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

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

doi:10.1074/jbc.m115.674507

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.

Highlights

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
Design and Expression of Recombinant Bacterial Collagen with a discoidin domain receptors (DDRs)-binding Motif—A major DDR-binding site contained in fibrillar collagens I–III is the six amino acid motif GVMGFO, which was identified using triple-helical synthetic collagen-mimetic peptides [29, 30]
These six amino acids are sufficient for binding recombinant DDR ectodomains, as assessed by solid-phase binding assays, the activation of fulllength receptors on the surface of cells requires the presence of additional amino acids N-terminal to the GVMGFO motif; peptides presenting GPRGQOGVMGFO induce DDR autophosphorylation with similar kinetics to full-length collagen [29]

Summary

Introduction

A bacterial collagen-like protein Scl 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. Identification of specific ligand binding sequences in human collagen presents an opportunity to insert defined biological activities in a stable triple-helical scaffold provided by recombinant bacterial collagens such as Scl. Short sequences of tripeptides (Gly-Xaa-Yaa)n, where n ϭ 2 to 6, define a ligand-binding site, and experiments introducing the sequences for recognizing integrins, fibronectin, and heparin into the Scl triple-helical domain have demonstrated the expected biological activity, both in solid state binding assays and cell culture studies (11, 18 –20). The DDR family consists of two closely related receptors, DDR1 and DDR2, that are both activated by a number of different collagen types, in particular fibrillar collagens [24, 25] Both receptors play important roles in embryo development, and alterations in DDR function have been related to organ fibrosis, osteoarthritis, and tumor progression [22, 26, 27]. Peptide studies indicated additional binding sites, with sequences other than GVMGFO, in the fibrillar collagens for DDR2 but not DDR1 [29, 30]

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