Ancient conservation of androglobin expression reveals its evolutionary link to ciliary processes

Androglobin (Adgb) is the most distinctive member of the globin superfamily. Its characteristic globin domain is permuted, interrupted by a calmodulin-binding motif, and embedded within a large multidomain protein of ∼1500 amino acids that also contains a calpain protease domain. Initially described as testis-specific in mammals, Adgb is also expressed in ciliated epithelia of the female reproductive tract, lung, and brain, and knockout studies reveal its pivotal role during spermatogenesis. To trace its evolutionary origin, we performed comprehensive phylogenetic analysis across diverse eukaryotic taxa. Adgb is present in all major flagellated eukaryotic lineages but absent from nonflagellated clades. Orthology analysis indicates Adgb has been maintained as a predominantly single-copy gene across >1 billion years of evolution-a pattern contrasting sharply with other globins that underwent repeated duplication and functional diversification. Analysis of publicly available transcriptomes from early-branching metazoans confirmed robust Adgb expression in ciliated cell types and provides evidence for its regulation by the ancient ciliogenic transcription factor cRFXa in the choanoflagellate Salpingoeca rosetta. RNA in situ hybridization validated these findings, and comparative analyses suggest that ancestral Adgb homologs lacked the permuted globin domain found in metazoans. Collectively, our results demonstrate that Adgb exemplifies a rare evolutionary trajectory where structural innovation (domain permutation and fusion) enabled functional specialization while being an integral part of the nonredundant ciliary machinery. Adgb thus illustrates how constraint and innovation combine to shape the long-term fate of a protein.