Changes in Thermal Stability and Microunfolding Pattern of Collagen Helix Resulting from the Loss of α2(I) Chain in Osteogenesis Imperfecta Murine

Abstract

Homozygous mutations resulting in formation of α1(I) 3 homotrimers instead of normal type I collagen cause mild to severe osteogenesis imperfecta (OI) in humans and mice. Limited studies of changes in thermal stability of type I homotrimers were reported previously, but the results were not fully consistent. We revisited this question in more detail using purified tendon collagen from wild-type (α1(I) 2α2(I) heterotrimers) and oim (α1(I) 3) mice as well as artificial α1(I) 3 homotrimers obtained by refolding of rat-tail-tendon collagen. We found that at the same heating rate oim homotrimers completely denature at ∼2.5 deg.C higher temperature than wild-type heterotrimers, as determined by differential scanning calorimetry. At the same, constant temperature, homotrimers denature ∼100 times slower than heterotrimers, as determined by circular dichroism. Detailed analysis of proteolytic cleavage at different temperatures revealed that microunfolding of oim homotrimers and wild-type heterotrimers occurs at similar rate but within a number of different sites. In particular, the weakest spot on the oim triple helix is located ∼100 amino acid residues from the C-terminal end within the cyanogen bromide peptide CB6. The same microunfolding site is also present in wild-type collagen, but the weakest spot of the latter is located close to the N-terminal end of CB8. Amino acid analysis and differential gel electrophoresis showed virtually no posttranslational overmodification of oim mouse tendon collagen. Moreover, thermal stability and microunfolding of artificial rat-tail-tendon homotrimers were similar to oim homotrimers. Thus, the observed changes are associated with difference in the amino acid composition of α1(I) and α2(I) chains rather than posttranslational overmodification.