Human Heparanase Is Localized within Lysosomes in a Stable Form Orit Goldshmidt,* ,3 Liat Nadav,§ ,3 Helena Aingorn,* Cohen Irit,* Naomi Feinstein, Neta Ilan, ¶, ‡ Eli Zamir,** Benjamin Geiger,** Israel Vlodavsky,* , ‡ ,1 and Ben-Zion Katz§ ,2 *Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem; §Department of Hematology, Tel-Aviv Sourasky Medical Center, Tel-Aviv; Electron Microscopy Unit, Institute of Life Sciences, Hebrew University of Jerusalem; ¶ InSight Ltd., Rehovot; **Department of Cellular and Molecular Biology, Weizmann Institute of Science, Rehovot; and ‡Vascular Biology Research Center, the Bruce Rappaport Faculty of Medicine, Technion, Haifa, 31096, Israel Heparanase is an endo--D-glucuronidase involved in degradation of heparan sulfate (HS) and extracellu- lar matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. The enzymatic activity of heparanase is characterized by specific intrachain cleavage of glycosidic bonds with a hydrolase mecha- nism. This enzyme facilitates cell invasion and hence plays a role in tumor metastasis, angiogenesis, inflam- mation, and autoimmunity. Although the expression pattern and molecular properties of heparanase have been characterized, its subcellular localization has not been unequivocally determined. We have previ- ously suggested that heparanase subcellular localiza- tion is a major determinant in regulating the enzyme’s biological functions. In the present study we examined heparanase localization in three different cell types, utilizing immunofluorescent staining and electron mi- croscopy. Our results indicate that heparanase is lo- calized primarily within lysosomes and the Golgi ap- paratus. A construct composed of heparanase cDNA fused to green fluorescent protein, utilized in order to visualize the enzyme within living cells, confirmed its localization in acidic vesicles. We suggest that follow- ing synthesis, heparanase is transported into the Golgi apparatus and subsequently accumulates in a stable form within the lysosomes, where it functions in HS turnover. The lysosomal compartment may also serve as a site for heparanase confinement within the cells, limiting its secretion and uncontrolled extracellular activities associated with tumor metastasis and angio- genesis. © 2002 Elsevier Science (USA) Key Words: heparan sulfate proteoglycans; hepara- nase; heparanase-GFP; lysosomes; signal peptide; MCF7 breast carcinoma. INTRODUCTION Heparan sulfate proteoglycans (HSPGs) on cell sur- faces and in basement membranes and extracellular matrices (ECM) affect biological processes by interact- ing with various ECM components and a large number of biologically active molecules [1, 2]. HSPGs can thus influence a variety of normal and pathological pro- cesses, involving cell adhesion, migration, and invasion [1– 4]. The importance and multifunctional roles of HSPGs in the physiology of cells and tissues make their cleavage an essential factor in the regulation of the integrity and functional state of organs. Enzymatic degradation of heparan sulfate (HS) is therefore likely to be involved in fundamental biological processes ranging from pregnancy, morphogenesis, and normal development to inflammation, angiogenesis, and can- cer metastasis [5–10]. Despite earlier reports on the existence of several distinct mammalian HS degrading endoglycosidases (heparanases), the cloning of the same gene by several groups suggests that mammalian cells express predominantly a single functional heparanase enzyme [6, 11–13]. The HS chains are cleaved by heparanase at only a few sites, yielding HS fragments of still appreciable size (10 –20 sugar units) [14]. The heparanase mRNA and protein are preferen- tially expressed in metastatic cell lines and human tumor tissues [6, 8 –12, 15]. Moreover, enhanced heparanase mRNA expression correlates with reduced postoperative survival of cancer patients [16, 17]. Overexpression of the heparanase cDNA in low-meta- static tumor cells conferred a high metastatic potential in experimental animals, resulting in an increased rate of mortality [6]. The heparanase enzyme also releases ECM-resident angiogenic factors in vitro and its over- expression induces an angiogenic response in vivo [18]. While several studies have reported heparanase clon- ing, molecular properties, expression, and involvement in cancer progression, heparanase regulation and sub- cellular localization were not thoroughly investigated. Heparanase activity was first isolated from rat liver lysosomes [19] and has been demonstrated in both 1 To whom correspondence and reprint requests should be addressed at Department of Oncology, Hadassah Hospital, POB 12000, Jerusalem 91120, Israel. Fax: 972-2-6422794. E-mail: vlodavsk@cc.huji.ac.il. 2 Dedicated to the memory of Professor Amiram Eldor (who died November 2001 in a tragic airplane crash), whose inspiration, wis- dom, and encouragement contributed to the accomplishment of this study. 3 These authors contributed equally to this study. 50 0014-4827/02 $35.00 © 2002 Elsevier Science (USA) All rights reserved. Experimental Cell Research 281, 50 – 62 (2002) doi:10.1006/excr.2002.5651