Metalloproteases of human articular cartilage that digest cartilage proteoglycan at neutral and acid pH.

Abstract

A B S T R A C T Extracts of human articular cartilage contain proteases capable of degrading the proteoglycan component of cartilage matrix at neutral and acid pH. These enzymes have been partially purified by ion exchange chromatography and characterized by disc electrophoresis, inhibition patterns, and action on proteoglycan. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit optimally at pH 7.25 has been purified up to 900-fold. It is strongly inhibited by o-phenanthroline, a-2-macroglobulin, and egg white, and to a lesser extent by D-penicillamine and EDTA. Inhibition by chelating agents is reversed by cobalt, zinc, and ferrous ions. Two acid metalloproteases, distinct from cathepsins Bi, D, and F, digest proteoglycan subunit at pH 4.5 and 5.5. Both are inhibited by o-phenanthroline and activity is restored by cobalt, zinc, or ferrous ions. With electron microscopy, it was found that cartilage slices were depleted of ruthenium red-staining matrix proteoglycan after incubation in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, sodium dodecyl sulfate-gel electrophoresis, and immunodiffusion studies of digests of isolated proteoglycan fraction produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage enzymes act only on the protein component of proteoglycan subunit, producing fragments with 5 to 12 chondroitin sulfate chains. The link proteins were not digested.

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen

“…pH 7 and is inhibited by metal-binding agents, but not by inhibitors of serine proteinases nor by thiol-blocking agents, It is thus likely to be due to a neutral metal-dep-endent proteinase. Proteoglycan-degrading neutral metalloproteinases similar in several respects to the present one have been found in directly active form in human cartilage tissue (Sapolsky et al, 1976) or as a component of the secretion of rabbit bone-marrow macrophages or fibroblasts in culture , 1978Vaes et al, 1977; Huybrechts-Godin & G. Vaes, unpublished work). These enzymes are different from the two proteoglycan-degrading neutral serine proteinases (elastase and the 'chymotrypsin-like' enzyme, cathepsin G) present in human polymorphonuclear leucocytes (for a review, see Janoff, 1975).…”

Further characterization of a neutral metalloprotease isolated from human articular cartilage

“…The only metals that form such a complex are Fe2+, Co2+, and Cu+ . These together with our previous observations (1) indicate that the metal involved is a heavy metal, more likely Zn2+ rather than Co2+ or Fe2+. EDTA inhibited more poorly than o-phenanthroline.…”

“…Chromatography on a 2x85-cm column was as previously described (1). The elution pattern with sodium acetate buffer was similar to that of the previous chromatography (1). Most of the neutral protease activity emerged as a single peak at 0.1M buffer strength.…”

Sodium NMR evaluation of articular cartilage degradation

“…In response to IL-1, chondrocytes release metalloproteinases that degrade proteoglycans in the cartilage (3)(4)(5)(6)(7). One of the earliest effects of degenerative osteoarthritis is the loss of proteoglycans from articular cartilage (5)(6)(7).…”