Identifying putative substrates of Calpain-15 in neurodevelopment.

Calpain 15 (CAPN15) is an intracellular cysteine protease belonging to the non-classical small optic lobe (SOL) family of calpains, which has an important role in developmental processes. Loss of Capn15 in mice leads to developmental eye anomalies and volumetric changes in the brain. Human individuals with biallelic variants in CAPN15 have developmental delay, neurodevelopmental disorders, as well as congenital malformations, including eye anomalies. However, the substrates of Capn15 are still unidentified. Here, using Capn15 KO P2 mice of both sexes, we have used RNA sequencing (RNA-SEQ), proteomics, and N-terminomics/terminal amino isotopic labelling of substrates (TAILS), to examine putative substrates of Capn15. There were few changes in the transcriptome profile, and we could not verify a protein change in one selected mRNA between Capn15-/- and WT mice, although a putative transcription factor linked to these changes, Pax2, did show a significant increase after the loss of Capn15. TAILS revealed a preference for cleavage at basic residues, and while no hits showed a significant change in cleavage, some were more abundant when Capn15 was removed. These included Doublecortin and Tubb3, and the Doublecortin predicted cleavage was at a lysine residue. Cleavages at lysine residues were enriched in peptides that were lost or reduced when Capn15 was removed, but not in cleavages that were unchanged when Capn15 was removed.