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

• Published in: Cell calcium (Edinburgh) 2000-06, Vol.27 (6), p.309-314
• Main Authors: Hou, J., Putkey, J.A., Hecht, J.T.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Calcium - metabolism; Cartilage - metabolism; Cartilage Oligomeric Matrix Protein; Centrifugation, Density Gradient; Circular Dichroism; Consensus Sequence; DNA Mutational Analysis; Edetic Acid - pharmacology; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix Proteins - genetics; Extracellular Matrix Proteins - metabolism; Gene Deletion; Genetic Vectors; Glycoproteins - genetics; Glycoproteins - metabolism; Humans; Matrilin Proteins; Molecular Sequence Data; Mutation; Protein Conformation; Protein Folding; Protein Structure, Secondary; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Structure-Activity Relationship
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1054/ceca.2000.0125

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.