin (denatured collagen type II) could inhibit the binding between collagen and fibronectin. When denatured collagen type II was incubated together with Fn in a collagen-coated plate, the binding signal (hatched green) observed were lower than the ones without gelatin inhibition (solid green).

FIGURE 5.

Dose-response of Fn binding to collagens adsorbed onto ELISA plates: SCl-Fn_G775-R792 (green), SCl-Fn_G769-R792 (red), bovine collagen type II (blue), and SCl with no insertion (black). Collagen in a native triple-helical state is represented by solid lines, whereas denatured collagen is represented by dashed lines.

FIGURE 6.

Megakaryocytes cultured on different SCl-Fn recombinant collagens. A, human mature MK were plated on two different SCl-Fn constructs: SCl-Fn_G769-R789, which did not bind Fn, and SCl-Fn_G769-R792, which did bind Fn. SCl-Fn-coated plates were preincubated with human cellular fibronectin prior to cell plating. After 16 h, MK showed higher adhesion and proplatelet formation on SCl-Fn_G769-R792 (right panel) compared with SCl-Fn_G769-R789 (left panel). In parallel controls were performed by seeding MK on uncoated and bovine type II collagen-coated plates. Arrows show proplatelet bearing MK. Scale bar = 100 μm. B, adherent cells were counted by phase-contrast microscopy (magnification ×40). Reported results are the mean ± S.D. of at least 10 randomly chosen fields in three independent experiments.