Label-Free and Direct Visualization of Multivalent Binding of Bone Morphogenetic Protein-2 with Cartilage Oligomeric Matrix Protein

Abstract

This work presents the first direct evidence of multivalent binding between bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein (COMP) using highresolution atomic force microscopy (AFM) imaging. AFM topographic images reveal the molecular morphology of COMP, a pentameric protein whose five identical monomer units bundle together at N-termini, extending out with flexible chains to C-termini. Upon addition of BMP-2, COMP molecules undergo conformational changes at the C-termini to enable binding with BMP-2 molecules. AFM enables local structural changes of COMP to be revealed upon binding various numbers, 1-5, of BMP-2 molecules. These BMP-2/COMP complexes exhibit very different morphologies from those of COMP: much more compact and thus less flexible. These molecular level insights deepen current understanding of the mechanism of how BMP-2/COMP complex enhances osteogenesis among osteoprogenitor cells: i.e., multivalent presentation of BMP-2 via the stable and relatively rigid BMP-2/COMP complex could form a lattice of interaction between multiple BMP-2 and BMP-2 receptors. These ligand-receptor clusters lead to fast initiation and sustained activation of the Smad signaling pathway, resulting in enhanced osteogenesis. This work is also of translational importance, as the outcome may enable usage of lower BMP-2 dosage for bone repair and regeneration.

Direct Visualization of the Binding of Transforming Growth Factor Beta 1 with Cartilage Oligomeric Matrix Protein via High-Resolution Atomic Force Microscopy

“…Mica(0001) surfaces were chosen as the support because they are atomically flat structures. 4,22,28,29 In Figure 1, the characteristic AFM topographical images for all three systems are displayed side-by-side, using the same scanning size of 500 x 500 nm 2 In Figure 1A, 100 βl of 2 nM COMP solution was deposited onto mica(0001) for 2 minutes, followed by washing with milli-Q water and drying with compressed air before AFM imaging. Individual COMP molecules were clearly separated and visualized.…”

“…Consistent with prior reports, COMP molecules exist as a homo-pentameric glycoprotein composed of five identical units assembling together with N-termini at the centre and C-termini at the distal end. 4,[30][31][32] Molecules exhibited various conformations upon immobilization owing to the flexibility of its monomer arms and assembly. 4,5,30 The AFM morphology of COMP molecules is also consistent with prior X-ray crystallography of two truncated COMP and prior TEM studies of pentameric COMP, in which the N-termini of pentameric COMP bundles at the center while the C-terminus extends outward.…”

“…4,[30][31][32] Molecules exhibited various conformations upon immobilization owing to the flexibility of its monomer arms and assembly. 4,5,30 The AFM morphology of COMP molecules is also consistent with prior X-ray crystallography of two truncated COMP and prior TEM studies of pentameric COMP, in which the N-termini of pentameric COMP bundles at the center while the C-terminus extends outward. 5,9,27,32 In Figure 1B, 100 μl of 20 nM TGF-β1 solution was pipetted onto mica(0001) for 2 minutes and then washed and dried prior to AFM imaging.…”

“…[21][22][23][24][25][26] In fact, our prior work has demonstrated that AFM enabled high-resolution imaging of BMP-2/COMP complexes. 4 This work utilizes AFM to investigate the COMP and TGF-β1 systems to reveal the binding and structure of the complexes. The measured outcomes include direct observation of the proteins, and protein complex formation including TGF-β1 molecules within each complex.…”

“…Transforming growth factor beta 1 (TGF-β1) belongs to a superfamily of multifunctional growth factors that regulate a variety of biological functions, including cell proliferation, differentiation, and maturation. , It has been implicated as an important regulatory molecule during differentiation of mesenchymal stem cells (MSC) into chondrocytes for cartilage formation . Recent studies have suggested that the manner in which growth factors are presented to cell surface receptors is vital for regulating and enhancing differentiation. , Prior investigation by our team discovered that the mixtures of TGF-β1 with cartilage oligomeric matrix protein (COMP), an extracellular matrix component, elicited a greater enhancement on the signaling transduction activity than TGF-β1 alone . COMP (524 kDa) is a disulfide-bonded homopentameric glycoprotein found in the extracellular matrix of cartilage, tendons, bone tissues, and ligaments. , Its structure is composed of five identical monomers, each consisting of an N-terminal coiled-coil domain, four epidermal growth factor (EGF) repeats, eight thrombospondin-3 repeats, and a C-terminal domain. , The pentameric structure of COMP allows its simultaneous interaction with multiple entities, such as growth factors, ,, fibronectin, and collagen .…”