Characterization of Protamine Modifications Using Newly Generated Modification Specific Antibodies

Protamines have traditionally been considered inert nuclear proteins serving as passive structural elements that condense the paternal genome. However, emerging evolutionary, developmental, and biochemical evidence calls for a need to revisit protamine protein’s presumed biological function. Importantly, in 2014 mass spectrometry analysis revealed that mouse protamines bear a number of post-translational modifications (PTMs). This raises the possibility that these modifications may bear a “protamine code” analogous to the “histone code”, however, to date none of these novel modifications have been further explored. Our objective was to generate modification specific antibodies to a subset of protamine modifications and begin to explore their functional significance. Laboratory experiments utilizing murine testes and sperm. Modification specific antibodies were generated through Genemed Synthesis Inc using a rabbit host. Specificity of polyclonal antibodies were tested via western blot, testing of pre-bleed serum, peptide competition and phosphatase treatment. The validated antibodies were subsequently tested with immunofluorescence (IF) on cross-sections of C57BL6 mouse testes and mature sperm. Antibodies to Protamine 1 K50 acetyl (P1K50Ac) and Protamine 1 S43, T45 Phospohorylation (P1S43/T45Ph) are both efficient and specific for the modified form of protamine 1. P1S43/T45Ph was noted in high abundance on acid extracted protamines, and throughout the testes, however, did demonstrate some stage-specific staining. P1K50Ac was found in lower abundance and was also detected in a stage-specific manner. We provide validation of previously described post-translational modifications on mouse protamine 1 using newly generated modification-specific polyclonal antibodies. Furthermore, we report the first histologic description of these modified proteins in testes and sperm.