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Disease-causing mutations in cartilage oligomeric matrix protein cause an unstructured Ca2+ binding domain
Quinn Kleerekoper1, Jacqueline T Hecht, John A Putkey
1Department of Biochemistry, Structural Biology Research Center, University of Texas, Houston Medical School, Houston, Texas 77030, USA.
The Journal of Biological Chemistry
|January 10, 2002
Summary
Calcium binding is crucial for cartilage oligomeric matrix protein (COMP) structure in pseudoachondroplasia and EDM1. Mutations disrupt calcium binding, leading to unfolded COMP and endoplasmic reticulum retention in chondrocytes.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Pseudoachondroplasia (PSACH) and multiple epiphyseal dysplasia (EDM1) are skeletal dysplasias.
- Chondrocytes in these conditions exhibit enlarged endoplasmic reticulum with accumulated extracellular matrix proteins, including COMP.
- Mutations are localized to the calcium (Ca2+) binding type 3 repeat domain of COMP.
Purpose of the Study:
- To investigate the role of Ca2+ binding in the structural integrity of the COMP type 3 repeat domain.
- To elucidate the structural consequences of disease-causing mutations in this domain.
- To understand the molecular basis of COMP mislocalization in PSACH and EDM1.
Main Methods:
- Expression of recombinant COMP type 3 repeat domain (T3) and its C-terminal region (T3-Cterm) in native and mutant forms.
- Biochemical characterization of Ca2+ binding capacity.
- Structural analysis using Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopy (1D proton, 2D 1H-15N HSQC).
Main Results:
- T3 and T3-Cterm bind significant amounts of Ca2+, with T3-Cterm adopting a distinct, largely non-helical conformation upon Ca2+ binding.
- Ca2+ removal leads to loss of structure, while Ca2+ binding promotes folding and stabilizes beta-sheet structures.
- A common mutation (Asp-470 deletion) significantly reduces Ca2+ binding and prevents Ca2+-dependent folding, resulting in an unstructured domain.
Conclusions:
- Ca2+ is essential for nucleating folding and maintaining the defined structure of the COMP type 3 repeat domain.
- Mutations impair Ca2+ binding and Ca2+-dependent folding, leading to an unstructured COMP.
- The persistent unstructured state of mutated COMP causes its retention in the endoplasmic reticulum of chondrocytes in PSACH and EDM1 patients.