mCLCA4 ER processing and secretion requires luminal sorting motifs

Abstract

Ca ϩ -activated Cl Ϫ channel (CLCA) proteins are encoded by a family of highly related and clustered genes in mammals that are markedly upregulated in inflammation and have been shown to affect chloride transport. Here we describe the cellular processing and regulatory sequences underlying murine (m) CLCA4 proteins. The 125-kDa mCLCA4 gene product is cleaved to 90-and 40-kDa fragments, and the NH2-and COOH-terminal fragments are secreted, where they are found in cell media and associated with the plasma membrane. The 125-kDa full-length protein is only found in the endoplasmic reticulum (ER), and specific luminal diarginine retention and dileucine forward trafficking signals contained within the CLCA4 sequence regulate export from the ER and proteolytic processing. Mutation of the dileucine luminal sequences resulted in ER trapping of the immaturely glycosylated 125-kDa peptide, indicating that proteolytic cleavage occurs following recognition of the trafficking motifs. Moreover, the mutated dileucine and diarginine signal sequences directed processing of a secreted form of enhanced green fluorescent protein in a manner consistent with the effects on mCLCA4. chloride channel; asthma; protein trafficking; epithelium; endoplasmic reticulum retention/trafficking signal CA 2ϩ -ACTIVATED CL Ϫ CHANNEL (CLCA) proteins are highly upregulated in human asthma and animal models of mucosal inflammation (15,17,23). Since their initial cloning in bovine epithelium (1) and endothelium (4), numerous mammalian CLCA genes have been described with distinct, tissue-specific expression patterns (2, 13, 18). Originally described as integral membrane proteins that may be chloride channels, CLCAs have recently been shown to undergo extensive processing, including proteolytic cleavage and secretion (3,6,14), suggesting a more complex role than the regulation of chloride permeability and mucous secretion. In the mouse, the highly homologous murine (m) CLCA genes are tightly clustered on chromosome 3 (H3) in a manner that may enable coordinated transcriptional regulation, and the marked alteration in expression of CLCA genes in several disease states has provoked further interest in their function (12,20,23). mCLCA4, which is most closely related to mCLCA2 and mCLCA1 [the orthologs of human (h) CLCA3], is expressed in epithelial and smooth muscle cells (2) and contains conserved family features, including predicted proteolytic cleavage and serine phosphorylation sites, although neither the processing nor phosphorylation of mCLCA4 has been demonstrated experimentally.Posttranslational processing in the endoplasmic reticulum (ER) is critical for the proper trafficking of proteins to their cellular targets, including the plasma membrane, secretory vesicles, and other organelles (21). Proper assembly and processing occurs through the folding and glycosylation of the native peptide and the recognition of specific motifs by chaperone proteins (5). With respect to proteins that traverse the secretory pathway, specific glycosylations occur that a...

IL-13–induced airway mucus production is attenuated by MAPK13 inhibition

“…Moreover, subsequent findings were more consistent with the proposal that CLCA1 effects on chloride transport may be based on increasing conductance of endogenous CaCCs (40). It further appears (based on analysis of crude membrane preparations) that secreted CLCA proteins are first processed intracellularly into N-terminal and C-terminal peptide fragments (18,39,(41)(42)(43)(44). The present findings support this alternative proposal as well, in that CLCA1 is colocalized within the cell along with MUC5AC in secretory granules and is secreted to the apical cell surface in response to IL-13.…”

“…In that regard, even the initial bioinformatic (16) and experimental data (17,18,39) indicated that CLCA proteins generally lack the structural features to form ion channels by themselves, since they either contain only a single transmembrane anchor or exhibit secretion from the cell in soluble form. Moreover, subsequent findings were more consistent with the proposal that CLCA1 effects on chloride transport may be based on increasing conductance of endogenous CaCCs (40).…”

Self-cleavage of Human CLCA1 Protein by a Novel Internal Metalloprotease Domain Controls Calcium-activated Chloride Channel Activation

“…1B). Because our data and previous studies indicated that CLCA proteins are processed intracellularly (7,8,21,22), we also analyzed CLCA1 retrieved from cell lysates to investigate the possibility of sequential processing. This sample displayed the same cleavage site as the fully secreted proteins found in supernatants.…”

The Role of CLCA Proteins in Inflammatory Airway Disease

“…A revised scheme for the domain structure for mouse and human CLCA proteins is provided in Figure 3, and an annotated analysis of amino acid sequence for these proteins is provided in Figure 4. This updated analysis implies that the CLCA proteins are soluble secreted molecules (29–31) with the exception of a subset of CLCA proteins that contain a C-terminal membrane-anchoring region (32, 33). This anchoring region takes the form of a transmembrane alpha-helix or GPI anchor that lacks the structural requirements to function as an ion channel itself (15, 29).…”

“…In each case, a precursor 120-kDa glycoprotein is cleaved to produce approximately 85-kDa N-terminal and 35-kDa C-terminal products, both of which contain numerous N-linked glycosylation sites (30, 33, 38). For hCLCA1, mClca3, and mClca4 (which have no identifiable transmembrane region), both the N- and C-terminal products are secreted into the media when expressed in human cell lines (29–31). In similar experiments with hCLCA2 (which has a transmembrane region), the N-terminal product is released into the media while the C-terminal product remains cell surface associated (32).…”