Definition of the Native and Denatured Type II Collagen Binding Site for Fibronectin Using a Recombinant Collagen System*

Background: Sequence requirements for triple-helical collagen to bind fibronectin are not fully understood. Results: Fibronectin affinity was conferred on a recombinant bacterial collagen by incorporating specific human collagen II sequences. Conclusion: The chimeric collagen defines the minimum collagen II sequence required for fibronectin affinity. Significance: This system is useful to investigate sequence/activity relationships for triple-helical collagen and to generate scalable bioactive materials. 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.