The Membrane Penetrating Ability of Opicalcin1 Is Mainly Derived from the Latter Segment in Its Primary Sequence

Background: Calcin is a group of globular peptides with 33−35 AAs that penetrate the cell membrane and specially target RyRs from scorpion venoms, however, which fragment in primary sequence for its transmembrane effect is unclear yet. Methods: eight different fragments of the typical Opicalcin1 (OpiCa1) were synthesized according to the distribution of charged amino acids in primary sequence, followed by the determination of both cell penetration activity and cytotoxic. Results: In all eight OpiCa1 fragments, OpiCa1₁₇₋₃₃, OpiCa1₁₋₁₁, and OpiCa1₂₃₋₃₃ were predicted to be CPPs by CellPPD, and their predicted scores were much smaller than that of TAT, which is consistent with the proportion of basic amino acids. Further fluorescent microscopic experiments found that three fragments FITC labeled OpiCa1₁₇₋₃₃, OpiCa1₁₂₋₂₂ and OpiCa1₂₃₋₃₃ displayed similar cell penetrating capacities to that of TAT. In contrast, flow cytometry found that FITC-OpiCa1₁₇₋₃₃ and FITC-OpiCa1₂₃₋₃₃ have even larger intracellular fluorescent intensities than that of TAT, indicating stronger cell penetrating capacity. Other OpiCa1 fragments displayed slightly cell penetrating effect, of which the fluorescent intensities were slightly larger than that of control but significantly lower than that of TAT, FITC-OpiCa1₁₇₋₃₃ and FITC-OpiCa1₂₃₋₃₃. Moreover, although it is not ruled out the impact on cell viability, overall, all OpiCa1 fragments exhibited no or lower cytotoxicity. Conclusion: The latter half of calcin e.g. OpiCa1 in primary sequence is the main responsible fragment for its membrane penetrating ability, and is also a potential new CPP for further utilization e.g. drug delivery.