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J. Biol. Chem., Vol. 280, Issue 14, 13568-13575, April 8, 2005

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Site-directed Mutagenesis, Proteolytic Cleavage, and Activation of Human Proheparanase^*

Ghada Abboud-Jarrous, Zehava Rangini-Guetta, Helena Aingorn, Ruth Atzmon, Sharona Elgavish, Tamar Peretz, and Israel Vlodavsky¶||

From the Department of Oncology, Hadassah-Hebrew University Hospital, Jerusalem 91120, the Bioinformatics Unit, Hebrew University-Hadassah Medical School, Jerusalem 91120, and the ^¶Cancer and Vascular Biology Research Center, The Bruce Rappaport Faculty of Medicine, Technion, P. O. Box 9649, Haifa 31096, Israel

Heparanase is an endo--D-glucuronidase that degrades heparan sulfate in the extracellular matrix and cell surfaces. Human proheparanase is produced as a latent 65-kDa polypeptide undergoing processing at two potential proteolytic cleavage sites, located at Glu¹⁰⁹-Ser¹¹⁰ (site 1) and Gln¹⁵⁷-Lys¹⁵⁸ (site 2). Cleavage of proheparanase yields 8- and 50-kDa subunits that heterodimerize to form the active enzyme. The fate of the linker segment (Ser¹¹⁰-Gln¹⁵⁷) residing between the two subunits, the mode of processing, and the protease(s) engaged in proheparanase processing are currently unknown. We applied multiple site-directed mutagenesis and deletions to study the nature of the potential cleavage sites and amino acids essential for processing of proheparanase in transfected human choriocarcinoma cells devoid of endogenous heparanase but possessing the enzymatic machinery for proper processing and activation of the proenzyme. Although mutagenesis at site 1 and its flanking sequences failed to identify critical residues for proteolytic cleavage, processing at site 2 required a bulky hydrophobic amino acid at position 156 (i.e. P2 of the cleavage site). Substitution of Tyr¹⁵⁶ by Ala or Glu, but not Val, resulted in cleavage at an upstream site in the linker segment, yielding an improperly processed inactive enzyme. Processing of the latent 65-kDa proheparanase in transfected Jar cells was inhibited by a cell-permeable inhibitor of cathepsin L. Moreover, recombinant 65-kDa proheparanase was processed and activated by cathepsin L in a cell-free system. Altogether, these results suggest that proheparanase processing at site 2 is brought about by cathepsin L-like proteases. The involvement of other members of the cathepsin family with specificity to bulky hydrophobic residues cannot be excluded. Our results and a three-dimensional model of the enzyme are expected to accelerate the design of inhibitory molecules capable of suppressing heparanase-mediated enhancement of tumor angiogenesis and metastasis.

Received for publication, November 29, 2004 , and in revised form, January 18, 2005.

^* This work was supported by Grant 532/02 from the Israel Science Foundation; by United States Public Health Services Grant RO1-CA106456-01 from NCI, National Institutes of Health; by The Susan G. Komen Breast Cancer Foundation; by The European Commission (5th Framework program, contract #QLKCT-2002-02049), and by a grant in the memory of Herbert and Frances Brody from Harvey M. Krueger, as trustee of a charitable trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed. Tel.: 972-4-829-5410; Fax: 972-4-852-3947; E-mail: vlodavsk{at}cc.huji.ac.il.

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