S-nitrosation of Ca~(2+)-loaded and Ca~(2+)-free recombinant calbindin D_(28k) from human brain

摘要

Calbindin D_(28k) is noted for its abundance and specific distribution in mammalian brain and sensory neurons.It can bind three to five Ca~(2+) ions and may act as a Ca~(2+) buffer to maintain intracellular Ca~(2+0 homeostasis,but its exact role is still unknown.In the presentstudy,mass spectrometric analysis reveals that the five cysteine residues in recombinant human brian calbindin D_(28k) (rHCaBP)are derivatized with N-ethylmaleimide,consistent with the determination of 5.3+-0.4 and 4.7+-0.4 free thiolsin the protien using the thiol-specific reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 5-(octyldithio)-2-nitrobenzoic acid,respectivley.The results of UV-vis and circular dichrosim absorption,intrinsic fluorescence,and mass spectrometry measurements indicate that both Ca~92+)-loaded (holo) and Ca~(2+)-free (apo)rHCaBP are S-nitrosated by S-nitrosocysteine (CysNO0.The number of cysteine residues S-nitrosated in holorHCaBP and aporHCaBP are 2.6+-0.05 and 3.4+-0.09,respectivley,as determined by the Saville assay.HolorHCaBP also undergoes S-nitrosation at one tothree cysteine residues when exposed to S-nitrosoglutathione (GSNO),and Cys 100 was found to be an S-nitrosationsite by peptide mass mapping.Treatment of holorHCaBP with free NO resulted in a mass increase of (59+-2Da,corresponding to two NO adducts.Since up to four cysteine residues can be S-nitrosated in rHCaBP,it is propsoed that the proteinmay act as a NO buffer or reservoir in the brain in a manner similar to serum albumin in blood.It is significant in this context that rHCaBP is found coexistent with nitric oxide synthase in cerebellum and that S-nitrosation varies with Ca~(2+) binding,with S-nitrosation occurring to a greater extent in aporHCaBP thanin the holoprotien.Furthermore,exposure of rHCaBP to either CysNO or GSNO also leads to rapid S-thiolation of Cys187.We demonstrate here for the first time that intrinsic portein fluorescence is a sensitive probe of protein S-nitrosation.This is due to efficient Forster energy transfer (R_0 approx 17A) between tryptophan donors and S-nitrosothiol acceptors.