Androglobin: a chimeric globin in metazoans that is preferentially expressed in Mammalian testes

Abstract

Comparative genomic studies have led to the recent identification of several novel globin types in the Metazoa. They have revealed a surprising evolutionary diversity of functions beyond the familiar O(2) supply roles of hemoglobin and myoglobin. Here we report the discovery of a hitherto unrecognized family of proteins with a unique modular architecture, possessing an N-terminal calpain-like domain, an internal, circular permuted globin domain, and an IQ calmodulin-binding motif. Putative orthologs are present in the genomes of many metazoan taxa, including vertebrates. The calpain-like region is homologous to the catalytic domain II of the large subunit of human calpain-7. The globin domain satisfies the criteria of a myoglobin-like fold but is rearranged and split into two parts. The recombinantly expressed human globin domain exhibits an absorption spectrum characteristic of hexacoordination of the heme iron atom. Molecular evolutionary analyses indicate that this chimeric globin family is phylogenetically ancient and originated in the common ancestor to animals and choanoflagellates. In humans and mice, the gene is predominantly expressed in testis tissue, and we propose the name "androglobin" (Adgb). Expression is associated with postmeiotic stages of spermatogenesis and is insensitive to experimental hypoxia. Evidence exists for increased gene expression in fertile compared with infertile males.

FIG. 1.

Diagrammatic representation of the phylogenetic distribution of Adgb orthologs. The Adgb domain structure, the conserved Cys (C) residues of the calpain-like domains and essential residues of the globin domain (CD1, E7, F8) are indicated by the amino acid one-letter code (for details see fig. 2). If a taxon contains different Adgb variants, the Adgb structure is shown for the species displayed in bold letters.

FIG. 2.

(A) The chimeric domain structure of human ADGB (HsaADGB). The calpain-like protease domain, the rearranged globin domain, the IQ motif, a C-terminal coiled-coil region, and candidate nuclear localisation (NLS) and ER membrane retention signals are indicated. The ADGB IQ motif may mediate binding to calmodulin, which reacts to Ca²⁺ levels via its EF hand (EFh) motifs. Small triangles indicate the position of introns in the human ADGB gene and intron phases are given (e.g., ‘.1’ indicates insertion of an intron in phase 1 between codon positions 1 and 2). For comparison, the structure of human calpain-7 (HsaCAPN7) is shown below (MIT, microtubule-interacting and trafficking domain; III’’ and III’, domains moderately similar to the Ca²⁺ binding domain of other calpains). (B) Amino acid sequence alignment of the concatenated human ADGB globin domain (helices A/B plus C-H) with human NGB, CYGB and Mb. The globin α-helical structure is drawn on top of the alignment. The functionally conserved Phe (F) in the C-D region and the proximal and distal His (H) residues at positions F8 and E7 are indicated. Note that ADGB contains a Gln (Q) instead of the distal His. Gray-scale shading indicates conservation of iso-functional amino acid residues. Intron positions within the globin domain of the ADGB gene are shown by arrows below the alignment.

FIG. 3.

Phylograms describing relationships among (A) the calpain domain of Adgb and the repertoire of human calpains, (B) the complete Adgb sequences, and (C) the globin domain of Adgb and representatives from other metazoan, fungal, plant, and bacterial globin lineages. Bayesian posterior probabilities and bootstrap support values are provided next to the nodes. For species abbreviations, see figure 1.

FIG. 4.

Adgb mRNA expression analysis. (A) Quantitative RT-PCR showing Adgb expression in normal mouse tissues. Bars indicate normalized mRNA levels with standard errors of three different mice. (B) Quantitative RT-PCR of Adgb in testes of younger and older mice (d = days, m = months after birth). (C) In situ hybridization of Adgb antisense RNA to mouse testis cryosections (1,2). Dark signal is concentrated towards the lumen of the seminiferous tubules. A hematoxylin/eosin-stained section (3) is shown for comparison. Smooth muscle cells (a), spermatogonia (b), and Sertoli cells (c) are indicated (magnification 40-fold).