S-nitrosation of Ca(2+)-loaded and Ca(2+)-free recombinant calbindin D(28K) from human brain.

Abstract

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+) homeostasis, but its exact role is still unknown. In the present study, mass spectrometric analysis reveals that the five cysteine residues in recombinant human brain calbindin D(28K) (rHCaBP) are derivatized with N-ethylmaleimide, consistent with the determination of 5.3 +/- 0.4 and 4.7 +/- 0.4 free thiols in the protein using the thiol-specific reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 5-(octyldithio)-2-nitrobenzoic acid, respectively. The results of UV-vis and circular dichroism absorption, intrinsic fluorescence, and mass spectrometry measurements indicate that both Ca(2+)-loaded (holo) and Ca(2+)-free (apo) rHCaBP are S-nitrosated by S-nitrosocysteine (CysNO). The number of cysteine residues S-nitrosated in holorHCaBP and aporHCaBP are 2.6 +/- 0.05 and 3.4 +/- 0.09, respectively, as determined by the Saville assay. HolorHCaBP also undergoes S-nitrosation at one to three cysteine residues when exposed to S-nitrosoglutathione (GSNO), and Cys100 was found to be an S-nitrosation site by peptide mass mapping. Treatment of holorHCaBP with free NO resulted in a mass increase of 59 +/- 2 Da, corresponding to two NO adducts. Since up to four cysteine residues can be S-nitrosated in rHCaBP, it is proposed that the protein may 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 than in the holoprotein. 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 protein fluorescence is a sensitive probe of protein S-nitrosation. This is due to efficient Förster energy transfer (R(0) approximately 17 A) between tryptophan donors and S-nitrosothiol acceptors.