Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72-kDa type IV collagenase.

We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.