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

Abstract

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 ex- change chromatography and characterized by disc elec- trophoresis, inhibition patterns, and action on proteogly- can. Three distinct metalloproteases are described. A neutral protease that digests proteoglycan subunit opti- mally 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 re- versed by cobalt, zinc, and ferrous ions. Two acid metal- loproteases, distinct from cathepsins Bi, D, and F, di- gest 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 ru- thenium red-staining matrix proteoglycan after incuba- tion in vitro with a partially purified cartilage extract at neutral pH. Sedimentation, gel chromatography, so- dium dodecyl sulfate-gel electrophoresis, and immuno- diffusion studies of digests of isolated proteoglycan frac- tion produced by the partially purified cartilage extract at neutral and acid pH confirmed that the cartilage en- zymes act only on the protein component of proteogly- can subunit, producing fragments with 5 to 12 chon- droitin sulfate chains. The link proteins were not digested.