Δ 469 mutation in the type 3 repeat calcium binding domain of cartilage oligomeric matrix protein (COMP) disrupts calcium binding

Cartilage oligomeric matrix protein (COMP/TSP5), a large glycoprotein found in the territorial matrix surrounding chondrocytes, is the fifth member of the thrombospondin (TSP) gene family. While the function of COMP is unknown, its importance is underscored by the finding that mutations in the highly conserved type 3 repeat domain causes two skeletal dysplasias. Pseudoachondroplasia (PSACH) and Multiple Epiphyseal Dysplasia, Fairbanks type (EDM1). The type 3 repeats are highly conserved low-affinity Ca²⁺binding domains that are found in all TSP genes. This study was undertaken to determine the effects of mutations on calcium binding and structure of the type 3 repeat domains. Wild-type (WT) and Δ469 recombinant COMP (rCOMP) proteins containing the entire calcium-binding domain were expressed in E. coli and purified. Equilibrium dialysis demonstrated that WT bound 10–12 Ca²⁺ions/molecule while Δ469 bound approximately half the Ca²⁺ions. Circular dichroism (CD) spectrometry had striking spectral changes for the WT in response to increasing concentrations of Ca²⁺. These CD spectral changes were cooperative and reversible. In contrast, a large CD spectral change was not observed at any Ca²⁺concentration for Δ469. Moreover, both WT and Δ469 proteins produced similar CD spectral changes when titrated with Zn²⁺, Cu²⁺and Ni²⁺indicating that the Δ469 mutation specifically affects only calcium binding. These results suggest that the Δ469 mutation, in the type 3 repeat region, interferes with Ca²⁺binding and that filling of all Ca²⁺binding loops may be critical for correct COMP protein conformation.