Comparative evolutionary and structural analysis of the avian and mammalian CSF1R systems

Abstract

Macrophages, phagocytic cells of the immune system involved in host defence, homeostasis and development, are controlled and influenced by a variety of growth factors. In mammals, the colony stimulating factor 1 (CSF1) is a secreted cytokine that controls macrophages survival, proliferation and differentiation. It acts through the CSF1 receptor (CSF1R), a transmembrane receptor tyrosine kinase, expressed mainly in mononuclear phagocytes. Mammalian CSF1R is found exclusively at the surface of the mononuclear phagocytes and their progenitors. CSF1R-/- knockout mice display more severe phenotypes than the CSF1-deficient mice, thus suggesting the existence of another CSF1R ligand. Indeed, recent studies have shown that interleukin 34 (IL34) also binds to and activates CSF1R and regulates monocyte viability in vitro. While the exact role of this protein is yet to be fully elucidated, studies in mammals thus far implied its involvement in embryogenesis and development. CSF1R system is highly conserved within vertebrates and has been identified in variety of mammals. Chicken has been used extensively as a model for vertebrate development and to identify fundamental biological processes. Previous studies by colleagues in the lab demonstrated that the CSF1R system is conserved in the chicken, where it controls the generation of monocytes and tissue macrophages. This thesis provides a thorough evolutionary and structural analysis to fully demonstrate the similarities and differences between avian and mammalian CSF1R systems. The primary objective of this thesis was the comparative functional and structural analyses of the three proteins in birds and mammals, using evolutionary and experimental approaches. Here the presence of CSF1, CSF1R and IL34 genes and protein products is identified in a number of evolutionary diverse birds, indicating that the system is well maintained within the group. Avian genes were cloned and sequenced or otherwise extracted from different databases, and the mammalian sequences were gathered from available online sources. Whilst the gene regulation and the differential expression of the mammalian CSF1R, CSF1 and IL34 are reasonably well understood, they have not been extensively studied in birds. Preliminary comparison between these two groups provided in this thesis suggests a number of similar patterns are involved in regulation of avian CSF1R system. The mammalian CSF1/CSF1R and IL34/CSF1R ligand:receptor peptide interface has been previously resolved and was used to model similar structures in the chicken. The models were then utilised to determine which amino acids are involved in receptor binding in birds. The apparent lack of cross-species reactivity between the chicken CSF1 and zebra finch CSF1R provided a basis for an experimental validation of the in silico binding site predictions. Altogether the structural modelling, evolutionary analysis and experimental confirmation provided sufficient proof for the location of avian CSF1/CSF1R interface. Finally, an extensive bioinformatics analysis has been performed on both the coding DNA and the protein structures of the CSF1R system. The results uniformly showed that IL34 remains under purifying selection in both groups. CSF1 is diverse amongst most mammalian species, while avian CSF1 is only positively selected along particular lineages. This implies the rapid evolution of mammalian CSF1, probably in response to the selection pressure from pathogens. Contrasting situation is found in the CSF1R. Whilst mammalian CSF1R remains positively selected only along particular branches, avian CSF1R presents a number of pervasively positively selected sites, found mostly in the extracellular domains of the receptor. That suggests that in birds it is the receptor, not CSF1, which remains under strong selective pressure. These indicates that birds employ a unique way of competing in the hostpathogen arms race, suggesting the existence of yet unknown pathogen-encoded protein interacting with the avian receptor.