Cartilage oligomeric matrix protein is a calcium-binding protein, and a mutation in its type 3 repeats causes conformational changes

Mutations in residues in the type 3 calcium-binding repeats and COOH-termin al globular region of cartilage oligomeric matrix protein (COMP) lead to tw o skeletal dysplasias, pseudoachondroplasia and multiple epiphyseal dysplas ia, It has been hypothesized that these mutations cause COMP to misfold and to be retained in the endoplasmic reticulum, However, this hypothesis is n ot supported by previous reports that COMP, when purified in the presence o f EDTA, shows no obvious difference in electron microscopic appearance in t he presence or absence of calcium ions. Since this discrepancy may be due t o the removal of calcium during purification, we have expressed wild-type C OMP and the most common mutant form found in pseudoachondroplasia, MUT3, us ing a mammalian expression system and have purified both proteins in the pr esence of calcium. Both proteins are expressed as pentamers, Direct calcium binding experiments demonstrate that wild-type COMP, when purified in the presence of calcium, is a calcium-binding protein, Rotary shadowing electro n microscopy and Limited trypsin digestion at various calcium concentration s show that there are conformational changes associated with calcium bindin g to COMP, Whereas COMB exists in a more compact conformation in the presen ce of calcium, it shows a more extended conformation when calcium is remove d. MUT3, with a single aspartic acid deletion in the type 3 repeats, binds less calcium and presents an intermediate conformation between the calcium- replete and calcium depleted forms of COMP, In conclusion, we show that a s ingle mutation in the type 3 repeats of COMP causes the mutant protein to m isfold, Our data demonstrate the importance of calcium binding to the struc ture of COMP and provide a plausible explanation for the observation that m utations in the type 3 repeats and COOH-terminal globular region lead to ps eudoachondroplasia.