Calcium-dependent proteolysis of the chimeric androglobin reveals altered localization of its isolated globin domain.

Androglobin (ADGB), a protein essential for spermatogenesis, is one of the most structurally unusual members 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, as described for other calpains. ADGB displayed a characteristic proteolytic processing pattern following 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 enhanced by Ca2+. Broad protease inhibitors with activity against calpains reduced ADGB processing, whereas the more selective inhibitor PD150606 and siRNA-mediated depletion of CAPN1 or CAPN2 did not substantially affect cleavage, arguing against these classical calpains as its principal mediators. In contrast, CAPNS1 downregulation reduced calcium-dependent ADGB processing in both cellular and cell-free assays, indicating a contribution of CAPNS1 without establishing a direct role in the proteolytic reaction. Domain-mapping analyses localized major cleavage regions between the N-terminal calpain-like and globin-containing regions of ADGB. The experimentally isolated globin domain displayed enhanced interaction with calmodulin compared with full-length ADGB, and preferential localization to centrosomal structures. Collectively, these findings identify calcium-dependent cytoplasmic proteolytic processing as a previously unrecognized property of ADGB and suggest that such processing may influence the biochemical accessibility and cellular behavior of its constituent domains.