Partial purification of heparanase activities in Chinese hamster ovary cells: evidence for multiple intracellular heparanases

Heparanases are mammalian endoglycosidases that cleave heparan sulphate gly cosaminoglycans from proteoglycan core proteins and degrade them into short er chains. The enzymes have been proposed to act in a variety of cellular p rocesses, including proteoglycan catabolism, remodelling of basement membra nes and release of heparan sulphate-binding ligands from their extracellula r storage sites. Additional functions for heparanases may be to generate sh ort heparan sulphate chains that stabilize or activate other proteins. Whil e heparanase activities have been described in a number of tissues and cell lines, it is not known how many different enzymes are responsible for thes e activities. Our recent studies characterizing the short glycosaminoglycan s produced in Chinese hamster ovary (CHO) cells suggested that multiple hep aranases are necessary for the formation of the short heparan sulphate chai ns [Bame and Robson (1997) J. Biol. Chem. 272, 2245-2251]. We examined whet her this is the case by purifying heparanase activity from CHO cell homogen ates. Based on their ability to bind ion-exchange resins and their elution from gel-filtration columns, four separate heparanase activities were parti ally purified. All four activities cleave free glycosaminoglycans over a br oad pH range of 3.5-6.0 or 6.5, suggesting that they act in the endosomal/l ysosomal pathway. The sizes of the short heparan sulphate chains generated by the partially purified heparanases ranged from 6 to 9 kDa, and for two o f the activities the product size is pH-dependent. Three of the four activi ties degrade proteoglycans as well as the free glycosaminoglycan chain. Int erestingly, all four enzymes generate short glycosaminoglycans with a sulph ate-rich, modified domain at the non-reducing end of the newly formed chain . Since our previous studies showed that in CHO cells there is also a popul ation of short heparan sulphates with a modified domain at the reducing end of the chain, this suggests that there may be another heparanase in CHO ce lls that was not purified. Alternatively, our findings suggest that the for mation of short heparan sulphate glycosaminoglycans inside CHO cells may be a result of the concerted action of multiple heparanases, and may depend o n the proportions of the different enzymes and the environment in which the chains are degraded.