The human androglobin globin domain was recombinantly expressed and shows an absorption spectrum characteristic of hexacoordinate heme iron, establishing heme binding for the human protein directly rather than by inference from the family.

The N-terminal region is homologous to the catalytic domain II of human calpain-7, which is the origin of the InterPro IPR001300 match and hence of the two proteolysis-related IEA rows.

The globin domain is a genuine myoglobin-like fold that has been rearranged and split, which is why the two halves receive separate UniProt Domain features.

Oxygen binding by the androglobin globin domain is demonstrable but transient, and the authors conclude from the autoxidation rate that it is not a physiological function. This is the evidence behind keeping GO:0019825 as non-core and behind not proposing GO:0005344 oxygen carrier activity or GO:0019826 oxygen sensor activity.

The measured chemistry points to nitric oxide handling rather than oxygen, which is the basis for the two proposed NEW molecular-function annotations.

Androglobin binds nitric oxide as a five-coordinate complex, which is highly unusual among globins and characteristic of nitric-oxide storage hemoproteins.

The distal heme-iron ligand of androglobin is a glutamine, not a histidine, stated explicitly in contrast with cytoglobin. This contradicts UniProt's bis-histidyl FUNCTION text and agrees with UniProt's own feature table.

A tyrosine replaces the otherwise invariant CD1 phenylalanine, a further departure from canonical eukaryotic globin architecture that affects heme stabilisation.

Calmodulin binding at the IQ motif increases the nitrite reductase catalytic efficiency of the androglobin globin domain roughly two-fold, linking the calcium-sensor module directly to the heme chemistry.

Independent confirmation that the distal heme ligand is a glutamine and the proximal ligand a histidine, in a construct whose numbering maps onto UniProt Q792 and H824.

The circularly permuted globin domain folds correctly with heme and engages calmodulin through the IQ motif, supporting both the heme-binding and the proposed calmodulin-binding annotations.

The authors themselves qualify whether nitrite reduction is a natural function of androglobin, which is recorded as an explicit caveat on the proposed GO:0098809 annotation.

A third independent confirmation that the heme distal residue of human androglobin is a glutamine, here established by mutagenesis rather than by inference - the authors generate Q12H and Q12Y substitutions and show that the distal residue controls both stability and nitrite reductase activity, and that deleting the IQ motif abolishes calmodulin-mediated activation.

Adgb-null mice are infertile and androglobin localises to the sperm flagellum, annulus and midpiece. This is the source of the mouse IDA annotations that both the PAINT IBA rows and the Ensembl Compara rows on human ADGB are transferred from.

The proteolysis claim is stated by the authors as a suggestion from in vitro data about overexpressed proteins, not as a demonstration that androglobin catalyses the cleavage. This is the wording the MARK_AS_OVER_ANNOTATED call on GO:0006508 rests on.

A construct spanning the globin and IQ domains binds calmodulin robustly by co-immunoprecipitation, whereas full-length androglobin did not under basal conditions, suggesting the interaction is conformationally gated by the long C-terminal region.

Human ADGB variants causing asthenozoospermia disrupt binding of androglobin to calmodulin. This is the human-specific evidence behind the proposed GO:0005516 annotation, and UniProt records the same interaction with ECO:0000269 against the human entry.

CFAP69 and SPEF2 were confirmed as androglobin-binding proteins, connecting androglobin to the ciliary and flagellar assembly machinery.

Adgb-null mice show abnormal spermatogenesis with malformation of elongating and elongated spermatids, the IMP that both the GO:0007286 IBA and Compara rows derive from.

Bi-allelic ADGB variants in three unrelated Han Chinese men cause severe asthenoteratozoospermia with acrosome and flagellum malformations on light and electron microscopy, providing human loss-of-function corroboration for the spermatogenesis and flagellum annotations transferred from mouse.

Immunofluorescence places androglobin in the acrosome and flagella of human and mouse spermatogenic cells, with expression rising after puberty. The flagellar localisation corroborates the transferred GO:0036126 annotation directly in human; the acrosomal localisation is not currently captured by GOA and is raised as a question rather than proposed, since it rests on this single study.

TTC29 and CFAP47 were confirmed as androglobin interactors by co-immunoprecipitation, extending the set of ciliary and flagellar partners beyond septin 10, CFAP69 and SPEF2.

Androglobin is a component of the C1b/C1f supercomplex of the ciliary central apparatus in Tetrahymena, alongside Spef2A and Cfap69 - the same proteins that were later found to bind androglobin in mammals. This supports the motile-cilium localisation outside the sperm context.

Deleting Adgb caused only minor changes in ciliary motility, unlike deletion of Spef2A or Cfap69 which abolished the entire C1b projection. Androglobin is therefore a resident of the structure rather than a core structural or motor component, which is consistent with annotating localisation but not a motility function.

Across human tissues, ADGB expression is specifically associated with motile-ciliated cell types beyond the testis, including the female reproductive tract, lung and brain, supporting the motile-cilium annotation as more than a sperm-flagellum artefact.

Overexpressing full-length ADGB increases the number of ciliated cells, and the ADGB promoter is a direct target of the ciliogenic transcription factors FOXJ1 and RFX2.