Calcium-Induced Exposure of a Hydrophobic Surface of Mouse ALG-2, Which Is a Member of the Penta-EF-Hand Protein Family

Abstract

ALG-2 is a 22 kDa EF-hand type Ca²⁺-binding protein associated with lymphocyte apoptosis. Comparison of the primary structure of ALG-2 with those of EF-hand type proteins revealed that it belongs to the penta-EF-hand (PEF) protein family including the small subunit of calpain. We established a convenient method for the purification of the recombinant mouse ALG-2 expressed in Escherichia coli. The recombinant protein was first pelleted from a lysate in the absence of a Ca²⁺-chelator, and then extracted with buffer containing EDTA/EGTA followed by purification by conventional column chromatographies. Estimation of the molecular mass by gel filtration suggested that the recombinant ALG-2 occurred as a monomeric form. Ca²⁺-dependent precipitation was blocked by inclusion of non-ionic detergent Triton X-100, suggesting hydrophobic self-aggregation at high concentrations of the protein. The N-terminal deletion mutant lacking the hydrophobic non-PEF region was found to be more soluble than the wild type in the presence of Ca²⁺. Analysis using a fluorescent hydrophobicity probe indicated that ALG-2 exposed a hydrophobic surface in a Ca²⁺-concentration dependent manner, the half-maximal effect occurring at approximately 6 μM. Mg²⁺ was not effective for the conformational change. On Western blotting, ALG-2 was detected in particulate fractions from cultured mammalian cells, suggesting the association of the protein with macromolecules in the cells.