Abstract
Tescalcin is a novel autosomal gene that was discovered to be preferentially expressed during mouse testis differentiation. The deduced amino acid sequence of this gene contains one consensus EF-hand domain and three other helix-loop-helix regions with partial EF-hand homology. I show that the tescalcin gene encodes a 24 kDa Ca2+-binding protein. During 45Ca2+ equilibrium dialysis, tescalcin binds one Ca2+ ion, which is blocked with a point mutation (D123A) of the consensus EF-hand. Ca2+ binding induces a conformational change, as evidenced by the Ca2+-induced quenching of tescalcin's intrinsic tryptophan fluorescence. The EF-hand D123A mutation prevented this Ca2+-induced fluorescence change. These results demonstrate that tescalcin has a single Ca2+-binding EF-hand, which alone is responsible for the Ca2+-induced conformational change. The Ca2+-mediated decrease in tryptophan fluorescence likely occurs from tertiary conformational changes, as circular dichroism revealed only minor changes in secondary structure. The Ca2+ affinity of tescalcin was found to be 0.8 muM. Tescalcin also binds Mg 2+ (Kd 73 muM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM MgCl2, tescalcin's Ca2+ affinity is shifted to 3.5 muM. Thus, tescalcin likely binds Mg2+ constitutively in a quiescent cell, replacing it with Ca2+ during increases of intracellular Ca2+ levels. I also found that tescalcin is most abundant in adult mouse heart, brain, stomach, as well as HeLa and HL-60 cells, with low expression in adult testis. Tescalcin levels are increased upon terminal differentiation of HL-60 cells. I also analyzed the subcellular localization of overexpressed tescalcin by immunofluorescence microscopy. Tescalcin shares sequence homology with calcineurin B homologous protein, and their functions may parallel, as I found that tescalcin, like CHP1, inhibits calcineurin A phosphatase activity. Tescalcin also has unique functions such as binding to the fourth subunit (CSN4) of the COPS signalosome. Overexpression of tescalcin increases the levels and half-life of ectopic CSN4. Thus, my research demonstrates that tescalcin is a novel 24 kDa protein with a single, functional Ca2+ -binding EF-hand and homology to the calcineurin B subfamily. In addition, since tescalcin has a unique expression pattern and novel protein interactions, I suggest that tescalcin may potentially have distinct functions.