Genomic, Tissue Expression, and Protein Characterization of pCLCA1, a Putative Modulator of Cystic Fibrosis in the Pig

Abstract

Recent studies have identified members of the CLCA (chloride channels, calcium-activated) gene family as potential modulators of the cystic fibrosis (CF) phenotype, but differences between the human and murine CLCA genes and proteins may limit the use of murine CF models. Recently established pig models of CF are expected to mimic the human disease more closely than the available mouse models do. Here, we characterized the porcine CLCA gene locus, analyzed the expression pattern and protein processing of pCLCA1, and compared it to its human ortholog, hCLCA1. The porcine CLCA gene family is located on chromosome 4q25, with a broad synteny with the human and murine clca gene loci, except for a pig-specific gene duplication of pCLCA4. Using pCLCA1-specific antibodies, the protein was immunohistochemically localized in mucin-producing cells, including goblet cells and mucinous glands in the respiratory and alimentary tracts. Similar to hCLCA1, biochemical characterization of pCLCA1 identified a secreted soluble protein that could serve as an extracellular signaling molecule or functional constituent of the protective mucous layers. The results suggest that pCLCA1 shares essential characteristics of hCLCA1, supporting the pig model as a promising tool for studying the modulating role of pCLCA1 in the complex pathology of CF.

Murine mCLCA5 is expressed in granular layer keratinocytes of stratified epithelia

“…Gradient gel electrophoresis using Novex Ò 4-20% Tris-glycine gel 1.0 mm (Invitrogen) was performed to better differentiate between similarly sized bands but failed to improve the resolution on the blots (not shown). Immunoprecipitation of mCLCA5 transfected HEK293 cells was performed as described previously (Plog et al 2009). Unfortunately, all antibodies failed to detect any specific band in the immunoblot after immunoprecipitation (data not shown).…”

“…For example, mCLCA3, its human ortholog hCLCA1, the equine ortholog eCLCA1 and the porcine ortholog pCLCA1 are expressed in goblet cells only (Anton et al 2005;Gruber et al 1998;Plog et al 2009), whereas mCLCA6 is exclusively expressed in nongoblet cell enterocytes (Bothe et al 2008). In contrast, more complex and partly contradictory expression patterns have been reported for several other CLCA family members, such as hCLCA2 (Agnel et al 1999;Connon et al 2005;Pauli et al 2000) and its murine ortholog mCLCA5 (Beckley et al 2004).…”

Impaired Autoproteolytic Cleavage of mCLCA6, a Murine Integral Membrane Protein Expressed in Enterocytes, Leads to Cleavage at the Plasma Membrane Instead of the Endoplasmic Reticulum

“…In each species analyzed to date, four to eight CLCA family members are predominantly expressed on the surfaces of mucous membranes and the skin (Braun et al, 2010;Loewen and Forsyth, 2005;Patel et al, 2009;Plog et al, 2009). All CLCA proteins are post-translationally cleaved into two subunits (Patel et al, 2009).…”

Tissue and Cellular Expression Patterns of Porcine CFTR: Similarities to and Differences From Human CFTR

“…Furthermore, the pCFTR signals obtained from goblet cells are clearly located between the nucleus and porcine goblet cell granule marker pCLCA1 (Plog et al 2009). Unfortunately, no other antibodies were identified that could serve for co-localization studies of pCFTR with specific subcellular structures, mostly due to their lack of crossreactivity on pig tissues (data not shown).…”

“…Analysis of the pCFTR mRNA Tissue Expression Pattern Total RNA was isolated from tissues as described previously (Plog et al 2009). PCR primers were designed using the Beacon Designer 3.0 software (Premier Biosoft International; Palo Alto, CA) and are listed in Table 1.…”