Identification of Avian Corticosteroid-binding Globulin (SerpinA6) Reveals the Molecular Basis of Evolutionary Adaptations in SerpinA6 Structure and Function as a Steroid-binding Protein

Abstract

Corticosteroid-binding globulin (CBG) was isolated from chicken serum and identified by mass spectrometry and genomic analysis. This revealed that the organization and synteny of avian and mammalian SerpinA6 genes are conserved. Recombinant zebra finch CBG steroid-binding properties reflect those of the natural protein in plasma and confirm its identity. Zebra finch and rat CBG crystal structures in complex with cortisol resemble each other, but their primary structures share only ∼40% identity, and their steroid-binding site topographies differ in several unexpected ways. Remarkably, a tryptophan that anchors ligands in mammalian CBG steroid-binding sites is replaced by an asparagine. Phylogenetic comparisons show that reptilian CBG orthologs share this unexpected property. Glycosylation of this asparagine in zebra finch CBG does not influence its steroid-binding affinity, but we present evidence that it may participate in protein folding and steroid-binding site formation. Substitutions of amino acids within zebra finch CBG that are conserved only in birds reveal how they contribute to their distinct steroid-binding properties, including their high (nanomolar) affinities for glucocorticoids, progesterone, and androgens. As in mammals, a protease secreted by Pseudomonas aeruginosa cleaves CBG in zebra finch plasma within its reactive center loop and disrupts steroid binding, suggesting an evolutionarily conserved property of CBGs. Measurements of CBG mRNA in zebra finch tissues indicate that liver is the main site of plasma CBG production, and anti-zebra finch CBG antibodies cross-react with CBGs in other birds, extending opportunities to study how CBG regulates the actions of glucocorticoids and sex steroids in these species.

The stressed brain: regional and stress‐related corticosterone and stress‐regulated gene expression in the adult zebra finch (Taeniopygia guttata)

“…CBGs are widely expressed in rodent and human brain, 25,54‐56 and, although their function is still unclear, they may serve as cellular mediators of CORT action in the absence of CORT receptors or as mechanisms enhancing CORT transport to classical receptors 26,57 . The songbird CBG sequence has recently been identified 58 and our very preliminary work suggests that the protein is expressed, albeit at quite low levels, in the zebra finch brain (M.A. Rensel, B.A.…”

“…However, despite expressing central CBG, previous microdialysis experiments in rodents have documented free CORT stress responses in the brain 59‐61 . The reasons for this discrepancy between birds and mammals is unknown, although it is of note that songbird CBG is not identical to the mammalian protein, with structural differences and distinct patterns of affinity to additional steroid hormones 23,58 . However, if CBG is expressed and active in the songbird brain and regulates central CORT, it could potentially limit or prevent a free CORT response, much as CORT bound to CBGs would be unable to pass through a microdialysis probe.…”

Chicken domestication changes expression of stress-related genes in brain, pituitary and adrenals

“…In the liver, we saw no changes in gene expression of the investigated genes: serpin family A member 10 ( SERPINA10 ), serpin family A member 4 ( SERPINA4 ) and hydroxysteroid 11-beta dehydrogenase 2 ( HSD11B2 ) after stress. SERPINA10 was recently identified as a candidate gene for domestication effects on the function of the HPA axis in chickens ( Fallahsharoudi et al., 2016 ), the gene currently annotated as SERPINA4 has been identified as the gene encoding corticosterone-binding globulin (CBG) ( Vashchenko et al., 2016 ), while the enzyme HSD11B2 converts corticosterone to inactive cortisone ( Carsia, 2015 ). The lack of any effect of stress treatment on these genes suggests that neither the removal of glucocorticosteroids from the blood, nor the amount of CBG present in the blood are affected by acute stress.…”

Domestication and early experiences in chickens: Behavior, stress and gene expression

“…If this is the case, however, variations in the amount of CBG produced could affect the overall response to glucocorticoids. In chickens, CBG is encoded by the gene currently annotated as serpin family A member 4 (SERPINA4) (Vashchenko et al, 2016).…”