Alzheimer's disease is characterized by senile plaques composed of polymeric fibrils of beta amyloid (A␤), a 39 -42-amino acid peptide formed after proteolytic processing of the amyloid precursor protein (␤APP). Heparan sulfate proteoglycans have been shown to colocalize with A␤ in Alzheimer's disease brain, and experimental evidence indicates that the interactions between the proteoglycan and the peptide are important for the promotion, deposition, and/or persistence of the senile plaques. Studies in rat brain indicated that both the core protein and the heparan sulfate glycosaminoglycan chains are required for amyloid fiber formation and deposition in vivo (Snow, A. D., Sekiguchi, R., Nochlin, D., Fraser, P., Kimata, K., Mizutani, A., Arai, M., Schreier, W. A., and Morgan, D. G. (1994) Neuron 12, 219 -234), suggesting that one mechanism to prevent the formation of A␤-heparan sulfate proteoglycan complexes that lead to deposition of amyloid would be to degrade the proteoglycan. Normally, heparan sulfate proteoglycans are internalized and degraded to short glycosaminoglycans by intracellular heparanases. These reactions occur in the endosomal-lysosomal pathway, which is the same intracellular location where ␤APP is processed to A␤. Using partially purified heparanase activities from Chinese hamster ovary cells we examined whether A␤(1-40) affects the catabolism of Chinese hamster ovary heparan sulfate glycosaminoglycans and proteoglycans in vitro. A␤(1-40) binds to both the long heparan sulfate glycosaminoglycans attached to core proteins and the short, heparanase-derived chains in a concentration-dependent and pH-dependent manner. When A␤(1-40) is added to heparanase assays, it prevents the partially purified activities from releasing heparan sulfate chains from core proteins and degrading them to short glycosaminoglycans; however, a large molar excess of the peptide to heparan sulfate is required to see the effect. Our results suggest that normally the levels of A␤ in the endosomal pathway are not sufficient to interfere with heparanase activity in vivo. However, once the level of A␤-peptides are elevated, as they are in Alzheimer's disease, they could interact with heparan sulfate proteoglycans and prevent their catabolism. This could promote the formation and deposition of amyloid, since the binding of A␤ to the proteoglycan species will predominate.Histochemical and immunocytochemical studies have shown that heparan sulfate (HS) 1 proteoglycans (HSPGs) and glycosaminoglycans colocalize with ␤-amyloid protein (A␤) in the senile plaques characteristic of Alzheimer's disease (1). Although the precise role of the proteoglycans in the process of amyloidosis is not known, experimental evidence suggests that they may promote amyloid formation, deposition, and/or persistence (1) by binding to A␤ (2-5). HSPGs are anionic molecules consisting of one or more HS glycosaminoglycan chains covalently attached to a core protein (6). A␤ binds to HSPGs with high affinity, and interestingly, it interacts with both the core pr...

Heparanases are mammalian endoglycosidases that cleave heparan sulphate glycosaminoglycans from proteoglycan core proteins and degrade them into shorter chains. The enzymes have been proposed to act in a variety of cellular processes, including proteoglycan catabolism, remodelling of basement membranes and release of heparan sulphate-binding ligands from their extracellular storage sites. Additional functions for heparanases may be to generate short heparan sulphate chains that stabilize or activate other proteins. While heparanase activities have been described in a number of tissues and cell lines, it is not known how many different enzymes are responsible for these activities. Our recent studies characterizing the short glycosaminoglycans produced in Chinese hamster ovary (CHO) cells suggested that multiple heparanases are necessary for the formation of the short heparan sulphate chains [Bame and Robson (1997) J. Biol. Chem. 272, 2245–2251]. We examined whether this is the case by purifying heparanase activity from CHO cell homogenates. Based on their ability to bind ion-exchange resins and their elution from gel-filtration columns, four separate heparanase activities were partially purified. All four activities cleave free glycosaminoglycans over a broad pH range of 3.5–6.0 or 6.5, suggesting that they act in the endosomal/lysosomal pathway. The sizes of the short heparan sulphate chains generated by the partially purified heparanases ranged from 6 to 9 kDa, and for two of the activities the product size is pH-dependent. Three of the four activities degrade proteoglycans as well as the free glycosaminoglycan chain. Interestingly, all four enzymes generate short glycosaminoglycans with a sulphate-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 population 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 cells that was not purified. Alternatively, our findings suggest that the formation of short heparan sulphate glycosaminoglycans inside CHO cells may be a result of the concerted action of multiple heparanases, and may depend on the proportions of the different enzymes and the environment in which the chains are degraded.