title: 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.
authors.affiliation: From the Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155 and.
authors.forename: Bo
authors.initials: B
authors.lastname: An
authors.forename: Vittorio
authors.initials: V
authors.lastname: Abbonante
authors.forename: Sezin
authors.initials: S
authors.lastname: Yigit
authors.forename: Alessandra
authors.initials: A
authors.lastname: Balduini
authors.forename: David L
authors.initials: DL
authors.lastname: Kaplan
authors.forename: Barbara
authors.initials: B
authors.lastname: Brodsky
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002417:Cattle; D002448:Cell Adhesion; D049109:Cell Proliferation; D024043:Collagen Type II / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D024043:Collagen Type II; D005353:Fibronectins; D011994:Recombinant Proteins
keywords: Binding; Collagen; Extracellular Matrix Proteins; Fibronectin; Gelatin; Protein Chimeras; Recombinant Protein Expression; Triple Helix