Calcium-dependent proteolysis of the chimeric androglobin reveals altered localization of its isolated globin domain
Androglobin (ADGB), a protein essential for spermatogenesis, is the most structurally unusual member of the vertebrate globin superfamily. It combines a calpain-like domain with a circularly permuted globin domain containing an embedded calmodulin-binding IQ motif, an architecture suggesting complex regulatory functions that remain poorly understood. Here, we investigated whether ADGB undergoes calcium-dependent post-translational processing, like it has been described for other calpains. ADGB underwent robust proteolytic processing upon calcium stimulation, generating several stable cleavage products following ectopic expression in mammalian cells. In vitro proteolysis assays demonstrated that ADGB cleavage requires cytoplasmic factor(s) and is strongly enhanced by Ca2+. While this process is sensitive to pan-calpain inhibition, siRNA-mediated knockdown excluded calpain 1 (CAPN1) and calpain 2 (CAPN2) as primary mediators of ADGB cleavage. In contrast, depletion of the calpain small regulatory subunit CAPNS1 markedly reduced calcium-dependent ADGB proteolysis, implicating a CAPNS1-associated calcium-responsive proteolytic pathway. Domain-mapping analyses localized the major cleavage hotspot between the N-terminal calpain-like domain and the globin-containing C-terminal region, indicating that proteolysis separates the protease-like and globin modules of the ADGB chimera. The isolated globin domain displayed enhanced interaction with calmodulin compared with full-length ADGB, whereas the extended C-terminal region impeded this interaction. Furthermore, unlike full-length ADGB, the isolated globin domain exhibited preferential localization to centrosomal structures. Collectively, these findings identify calcium-dependent proteolysis and altered subcellular localization of the isolated globin domain as previously unrecognized properties of ADGB that may be relevant to its role in ciliary biology.