title: Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor.
abstract: A bacterial collagen-like protein Scl2 has been developed as a recombinant collagen model system to host human collagen ligand-binding sequences, with the goal of generating biomaterials with selective collagen bioactivities. Defined binding sites in human collagen for integrins, fibronectin, heparin, and MMP-1 have been introduced into the triple-helical domain of the bacterial collagen and led to the expected biological activities. The modular insertion of activities is extended here to the discoidin domain receptors (DDRs), which are collagen-activated receptor tyrosine kinases. Insertion of the DDR-binding sequence from human collagen III into bacterial collagen led to specific receptor binding. However, even at the highest testable concentrations, the construct was unable to stimulate DDR autophosphorylation. The recombinant collagen expressed in Escherichia coli does not contain hydroxyproline (Hyp), and complementary synthetic peptide studies showed that replacement of Hyp by Pro at the critical Gly-Val-Met-Gly-Phe-Hyp position decreased the DDR-binding affinity and consequently required a higher concentration for the induction of receptor activation. The ability of the recombinant bacterial collagen to bind the DDRs without inducing kinase activation suggested it could interfere with the interactions between animal collagen and the DDRs, and such an inhibitory role was confirmed in vitro and with a cell migration assay. This study illustrates that recombinant collagen can complement synthetic peptides in investigating structure-activity relationships, and this system has the potential for the introduction or inhibition of specific biological activities.
authors.affiliation: From the Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155.
authors.forename: Bo
authors.initials: B
authors.lastname: An
authors.affiliation: the Department of Molecular Medicine, Istituto di Ricerca e Cura a Carattere Scientifico San Matteo Foundation, University of Pavia, 27100 Pavia, Italy.
authors.forename: Vittorio
authors.initials: V
authors.lastname: Abbonante
authors.affiliation: the Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Huifang
authors.initials: H
authors.lastname: Xu
authors.affiliation: the Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom.
authors.forename: Despoina
authors.initials: D
authors.lastname: Gavriilidou
authors.affiliation: the Department of Microbiology and Infectious Diseases, Faculty of Medicine, Toho University School of Medicine, Tokyo 143-8540, Japan, and.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Yoshizumi
authors.affiliation: the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom.
authors.forename: Dominique
authors.initials: D
authors.lastname: Bihan
authors.affiliation: the Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, United Kingdom.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Farndale
authors.affiliation: From the Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155.
authors.forename: David L
authors.initials: DL
authors.lastname: Kaplan
authors.affiliation: From the Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, the Department of Molecular Medicine, Istituto di Ricerca e Cura a Carattere Scientifico San Matteo Foundation, University of Pavia, 27100 Pavia, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Balduini
authors.affiliation: the Molecular Medicine Section, National Heart and Lung Institute, Imperial College London, London SW7 2AZ, United Kingdom, b.leitinger@imperial.ac.uk.
authors.forename: Birgit
authors.initials: B
authors.lastname: Leitinger
authors.affiliation: From the Department of Biomedical Engineering, Tufts University, Medford, Massachusetts 02155, barbara.brodsky@tufts.edu.
authors.forename: Barbara
authors.initials: B
authors.lastname: Brodsky
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024061:Collagen Type III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000070818:Discoidin Domain Receptors; D005312:Fetal Blood / Q000166:cytology; D057809:HEK293 Cells; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013297:Streptococcus pyogenes
chemical_list: D001426:Bacterial Proteins; D024061:Collagen Type III; D055767:Immobilized Proteins; D008024:Ligands; D010446:Peptide Fragments; D011975:Receptors, Mitogen; D011993:Recombinant Fusion Proteins; C423453:Scl2 protein, Streptococcus; D003094:Collagen; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases
keywords: binding; collagen; discoidin domain receptor; inhibition mechanism; peptides; protein chimera; recombinant protein expression; triple-helix