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J. Biol. Chem., Vol. 281, Issue 51, 39588-39597, December 22, 2006

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RGD-dependent Binding of Procathepsin X to Integrin _v3 Mediates Cell-adhesive Properties^*

Annette M. Lechner, Irmgard Assfalg-Machleidt, Stefan Zahler^¶, Mechthild Stoeckelhuber^||, Werner Machleidt, Marianne Jochum, and Dorit K. Nägler¹

From the Division of Clinical Chemistry and Clinical Biochemistry in the Department of Surgery, Ludwig-Maximilians-University and Adolf-Butenandt Institute, Ludwig-Maximilians-University, 80336 Munich, Germany, ^¶Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University, 81377 Munich, Germany and ^||Institute of Anatomy, Ludwig-Maximilians-University, 80336 Munich, Germany

Secreted lysosomal cysteine proteases (cathepsins) are involved in degradation and remodeling of the extracellular matrix, thus contributing to cell adhesion and migration. Among the eleven human lysosomal cysteine proteases, only procathepsin X contains an RGD motif located in a highly exposed region of the propeptide, which may allow binding of the proenzyme to RGD-recognizing integrins. Here, we have tested procathepsin X for cell-adhesive properties and found that it supports integrin _v3-dependent attachment and spreading of human umbilical vein endothelial cells. Using site-directed mutants of procathepsin X, we proved that this effect is mediated by the RGD sequence within the proregion of the protease. Endogenous procathepsin X is transported to the plasma membrane, accumulates in vesicles at lamellipodia of the human umbilical vein endothelial cell, and is partly associated with the cell surface, as shown by immunofluorescence. In addition, procathepsin X is partly co-localized with integrin ₃, as detected by immunogold electron microscopy. A direct interaction between endogenous procathepsin X and _v3 was demonstrated by co-immunoprecipitation. Moreover, surface plasmon resonance analysis revealed significant and RGD-dependent binding of procathepsin X to integrin _v3. Our results provide for the first time evidence that the extracellular function of cathepsin X may include binding to integrins thereby modulating the attachment of migrating cells to ECM components.

Received for publication, December 19, 2005 , and in revised form, September 29, 2006.

^* This work was supported by the Friedrich-Baur-Stiftung (0031/2003) and a graduate scholarship (Ludwigs-Maximilians-University, Munich) (to A. M. L.). 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: Div. of Clinical Chemistry and Clinical Biochemistry in the Dept. of Surgery, Ludwig-Maximilians-University, Nussbaumstr. 20, D-80336 Munich, Germany. Tel.: 49-89-5160-2555; Fax: 49-89-5160 4740; E-mail: dorit.naegler{at}med.uni-muenchen.de.