COMPLETE DEGRADATION OF TYPE-X COLLAGEN REQUIRES THE COMBINED ACTION OF INTERSTITIAL COLLAGENASE AND OSTEOCLAST-DERIVED CATHEPSIN-B

We have studied the degradation of type X collagen by metalloproteinas es, cathepsin B, and osteoclast-derived lysates, We had previously sho wn (Welgus, H, G,, C, J, Fliszar, J. L, Seltzer, T, M, Schmid, and J, J, Jeffrey, 1990, J. Biol. Chem, 265:13521-13527) that interstitial co llagenase rapidly attacks the native 59-kD type X molecule at two site s, rendering a final product of 32 kD, This 32-kD fragment, however, h as a T-m of 43 degrees C due to a very high amino acid content, and th us remains helical at physiologic core temperature, We now report that the 32-kD product resists any further attack by several matrix metall oproteinases including interstitial collagenase, 92-kD gelatinase, and matrilysin, However, this collagenase-generated fragment can be readi ly degraded to completion by cathepsin B at 37 degrees C and pH 4.4, I nterestingly, even under acidic conditions, cathepsin B cannot effecti vely attack the whole 59-kD type X molecule at 37 degrees C, but only the 32-kD coIlagenase-generated fragment. Most importantly, the 32-kD fragment was also degraded at acid pH by cell lysates isolated from mu rine osteoclasts, Degradation of the 32-kD type X collagen fragment by osteoclast lysates exhibited the following properties: (a) cleavage o ccurred only at acidic pH (4.4) and not at neutral pH; (b) the cystein e proteinase inhibitors E64 and leupeptin completely blocked degradati on; and (c) specific antibody to cathepsin B was able to inhibit much of the lysate-derived activity, Based upon these data, we postulate th at during in vivo endochondral bone formation type X collagen is first degraded at neutral pH by interstitial collagenase secreted by resorb ing cartilage-derived cells, The resulting 32-kD fragment is stable at core temperature and further degradation requires osteoclast-derived cathepsin B supplied by invading bone.