Heparanases: endoglycosidases that degrade heparan sulfate proteoglycans

Karen J. Bame 0 0 Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City , Kansas City, MO 64110, USA Heparanases are endoglycosidases that cleave the heparan sulfate glycosaminoglycans from proteoglycan core proteins and degrade them to small oligosaccharides. Inside cells, these enzymes are important for the normal catabolism of heparan sulfate proteoglycans (HSPGs), generating glycosaminoglycan fragments that are then transported to lysosomes and completely degraded. When secreted, heparanases are thought to degrade basement membrane HSPGs at sites of injury or inflammation, allowing extravasion of immune cells into nonvascular spaces and releasing factors that regulate cell proliferation and angiogenesis. Heparanases have been described in a wide variety of tissues and cells, but because of difficulties in developing simple assays to follow activity, very little has been known about enzyme diversity until recently. Within the last 10 years, heparanases have been purified from platelets, placenta, and Chinese hamster ovary cells. Characterization of the enzymes suggests there may be a family of heparanase proteins with different substrate specificities and potential functions. - Interactions between the extracellular environment and the cell surface influence cell proliferation, differentiation, migration, and shape. Heparan sulfate proteoglycans (HSPGs) play an important role in these interactions, because they are components of both basement and plasma membranes (Iozzo et al., 1994; Bernfield et al., 1999). The anionic heparan sulfate (HS) glycosaminoglycan chains bind to extracellular matrix and cell surface proteins, providing a framework for matrix organization and cellcell or cellmatrix interactions. However, HSPGs play more than just a structural role. Both basement membrane and cell surface HSPGs bind a wide variety of protein ligands that are involved in wound repair, morphogenesis, host defenses, and lipid metabolism (Conrad, 1998; Bernfield et al., 1999). Numerous studies indicate that the proteinHS interactions are functionally important. Association of the ligand with HS glycosaminoglycans may activate or stabilize the ligand (Pillarisetti et al., 1997; Conrad, 1998; Lyon and Gallagher, 1998; Woods et al., 1998; Sperinde and Nugent, 1998) or be involved in directing the molecule to a different intracellular or extracellular location (Sperinde and Nugent, 1998; Tumova et al., 1999; Mahley and Ji, 1999). Therefore, to understand how these physiological processes are controlled, it is important to determine how the interactions between ligands and HSPGs are regulated. Much attention has been focused on the sequence of the HS glycosaminoglycan to which ligands bind. HS chains are originally synthesized as a polysaccharide of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid residues (GlcUA). In the Golgi, a series of enzymatic reactions occur that replace acetyl groups with sulfate groups, epimerize the glucuronic acid to iduronic acid (IdoUA), and add sulfate to the C-6 and C-3 hydroxyl groups of glucosamine and the C-2 hydroxyl groups of uronic acid residues (Lindahl et al., 1998). Because these modification reactions are incomplete, the final HS molecule has a domain structure (Lyon and Gallagher, 1998): N- and O-sulfate groups are clustered in IdoUA-rich sequences (S-domains or NS domains), which are separated by regions of [GlcNAc-GlcUA] disaccharide repeats (NA domains) that contain very little O-sulfate (Figure 1). Bridging these two domains are mixed sequences (or NA/NS domains) where GlcNAc disaccharides and GlcNS disaccharides alternate. In most HS species, S-domains range from 2 to 9 disaccharides and are separated by mixed and unmodified sequences that average 16 to 18 disaccharides. Ligands bind to the S-domains and mixed sequences (Conrad, 1998; Lyon and Gallagher, 1998); in some cases the ligand binds to a specific arrangement of sulfated glucosamine and uronic acid residues, and in others the interaction is primarily electrostatic. In either case, the formation of these S-domain sequences will determine whether a ligand can bind to the HSPG. Therefore, one way to regulate the interaction of a ligand with extracellular or cell surface proteoglycans is to regulate the synthesis of specific S-domains. Indeed, the ability of specific ligands to bind HSPGs can change on cell differentiation or age, Fig. 1. Heparan sulfate glycosaminoglycan structure and potential heparanase cleavage sites. Evidence suggests that Hpa1 heparanase cleaves a sequence within the highly modified S-domain (indicated by open arrow; Marchetti et al., 1997; Pikas et al., 1998), and studies with C1A heparanase suggest the enzyme cleaves within the mixed sequences (indicated by closed arrow; Bame and Robson, 1997; Bame et al., 2000). due to differences in the fine structure of the S-domains (Brickman et al., 1998; Feyzi et al., 1998; Molist et al., 1998). Another way to regulate the interaction of cell surface and matrix HSPGs with protein ligands is to degrade the HS glycosaminoglycans. This is done through the action of extracellular and intracellular endoglycosidases called heparanases. Heparanases can remove all binding sites from the proteoglycan by cleaving the HS chain from the core protein, or they can destroy specific ligand binding sites by cleaving within S-domains and/or mixed sequences. In addition to removing binding sequences, heparanases have been proposed to have other functions. Extracellular heparanases are believed to play roles in remodeling basement membranes after injury or at inflammation sites (Hoogewerf et al., 1995; Parish et al., 1998; Ihrcke et al., 1998; Dempsey et al., 2000) and in regulating cell growth and differentiation by releasing growth factors that are bound to extracellular HSPGs (Ishai-Michaeli et al., 1990). Heparanases in endosomes (Brauker and Wang, 1987; Yanagishita and Hascall, 1992; Tumova et al., 1999) are responsible for degrading internalized cell-associated HSPGs. In addition to generating short chain substrates for the lysosomal exoglycosidases (Kresse and Glssl, 1987), the intracellular heparanases may release bound ligands from their proteoglycan receptors once the complex is internalized (Tumova et al., 1999) or modulate the modification state of cell surface HSPGs by removing the HS chains from core proteins that recycle through the Golgi back to the plasma membrane (Edgren et al., 1997). The short HS oligosaccharides generated by either extracellular or intracellular heparanases may regulate the binding of ligands to cell surface or extracellular HSPGs, stabilize the ligand (Moscatelli, 1988; Pillarisetti et al., 1997), or facilitate the transport of the ligand to other sites of action (Sperinde and Nugent, 1998; Tumova et al., 1999). Degradation of HSPGs by heparanases may also be an important mechanism to prevent proteoglycans or long HS glyc (...truncated)