Aequorin mutations Asn 28 and Lys 30 enhance Ca2+ capture activity - A possibility for neurodegenerative disease treatment

Introduction: Photoprotein aequorin is a calcium binding protein (CaBp) that play a critical role in somediseases such as Alzheimer and Parkinson by regulating intracellular calcium concentration. The primarysequence of aequorin has three Ca2+ binding EF-hands containing one Ca2+ in each of them.Methods: To increase the calcium binding capacity of aequorin, we designed 4 mutants in its EF hand I by sitedirectmutagenesis quick change PCR method. To observe the change in the Ca binding activity, we used theneuronal cell line, PC12. After expression and purification of wild-type and mutants of aequorin, PC12 cellswere cultured in a CaCl2-rich medium. Wild-type and all of its mutants such as CaBps with coelenterazine wereadded to separate mediums; after 24 h, cell viability was assessed by MTT and AO/EB and Hoechst stain toobserve nuclear morphology. Measurement of intracellular calcium was done using fluorescent fura2-AM; thecell-permeable acetoxymethyl ester and the calcium amount were expressed as the ratio of fluorescence intensity.Results: We observed that all types of aeqourin have protective effects on cell stress. The features of its mutantsare not the same and also with wild type. The four mutants had higher affinitiy to Ca2+ as measured byflorescent.Conclusion: In vitro, aeqourin mutants with higher Ca2+ affinities can prevent cell damage, and is a useful toolto control neurodegeneration. In this study, by wild and mutated types of aeqourin, we evaluated how mutationcan change aeqourin tendency for Ca2+.

Keywords:

Aequorin , Neurodegenerative disease , Intracellular calcium