Comparative evolutionary and structural analysis of the avian and mammalian CSF1R systems
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Date
04/07/2015Author
Gutowska, Maria Weronika
Metadata
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.