Type I collagen triple helix destabilization caused by glycine and arginine mutations.

A, DSC denaturation thermograms of pepsin-purified collagen (solid lines) and procollagen (dotted lines) secreted into media by normal control (NC) and patient cells. B, Denaturation thermograms of pepsin-purified collagen from media (secreted, solid lines), cell layer (intracellular, dashed line), and extracellular matrix deposited by cells (matrix, dash-dotted lines). C,D, Analysis of α1(I)-R780C collagen thermal stability by enzymatic digestion and gel electrophoresis. In A and B, each thermogram peak represents denaturation of molecules with or without mutant chains. The peak maximum is the corresponding apparent denaturation temperature Tm, which is the same in collagen and procollagen molecules (for NC as well as most mutants) [27]. A heterozygous Gly substitution in the α1(I) chain might result in up to 3 peaks on the denaturation thermogram, representing molecules with 2, 1, and 0 mutant chains. In α1(I)-G766C collagen, the molecules with 1 and 2 mutant chains have the same Tm, producing one peak at ≈39.5 °C; the molecules without mutant chains produce the second peak at ≈42 °C. The same is true for α1(I)-G763S collagen. In α1(I)-R780L or α1(I)-R780C, molecules with 1 and 2 mutant chains produce a peak at ≈41 °C; the 42 °C peak of molecules without the mutant chains is too close to 41 °C, so that it is not resolved. The area under each peak is proportional to the fraction of the corresponding molecules. A change in this fraction upon secretion from cells or incorporation into matrix alters the shape of the denaturation thermogram [27]. A reduced intensity of the 39.5 °C peak in matrix vs. secreted collagen indicated that only 25% of α1(I)-G766C molecules in extracellular matrix contained mutant chains, but no effects of other mutations on collagen secretion or matrix incorporation were detected. In C and D, thermal stability of secreted collagen was measured by 2 min equilibration at different temperatures followed by 5 min at room temperature, 1 min digestion with trypsin/chymotrypsin mixture, and separation of chains by gel electrophoresis at non-reducing conditions as described in [31]. Panel C shows quantitative analysis of gel electrophoresis (D), each point representing an average of 4 experiments.