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J. Biol. Chem., Vol. 281, Issue 40, 30072-30080, October 6, 2006

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Both Cleavage Products of the mCLCA3 Protein Are Secreted Soluble Proteins^*

Lars Mundhenk, Marwan Alfalah, Randolph C. Elble^¶, Bendicht U. Pauli^||, Hassan Y. Naim, and Achim D. Gruber¹

From the Department of Veterinary Pathology, Freie Universitaet Berlin, Robert-von-Ostertag-Strasse 15, D-14163 Berlin, Germany, Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Bünteweg 17, D-30559 Hannover, Germany, ^¶Department of Pharmacology and Simmons Cooper Cancer Institute, Southern Illinois University School of Medicine, Springfield, Illinois 62702, and ^||Cornell University College of Veterinary Medicine, Department of Molecular Medicine, Cancer Biology Program, Ithaca, New York 14853

Members of the chloride channels, calcium-activated (CLCA) family of proteins and in particular the murine mCLCA3 (alias gob-5) and its human ortholog hCLCA1 have been identified as clinically relevant molecules in diseases with secretory dysfunctions including asthma and cystic fibrosis. Initial studies have indicated that these proteins evoke a calcium-activated chloride conductance when transfected into human embryonic kidney cells 293 cells. However, it is not yet clear whether the CLCA proteins form chloride channels per se or function as mediators of other, yet unknown chloride channels. Here, we present a systematic biochemical analysis of the posttranslational processing and intracellular trafficking of the mCLCA3 protein. Pulse-chase experiments after metabolic protein labeling of mCLCA3-transfected COS-1 or human embryonic kidney 293 cells revealed cleavage of a primary 110-kDa mCLCA3 translation product in the endoplasmic reticulum into a 75-kDa amino-terminal and a 35-kDa carboxyl-terminal protein that were glycosylated and remained physically associated with each other. Confocal fluorescent analyses identified both cleavage products in vesicles of the secretory pathway. Neither cleavage product was associated with the cell membrane at any time. Instead, both subunits were fully secreted into the extracellular environment as a soluble complex of two glycoproteins. These results suggest that the two mCLCA3 cleavage products cannot form an anion channel on their own but may instead act as extracellular signaling molecules. Furthermore, our results point toward significant structural differences between mCLCA3 and its human ortholog, hCLCA1, which is thought to be a single, non-integral membrane protein.

Received for publication, July 7, 2006 , and in revised form, July 25, 2006.

^* This study was supported by Deutsche Forschungsgemeinschaft Grants SFB 621 C8 (to H. Y. N.) and SFB 621 C6 (to A. D. G.). This work is part of the doctoral thesis of L. M. 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. Tel.: 49-30-838-62440; Fax: 49-30-838-62114; E-mail: gruber.achim{at}vetmed.fu-berlin.de.

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