RESUMEN
This work reports the first direct observations of binding and complex formation between transforming growth factor beta 1 (TGF-ß1) and cartilage oligomeric matrix protein (COMP) using high-resolution atomic force microscopy (AFM). Each COMP molecule consists of pentamers whose five identical monomeric units bundle at N-termini. From this central point, the five monomers' flexible arms extend outward with C-terminal domains at the distal ends, forming a bouquet-like structure. In commonly used buffer solutions, TGF-ß1 molecules typically form homodimers (majority), double dimers (minority), and aggregates (trace amount). Mixing TGF-ß1 and COMP leads to rapid binding and complex formation. The TGF-ß1/COMP complexes contain one to three COMP and multiple TGF-ß1 molecules. For complexes with one COMP, the structure is more compact and less flexible than that of COMP alone. For complexes with two or more COMP molecules, the conformation varies to a large degree from one complex to another. This is attributed to the presence of double dimers or aggregates of TGF-ß1 molecules, whose size and multiple binding sites enable binding to more than one COMP. The number and location of individual TGF-ß1 dimers are also clearly visible in all complexes. This molecular-level information provides a new insight into the mechanism of chondrogenesis enhancement by TGF-ß1/COMP complexes, i.e., simultaneous and multivalent presentation of growth factors. These presentations help explain the high efficacy in sustained activation of the signaling pathway to augment chondrogenesis.
Asunto(s)
Transducción de Señal , Factor de Crecimiento Transformador beta1 , Sitios de Unión , Proteína de la Matriz Oligomérica del Cartílago , Microscopía de Fuerza AtómicaRESUMEN
This work presents the first direct evidence of multivalent binding between bone morphogenetic protein-2 (BMP-2) and cartilage oligomeric matrix protein (COMP) using high-resolution 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 the 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 use of lower BMP-2 dosage for bone repair and regeneration.
Asunto(s)
Proteína Morfogenética Ósea 2/química , Proteína de la Matriz Oligomérica del Cartílago/química , Animales , Células Cultivadas , Células HEK293 , Humanos , Ratones , Microscopía de Fuerza Atómica , Unión Proteica , Conformación ProteicaRESUMEN
STUDY DESIGN: The aim of this study is to test the effect of cartilage oligomeric matrix protein (COMP) on enhancing rhBMP-2 induced spinal fusion in a prospective 8-week interventional trial of spinal fusion in rats. OBJECTIVE: To determine whether the amount of bone morphogenetic protein-2 (BMP-2) required to achieve spinal fusion in a pre-clinical model can be reduced by the addition of COMP. SUMMARY OF BACKGROUND DATA: BMPs are applied clinically at supraphysiological doses to promote spinal fusion by inducing osseous growth, but dose-related limitations include ectopic bone formation and local inflammatory reactions. COMP is a matricellular BMP-binding protein expressed during endochondral ossification and fracture healing. In vitro studies demonstrate enhanced activity of BMP bound to COMP. We hypothesized that BMP bound to COMP could achieve equivalent spinal fusion rates at lower doses and with fewer complications. METHODS: Posterolateral intertransverse process spinal fusion at L4 to L5 was performed in 36 Lewis rats. COMP (10âµg) was tested with or without "low-dose" rhBMP-2 (2âµg), and the results were compared with the "low-dose" (2âµg rhBMP-2) and "high-dose" (10âµg rhBMP-2) groups. All groups utilized insoluble collagen bone matrix carrier (ICBM). Fusion was evaluated by radiology, histology, and manual palpation. BMP release kinetics were evaluated in vitro. RESULTS: Fusion grading of microCT images demonstrated that the fusion rate with the COMP+LoBMP was statistically equivalent to HiBMP, and significantly better than LoBMP without COMP. These results were confirmed with radiographs and manual palpation. BMP release kinetics suggest that COMP increased local concentrations of BMP due to decreased growth factor retention on the scaffold. CONCLUSION: COMP enhances BMP-induced bone formation, enabling lower doses of BMP to achieve the same level of spinal fusion. COMP may function by affecting the availability and biological presentation of BMP-2. A decrease of BMP-2 required for fusion may reduce dose-related adverse effects, surgical costs, and improve clinical outcomes. LEVEL OF EVIDENCE: N/A.