Characterization of Tescalcin, a Novel EF-Hand Protein with a Single Ca2+-Binding Site: Metal-Binding Properties, Localization in Tissues and Cells, and Effect on Calcineurin

Christina Gutierrez-Ford, Konstantin Levay, Aldrin V Gomes, Erasmo M. Perera, Tapan Som, You Me Kim, Jeffrey L. Benovic, Gary D. Berkovitz, Vladlen Z. Slepak

Research output: Contribution to journalArticle

49 Scopus citations

Abstract

The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca 2+- and Mg2+-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with 45Ca2+ revealed that recombinant tescalcin binds approximately one Ca2+ ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca2+, indicative of a conformational change. The apparent K d for Ca2+ was 0.8 μM. A point mutation in the consensus EF-hand (D123A) abolished 45Ca2+ binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca2+-induced conformational change. Tescalcin also binds Mg2+ (Kd 73 μM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg2+, tescalcin's Ca2+ affinity is shifted to 3.5 μM. These results illustrate that tescalcin should bind Mg2+ constitutively in a quiescent cell, replacing it with Ca2+ during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca2+ ion.

Original languageEnglish (US)
Pages (from-to)14553-14565
Number of pages13
JournalBiochemistry
Volume42
Issue number49
DOIs
StatePublished - Dec 16 2003
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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    Gutierrez-Ford, C., Levay, K., Gomes, A. V., Perera, E. M., Som, T., Kim, Y. M., Benovic, J. L., Berkovitz, G. D., & Slepak, V. Z. (2003). Characterization of Tescalcin, a Novel EF-Hand Protein with a Single Ca2+-Binding Site: Metal-Binding Properties, Localization in Tissues and Cells, and Effect on Calcineurin. Biochemistry, 42(49), 14553-14565. https://doi.org/10.1021/bi034870f