Purification and characterisation of cysteine proteinases from human osteoclastomas

Abstract

The process of normal bone remodeling requires the continual resorption and replacement of bone mineral and bone matrix proteins. Excessive bone resorption is a feature of a number of pathological conditions. These include osteoporosis, rheumatoid arthritis and Paget's disease [l]. Bone resorption is accomplished by the osteoclast. Osteoclasts attached to bone are highly polarised cells [2]. The osteoclast attaches to bone through a peripheral attachment zone within which the surface (apical) membrane is thrown into complex ruffles. The space enclosed by the osteoclast apical surface and the bone surface is acidified, probably by the action of a proton pump, resulting in the dissolution of bone mineral. Lysosomal enzymes are released into the acidified subosteoclastic space where they are responsible for the degradation of bone matrix components. Bone matrix is largely composed of type I collagen with small amounts of types 111, V and VI collagens [31. In addition, bone contains a number of glycoproteins (e.g. osteopontin, osteonectin and bone sialoproteins) and proteoglycans [4]. In vitro studies using disaggregated osteoclasts have suggested that cysteine proteinases released by osteoclasts are responsible for the degradation of bone proteins 151. Investigations of the properties of osteoclast cysteine proteinases are hampered by the scarcity of osteoclasts in normal bone. As our starting material we have used osteoclastomas. These are primary bone tumours that contain large numbers of apparently normal osteoclasts. Using the synthetic peptide Z-PheArg-NHMec as substrate, we have separated six cysteine proteinase activities from osteoclastoma extracts by sequential chromatography on S-Sepharose, phenyl-Sepharose, heparin-Sepharose and Sephacryl S200HR. The proteinases have molecular weights ranging from 20,000 to 42,000 (determined by gel filtration). Z-Phe-Arg-NHMec was the preferred synthetic substrate. Four of the enzymes showed some activity towards Bz-Phe-Val-Arg-NHMec but only two demonstrated low activity against Z-Arg-Arg-NHMec. The kinetics of hydrolysis of these substrates gave values within the range expected for cathepsin B [61. All six activities were capable of degrading soluble and insoluble collagen. The pH optima for the hydrolysis of Z-Phe-Arg-NHMec and type I collagen by the six enzymes are shown in table 1. A variety of inhibitors of cysteine proteinases (eg. leupeptin, E-64, cystatin) were potent inhibitors of the osteoclastoma enzymes. The kinetics of inhibition by cystatin and the rate constants of inactivation by Z-PheTyr-(O-t-Bu)CHNz were similar to published values for cathepsin B [7,81. Immunolocalisation of cathepsin B in sections of osteoclastomas revealed that osteoclasts stained strongly with an antibody to cathepsin B whereas the neoplastic osteoblasts were only weakly stained. The Table 1 pH-Optima of osteoclastoma cysteiiie protciilases