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

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.