Native r/r collagen forms fibrils similar to WT collagen but, unlike WT collagen, cannot be cleaved by interstitial collagenases.

(A) Native WT or r/r collagens (20 μg) were incubated in CAB for 18 hours at 24°C with or without 0.5 μg MMP-1 or MMP-2 that had been preactivated for 1 hour with 2 mM APMA at 37°C. Following incubation, the proteins were separated by reducing 8% SDS-PAGE and visualized with Coomassie blue. Three-quarter length fragments (TC^Afragments) appear in the collagenase-treated WT samples (lanes 2 and 3) but not in r/r samples (lanes 5 and 6). (B) WT or r/r collagens (20 μg) were heated to 60°C for 15 minutes to form gelatin prior to incubation with preactivated MMP-2 (0.5 μg) in CAB for the indicated times. Samples (20 μg) were separated by 12% SDS-PAGE under reducing conditions and visualized with Coomassie blue. A similar pattern of proteolytic fragments appears when heat-denatured WT or r/r collagen was treated with the MMP-2 gelatinase, indicating that the unique specificity in the r/r collagen lies in its resistance only to interstitial collagenases shown in panel A.