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 metalloproteinases, cathepsin B, and osteoclast-derived lysates. We had previously shown (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 collagenase rapidly attacks the native 59-kD type X molecule at two sites, rendering a final product of 32 kD. This 32-kD fragment, however, has a Tm of 43 degrees C due to a very high amino acid content, and thus remains helical at physiologic core temperature. We now report that the 32-kD product resists any further attack by several matrix metalloproteinases including interstitial collagenase, 92-kD gelatinase, and matrilysin. However, this collagenase-generated fragment can be readily degraded to completion by cathepsin B at 37 degrees C and pH 4.4. Interestingly, even under acidic conditions, cathepsin B cannot effectively attack the whole 59-kD type X molecule at 37 degrees C, but only the 32-kD collagenase-generated fragment. Most importantly, the 32-kD fragment was also degraded at acid pH by cell lysates isolated from murine osteoclasts. Degradation of the 32-kD type X collagen fragment by osteoclast lysates exhibited the following properties: (a) cleavage occurred only at acidic pH (4.4) and not at neutral pH; (b) the cysteine proteinase inhibitors E64 and leupeptin completely blocked degradation; and (c) specific antibody to cathepsin B was able to inhibit much of the lysate-derived activity. Based upon these data, we postulate that during in vivo endochondral bone formation type X collagen is first degraded at neutral pH by interstitial collagenase secreted by resorbing 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.
Sires
U I
UI
Department of Medicine, Washington University School of Medicine, Jewish Hospital, St. Louis, Missouri 63110, USA.
Schmid
T M
TM
Fliszar
C J
CJ
Wang
Z Q
ZQ
Gluck
S L
SL
Welgus
H G
HG
D000818:Animals; D002358:Cartilage, Articular / Q000378:metabolism; D002401:Cathepsin B / Q000378:metabolism*; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1; D008970:Molecular Weight; D010010:Osteoclasts / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D014162:Transfection
D015853:Cysteine Proteinase Inhibitors; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D002401:Cathepsin B; D017364:Collagenases; D020781:Matrix Metalloproteinase 1; D007930:Leucine; C024974:E 64