Abstract
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.
Highlights
Several members of the CLCA2 gene family have been shown to mediate an anion current that is activated by intracellular calcium
Prediction of CLCA transmembrane domains has mainly been based on Kyte-Doolittle analyses, leading consistently to CLCA protein conformation models with a single hydrophobic, amino-terminal, cleavable signal sequence, and four or five potential transmembrane domains [2, 4, 6, 7, 16, 18]
Endoglycosidase treatments of the carboxyl-terminal cleavage products revealed similar results; the 65-kDa carboxyl terminus in the cell lysate was sensitive to both Endo-acetylglucosaminidase H (endo H) and PNGase F treatments (Fig. 7B, left panels), and the 68-kDa carboxyl terminus released into the medium was sensitive to PNGase F only (Fig. 7B, right panel)
Summary
Computer-aided Analyses—The mCLCA3 amino acid sequence (GenBankTM accession number NP_059502) was screened for a signal sequence using the SignalP 3.0 software [25,26,27]. For immunoprecipitation and Western blot analysis of untagged mCLCA3 protein and for immunofluorescence microscopy, the rabbit polyclonal antibody anti-p3b was used. Western blot analyses of YFP-tagged mCLCA3 protein were carried out using the rabbit polyclonal antibodies anti-p3a and p3bs used which were generated against synthetic oligopeptides corresponding to amino acids 83 to 97 (p3a) or 253 to 267 (p3b) of the mCLCA3 protein [11]. After a preclearing step of medium and cell lysates with protein A-Sepharose beads, immunoprecipitation was performed using anti-YFP or antip3b antibodies. Endoglycosidase Treatment—Digestion of the immunoprecipitates of mCLCA3 protein or mCLCA3-YFP tagged with endo H or PNGase F before SDS-PAGE analysis and Western blotting was performed as described previously [36].
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