• 2018-07
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  • 2019-10
  • Collagen type is the most abundant extracellular matrix


    Collagen type 1 is the most abundant extracellular matrix (ECM) protein found in vertebrates. Collagen exerts its role as a load bearing structure and regulator of cell function, only after it has assembled into fibrils., The equilibrium between the synthesis and degradation of collagen fibrils and fibers is critical for several physiological processes. An imbalance in the collagen fibril formation process (fibrillogenesis) can lead to pathological conditions such as fibrosis or increased cell migration as in AM 281 of tumor cells. It has recently being discovered that collagen-binding proteins can play a critical role in regulating the formation of fibrillar collagen type 1. Cell surface receptors such as integrins and secreted proteins such as decorin, fibromodulin and lumican have been reported to influence fibrillogenesis of collagen type 1.[3], 4., 5., 6., 7., 8. Discoidin domain receptors (DDR1 and DDR2) are cell-surface receptors that bind to and are activated by collagens, including collagen type 1., Both DDR1 and DDR2 are widely expressed in normal human tissues, and overexpressed in several malignancies. The extracellular domain (ECD) of DDRs is both necessary and sufficient for their binding to collagen.14., 15., 16. DDR1 and DDR2 are known to differ in their ligand specificities. While both DDRs bind to the native triple-helical structure of fibrillar collagens (types 1–3 and 5), only DDR1 binds to non-fibrillar collagen type 4. However, very little is known about how and where the ECDs of DDRs bind to collagen and their direct consequences on collagen regulation. In our previous studies, we established that DDR2 binds at a single site on the collagen type 1 molecule. AM 281 Recently another group has reported that the DDR2 binding site is confined to the D2 domain of collagen type 2 molecules. Here we investigated a functional significance of binding of DDR2 to collagen type 1. We have found that by binding to collagen, DDR2 affects the fibrillogenesis of collagen type 1. Our in vitro assay utilized DDR2-Fc fusion proteins, which contain only the ECD of DDR2. We employed surface plasmon resonance (SPR), turbidity measurements, biochemical assays, atomic force microscopy (AFM) and transmission electron microscopy (TEM) to determine the kinetics of DDR2–collagen interaction and analyze the role of DDR2 ECD in collagen fibrillogenesis. We confirmed that further oligomerization of DDR2-Fc greatly enhanced its binding to immobilized collagen type 1. DDR2 oligomers were found to have a direct impact on the fibrillogenesis of collagen type 1 by affecting the “lag-time” and by disrupting the native banded structure of collagen fibers. Further, using a cell-based assay we demonstrate that expression of DDR2 significantly affects the collagen fibrillogenesis process. We thus elucidate a novel mechanism by which the expression alone of DDR2 ECD may contribute to regulation of collagen via modulation of the fibrillogenesis process.
    Discussion Our work involves in vitro studies conducted by using purified DDR2-Fc proteins consisting of only the ECD of DDR2. Similar in vitro investigations carried out by other groups,,, using purified DDR ECD native or mutated proteins have established that binding of DDRs to collagen is controlled only by the DDR ECD and is in agreement with receptor activation studies. Our cell-based studies involving overexpression of DDR2 are in agreement with our in vitro studies and our results help elucidate a novel role of DDR2 on collagen fibrillogenesis. Our AFM data suggest that the DDR2 oligomers are nearly 3.2 times the size (volume) of DDR2-Fc proteins (which is more than the net size arising from an oligomer formed with two DDR2-Fc proteins bound to one antibody). Further, the SEC data show that 63% of DDR2-Fc and 78% of anti-Fc antibody is incorporated in the oligomer form. Based on these results we propose that each DDR2 oligomer is likely comprised of two DDR2-Fc (dimeric) proteins bound to one anti-Fc antibody with an additional antibody molecule attached to an unbound Fc portion of a DDR-Fc dimer. This oligomeric stoichiometry and molecular mass of DDR2-Fc dimers (determined using Western blotting) have been used to calculate the molarity of DDR2 in oligomeric solutions and to determine their dissociation constants by SPR.