Collagenase Digestion—To generate avian collagen XII molecules consisting only of the trimeric NC3 domain, i.e. lacking their collagenous parts as well as the NC1 and NC2 domains, the protein was subjected to collagenase digestion. A solution of 77 n collagen XII was incubated with 100 units/ml highly purified collagenase (CLSPA; Worthington Biochemicals) in TBS containing 5 m CaCl2 and 1 m 4-(2-aminoethyl)benzylsulfonyl fluoride (Roche Applied Science) for 4 h at37°C. Collagenase-treated collagen XII was immediately used for SDS-PAGE and solid phase binding assay. Alternatively, the NC3 domain was separated from collagenase and digestion fragments by running the sample over a gel filtration column (Superose 12 HR 10/30; GE Healthcare).
Surface Plasmon Resonance Spectroscopy—Surface plasmon resonance spectroscopy was performed using a Biacore 2000 (BIAcore AB) system. Avian tenascin-X, fulllength avian collagen XII, and the purified collagen XII NC3 domain were coupled in 25 m sodium acetate, pH 4.7, with a flow rate of 5 μl/min to a CM5 chip. The chip was previously activated with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride. After coupling the required amount of protein (∼1000 response units), unbound reactive groups were saturated with 1 ethanolamine hydrochloride, pH 8.5. Experiments were carried out using different concentrations (3, 10, 30, and 100 n) of avian tenascin-X and full-length collagen XII diluted in running buffer (20 m Hepes, 150 m NaCl, 2 m CaCl2, 0.005% P20). The analyte was passed over the sensor chip with a constant flow rate of 30 μl/min for 120 s, and dissociation was measured over 350 s. Fittings of the data, overlay plots, and calculation of KD values were done with BIAevaluation software 3.2.
Solid Phase Binding Assay—The production of recombinant avian tenascin-X (formerly tenascin-Y) has been described previously (). Tenascin-C and collagen XII were purified from chick embryo fibroblast-conditioned medium by monoclonal antibody affinity chromatography following established procedures (, ). To calculate the molar concentrations of the proteins utilized for titration measurements, the molecular masses of the monomeric forms of avian tenascin-X (205 kDa), avian tenascin-C (200 kDa), and the mean value of the small and large splice variants of avian collagen XII, estimating a molar ratio of 1:1 (274.5 kDa), were used. Purified proteins were diluted in TBS, pH 7.4, and 5 μg/ml (250 ng/well) were coated onto 96-well plates (Nunc Maxisorb) at 4 °C overnight. After washing with TBS, unspecific binding sites were blocked at room temperature with 5% skimmed milk powder in TBS for 2 h. Ligands were diluted in blocking buffer to concentrations from 0.03 to 300 n for the tenascins or 0.023 to 230 n for collagen XII and incubated for 1.5 h. For competition experiments the competitor was added to the ligand solution before incubation. After removing excess ligand by washing twice with TBS, bound ligand was fixed with 2.5% (v/v) glutaraldehyde for 10 min. Bound ligands were detected with specific affinity-purified antibodies, rabbit against chick collagen XII (522), chick tenascin-X (KX8), or chick tenascin-C (TN474) followed by swine anti-rabbit horseradish peroxidase-coupled IgG (Dako Cytomation). For enzymatic reaction, wells were incubated with 50 μl of 0.25 m tetramethylbenzidine and 0.005% (v/v) H2O2 in 0.1 sodium acetate, pH 6.0, for 10 min. The reaction was stopped with 50 ml/well 2.5 H2SO4, and the absorbance was measured at 450 nm using a microplate reader (Labsystems Multiscan MS). For analysis, measurements of wells treated equally, except for the addition of ligand, were subtracted as blank values. All buffers contained 2 m CaCl2.
For collagen XII, the function as a modulator of tissue biomechanical properties by bridging collagen I-containing fibrils to other extracellular matrix components has been suggested (, ). Our findings reported here support this concept. Avian tenascin-X interacts with the NC3 domain of avian collagen XII, thereby establishing a mechanical coherence of banded collagen fibrils with their extrafibrillar environment.