Publikationsdatenbank

Evolutionary diversity of CLCA genes among birds and mammals (2023)

Art

Hochschulschrift

Autor

Bartenschlager, Florian (WE 12)

Quelle

Berlin, 2023 — IX, 116 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/39362

Kontakt

Institut für Tierpathologie

Robert-von-Ostertag-Str. 15
14163 Berlin
+49 30 838 62450
pathologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The chloride channel regulators, calcium activated (CLCA) gene family has mainly been associated with cancer and chronic inflammatory airway diseases but the presumably complex cellular functions of these gene products are still widely unknown. The family comprises four distinct genetic clusters in mammals, termed CLCA1 to CLCA4. It is highly complex and diverse and includes amplified or inactivated genes with a high degree of variation between species. For example, in contrast to mice with eight CLCAs including one inactivated gene, humans have only three intact CLCAs and one inactivated gene. The tissue and cellular expression patterns of different CLCA homologues within a species are also often redundant. This complexity and redundancy of the CLCA members might be a reason, why the function of this gene family has not been revealed yet based on a mammalian model organism. The complexity and redundancy of mammalian CLCAs raise two questions: Are the complexity and diversity of these genes unique features of mammals? And second, what is the evolutionary background of these peculiar developments? To address these questions, data on CLCA homologues obtained from evolutionarily more distant species are needed. Recently, a rather simple CLCA gene locus was predicted for the chicken, comprising only two CLCA genes. In a phylogenetic analysis, the first galline CLCA gene product, gCLCA1, was found closely related to mammalian CLCA1, 3 and 4. In contrast, the second one, gCLCA2, seemed more closely related to mammalian CLCA2 than to gCLCA1 or mammalian CLCA1, 3 and 4. In this cumulative thesis, the galline CLCA genes and their genomic structures were analyzed and both members were cloned. Their protein architecture and biochemical properties were investigated in silico and in vitro. In addition, their mRNA as well as their cellular protein expression patterns were analyzed. All data were compared to mammalian CLCA1, 2, 3, and 4. Both avian proteins are encoded by 14 exons and are located in a conserved locus between the Outer Dense Fiber of Sperm Tails 2-Like (ODF2L) and SH3-Domain GRB2-Like Endophilin B1 (SH3GLB1) genes. gCLCA1 combines many properties of mammalian CLCA1, 3 and 4 as it was shown to be a cleaved protein with a typical CLCA domain architecture. Despite its relatively high phylogenetic distance to mammalian CLCA4, it shares most common traits with this member. This includes heavy asparagine-linked glycosylation, the presence of a transmembrane domain in the carboxy-terminal cleavage product and protein expression in the apical membrane of enterocytes. In contrast, gCLCA2 was highly similar to mammalian CLCA2 in terms of its protein architecture, cleavage and glycosylation. These findings were in line with results from in silico analyses of CLCA2 sequences from other avian species.Furthermore, the presence of a transmembrane domain in the carboxy-terminal cleavage product and its expression in keratinocytes are traits of avian CLCA2, which are also found in mammalian CLCA2. Interestingly, and as opposed to the expression patterns of mammalian CLCA proteins, no overlapping tissue or cellular expression patterns were detected for the two galline CLCA members. Based on these findings, CLCA2 appears to be highly conserved among birds and mammals. The results allow to speculate that a hypothetical gene ancestor was expressed in keratinocytes of a common ancestral species before mammalian and avian lineages diverged. This high degree of CLCA2 conservation is in contrast to gCLCA1 and mammalian CLCA1, 3 and 4. During evolution, a hypothetical ancient ancestor of gCLCA1 / mammalian CLCA1, 3, and 4, was likely expressed by enterocytes of a common ancestral species of mammals and birds. The hypothetical ancestral gene seems to have expanded by gene duplications in the mammalian lineage, which did not occur in birds. Besides that, these findings cannot only be used to unveil the evolutionary history of the CLCA family but should be taken into account with regard to the selection of an animal model for the functional analysis of these genes. The chicken might serve as a promising species for knockout models to study CLCA2 functions in vivo. Results obtained from such a knockout chicken are likely transferable to mammalian CLCA2 due to the high degree of conservation. A chicken gCLCA1 knockout model might provide data, which might be transferred most likely to mammalian CLCA4 genes in the gut, as both share a similar cell type specific protein expression in this microenvironment, a similar protein architecture as well as similar biochemical properties. At the transition to the post-genomic era with publically accessible information on gene structures as well as nucleotide and protein sequences of various species, comprehensive analyses of gene families across species have become possible. The comparison of such data in combination with the comparison of gene related information, including cellular expression patterns and biochemical properties, is a powerful approach to enlighten the evolutionary background of proteins. Furthermore, it might be beneficial to identify the most suitable animal model for further functional and biomedical studies.