Recombinant Collagen Engineered to Bind to Discoidin Domain Receptor Functions as a Receptor Inhibitor

Bo Løfgren, Vittorio Abbonante, Huifang Xu, Despoina Gavriilidou, Ayumi Yoshizumi, Dominique Bihan, Richard W Farndale, David L. Kaplan, Alessandra Balduini, Birgit Leitinger, Barbara Brodsky

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)4343-55
Number of pages13
JournalJournal of Biological Chemistry
Volume291
Issue number9
DOIs
Publication statusPublished - Feb 26 2016

Keywords

  • Bacterial Proteins
  • Binding Sites
  • Cell Movement
  • Cells, Cultured
  • Collagen
  • Collagen Type III
  • Discoidin Domain Receptors
  • Fetal Blood
  • HEK293 Cells
  • Humans
  • Immobilized Proteins
  • Ligands
  • Megakaryocytes
  • Models, Molecular
  • Peptide Fragments
  • Protein Engineering
  • Protein Interaction Domains and Motifs
  • Receptor Protein-Tyrosine Kinases
  • Receptors, Mitogen
  • Recombinant Fusion Proteins
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Streptococcus pyogenes
  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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