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J. Biol. Chem., Vol. 280, Issue 41, 34513-34520, October 14, 2005

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Identification of Hepatocyte Growth Factor Activator Inhibitor-1B as a Potential Physiological Inhibitor of Prostasin^*

Bin Fan, Thomas D. Wu, Wei Li, and Daniel Kirchhofer1

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

Prostasin is a trypsin-like serine protease that is glycosylphosphatidylinositol-anchored to the epithelial cell surface, from where it can be released in a soluble form. We undertook a co-expression search using the Genesis Enterprise System Database from Gene Logic to identify prostasin inhibitors, on the assumption that prostasin and its natural inhibitors may have a similar gene expression pattern. We found the expression profile of prostasin in normal human tissues to correlate highly with hepatocyte growth factor activator inhibitor-1B (HAI-1B) and its splice variant HAI-1. Soluble HAI-1B (sHAI-1B), comprising the entire extracellular domain, formed a 1:1 complex with purified prostasin in protein binding assays and inhibited prostasin enzymatic activity with an IC₅₀ of 66 ± 15 nM. Two sHAI-1B mutants with inactivated N- and C-terminal Kunitz domains (KD1 and KD2) were used to show that the interaction of sHAI-1B with prostasin is mediated by KD1. In agreement, KD1 (Thr²⁴⁶-Val³⁰³) alone potently inhibited prostasin activity (IC₅₀ = 4.7 ± 0.5 nM). Furthermore, prostasin was isolated with two major HAI-1/1B fragments (40 and 58 kDa) from OVCAR3 cell medium, demonstrating that prostasin·HAI-1/1B complexes are formed naturally. Moreover, when prostasin and HAI-1B were co-expressed in Chinese hamster ovary cells, complexes of prostasin with HAI-1B were detected on the cell membrane as well as in the culture medium, suggesting that preformed complexes were shed from the cell surface. The identification of HAI-1B as a potential physiological regulator of prostasin function, as described herein, may further the investigation of the role of prostasin in normal physiology and cancer.

Received for publication, February 24, 2005 , and in revised form, July 26, 2005.

^* 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. E-mail: dak{at}gene.com.

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