A Cartilage Oligomeric Matrix Protein Mutation Associated with Pseudoachondroplasia Changes the Structural and Functional Properties of the Type 3 Domain

Abstract

Cartilage oligomeric matrix protein (COMP) is a member of the thrombospondin family of extracellular matrix glycoproteins. All members of the family contain a highly conserved region of thrombospondin type 3 sequence repeats that bind calcium. A mutation in COMP previously identified in a patient with pseudoachondroplasia resulted in abnormal sequestration of COMP in distinctive rER vesicles. The mutation, Asp-446 3 Asn, is located in the type 3 repeats of the molecule. This region was expressed in a mammalian culture with and without the mutation to study the structural or functional properties associated with the mutation. The biophysical parameters of the mutant peptide were compared with those of the wild type and revealed the following difference: secondary structural analysis by circular dichroism showed more ␣-helix content in the wild-type peptides. The calcium binding properties of the two peptides were significantly different; there were 17 calcium ions bound/wild-type COMP3 peptide compared with 8/mutant peptide. In addition, wild-type COMP3 had a higher affinity for calcium and bound calcium more cooperatively. Calcium bound by the wild-type peptide was reflected in a structural change as indicted by velocity sedimentation. Thus, the effect of the COMP mutation appears to profoundly alter the calcium binding properties and may account for the difference observed in the structure of the type 3 domain. Furthermore, the highly cooperative binding of calcium to COMP3 suggests that these type 3 sequence repeats form a single protein domain, the thrombospondin type 3 domain.

Disulfide Connectivity of Recombinant C-terminal Region of Human Thrombospondin 2

“…The structure of TSP2 is also Ca 2ϩ -sensitive based on its susceptibility to trypsin proteolysis (19). Sedimentation velocity experiments of TSP1 (13) and recombinant Ca 2ϩ -binding repeats of TSP5/COMP (20) revealed an increase in the sedimentation coefficient upon the addition of Ca 2ϩ , which is consistent with a change in structure upon Ca 2ϩ binding. Adhesion of cells to TSP1 (21,22) and TSP2 (19) is Ca 2ϩ -dependent, as is TSP1 inhibition of cathepsin G (23) and neutrophil elastase (24).…”

“…1); ESI, electrospray ionization; G2, recombinant C-terminal globule of TSP2 (residues 947-1172); LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; tCaG2, recombinant C-terminal portion of hTSP2 from within the Ca 2ϩ -binding repeats to the end of TSP2 (residues 718 -1172, Fig. 1 (18,20,29).…”

A Polymorphism in Thrombospondin-1 Associated with Familial Premature Coronary Artery Disease Alters Ca2+ Binding

“…Equilibrium dialysis of E3Ca indicated that 23-25 Ca 2ϩ ions bind to the Ca 2ϩ -binding repeats. This is higher than what has been observed for TSP-1, TSP-5, and TSP-5 constructs (13)(14)(15)(16)21) but is in agreement with the values predicted by the crystal structure (9) and what has been determined for TSP-2 constructs by atomic absorption spectroscopy (22). Trp-698, which is quenched upon the addition of Ca 2ϩ , is at position 7 of a putative motif (Fig.…”

COMP-assisted collagen secretion - a novel intracellular function required for fibrosis

“…Evidence for such intracellular protein complexes also comes from cartilage biopsies of individuals with chondrodysplasia caused by mutations in COMP (Maddox et al, 2000;Merritt et al, 2007). In these individuals and in cell culture models expressing selected mutant variants, the retention of misfolded COMP leads to coretention of other ECM components, including collagens (types II, IX and XI) (Hecht et al, 2005;Maddox et al, 1997;Vranka et al, 2001).…”