Evolutionarily conserved properties of CLCA proteins 1, 3 and 4, as revealed by phylogenetic and biochemical studies in avian homologues

Abstract

Species-specific diversities are particular features of mammalian chloride channel regulator, calcium activated (CLCA) genes. In contrast to four complex gene clusters in mammals, only two CLCA genes appear to exist in chickens. CLCA2 is conserved in both, while only the galline CLCA1 (gCLCA1) displays close genetic distance to mammalian clusters 1, 3 and 4. In this study, sequence analyses and biochemical characterizations revealed that gCLCA1 as a putative avian prototype shares common protein domains and processing features with all mammalian CLCA homologues. It has a transmembrane (TM) domain in the carboxy terminal region and its mRNA and protein were detected in the alimentary canal, where the protein was localized in the apical membrane of enterocytes, similar to CLCA4. Both mammals and birds seem to have at least one TM domain containing CLCA protein with complex glycosylation in the apical membrane of enterocytes. However, some characteristic features of mammalian CLCA1 and 3 including entire protein secretion and expression in cell types other than enterocytes seem to be dispensable for chicken. Phylogenetic analyses including twelve bird species revealed that avian CLCA1 and mammalian CLCA3 form clades separate from a major branch containing mammalian CLCA1 and 4. Overall, our data suggest that gCLCA1 and mammalian CLCA clusters 1, 3 and 4 stem from a common ancestor which underwent complex gene diversification in mammals but not in birds.

Genomic, biochemical and expressional properties reveal strong conservation of the CLCA2 gene in birds and mammals

“…Chloride channel regulators, calcium activated ( CLCA ) constitute a family of genes that has been correlated to various disease conditions, including chronic inflammatory airway diseases ( Hoshino et al, 2002 ; Nakanishi et al, 2001 ; Patel, Brett & Holtzman, 2009 ; Range, Mundhenk & Gruber, 2007 ), cystic fibrosis ( Hauber et al, 2003 ; Ritzka et al, 2004 ; Young et al, 2007 ) and cancer ( Chen et al, 2019 ; Hou et al, 2017 ; Walia et al, 2009 ; Walia et al, 2012 ; Yu, Walia & Elble, 2013 ) and shows striking evolutionary dynamics ( Bartenschlager et al, 2022 ; Mundhenk et al, 2018 ). In general, CLCAs comprise a prototypical protein domain architecture of a CLCA N-terminal (N-CLCA), a von Willebrand factor type A (vWA), a beta sheet rich (bsr) as well as a carboxy (C)-terminal fibronectin type III domain (fn3) that is separated from N-CLCA, vWA and bsr by proteolytical cleavage ( Patel, Brett & Holtzman, 2009 ).…”

“…In general, CLCAs comprise a prototypical protein domain architecture of a CLCA N-terminal (N-CLCA), a von Willebrand factor type A (vWA), a beta sheet rich (bsr) as well as a carboxy (C)-terminal fibronectin type III domain (fn3) that is separated from N-CLCA, vWA and bsr by proteolytical cleavage ( Patel, Brett & Holtzman, 2009 ). Some CLCAs contain a transmembrane (TM) domain that anchors the C-terminal cleavage product in the plasma membrane ( Bartenschlager et al, 2022 ; Braun et al, 2010 ; Elble et al, 2006 ; Patel, Brett & Holtzman, 2009 ; Plog et al, 2012a ). CLCAs lacking the TM appear completely secreted ( Gibson et al, 2005 ; Mundhenk et al, 2006 ; Patel, Brett & Holtzman, 2009 ; Plog et al, 2009 ).…”

“…On the genomic level, all CLCA genes in a given species are located within in a single locus, which is consistently flanked by the Outer dense fibre of sperm tails 2-like ( ODF2L ) and SH3-domain GRB2-like endophilin B1 ( SH3GLB1 ) genes ( Bartenschlager et al, 2022 ; Mundhenk et al, 2018 ; Plog et al, 2009 ). Although CLCA genes have been identified in numerous avian, reptile, amphibian or fish species in the recent past, the main research has been focused on CLCA homologues in mammalian species so far ( Cunningham et al, 2021 ).…”

“…From the phylogenetic perspective, mammalian CLCA genes have been assigned to four clearly distinct clusters ( Mundhenk et al, 2018 ; Plog et al, 2009 ). While clusters 3 and 4 exhibit a complex arrangement of multiple genes that seemingly arose from several independent duplication and inactivation events during the emergence of mammalian species ( Bartenschlager et al, 2022 ; Mundhenk et al, 2018 ), both CLCA clusters 1 and 2 comprise only one intact single gene in each mammalian species. Recently, we described a CLCA locus in the genome of chicken ( Gallus gallus ) which is similarly flanked by the galline ODF2L and the SH3GLB1 genes ( Bartenschlager et al, 2022 ; Mundhenk et al, 2018 ).…”

“…While clusters 3 and 4 exhibit a complex arrangement of multiple genes that seemingly arose from several independent duplication and inactivation events during the emergence of mammalian species ( Bartenschlager et al, 2022 ; Mundhenk et al, 2018 ), both CLCA clusters 1 and 2 comprise only one intact single gene in each mammalian species. Recently, we described a CLCA locus in the genome of chicken ( Gallus gallus ) which is similarly flanked by the galline ODF2L and the SH3GLB1 genes ( Bartenschlager et al, 2022 ; Mundhenk et al, 2018 ). In contrast to the four mammalian CLCA (ma CLCA) clusters, however, chickens possess only two CLCA homologous genes, the galline CLCA1 ( gCLCA1 ) and gCLCA2 .…”