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J. Biol. Chem., Vol. 281, Issue 41, 30439-30446, October 13, 2006

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Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin^*

Paul Moran, Wei Li, Bin Fan, Rajesh Vij, Charles Eigenbrot, and Daniel Kirchhofer¹

From the Departments of Protein Engineering and Antibody Technology, Genentech, Inc., South San Francisco, California 94080

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys¹⁵⁸-Ile¹⁵⁹ (P₁-P₁') peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (k_cat/K_m 4.8 x 10⁵ M^–1 s^–1) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ± 1.9 nM uPA h^–1, which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.

Received for publication, June 7, 2006 , and in revised form, July 28, 2006.

^* 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.

¹ To whom correspondence should be addressed: Dept. of Protein Engineering, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080. Tel.: 650-225-2134; Fax: 650-225-6327; E-mail: dak{at}gene.com.

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