Calcium Binding Properties of γ-Crystallin

Abstract

The ␤-and ␥-crystallins are closely related lens proteins that are members of the ␤␥-crystallin superfamily, which also include many non-lens members. Although ␤-crystallin is known to be a calcium-binding protein, this property has not been reported in ␥-crystallin. We have studied the calcium binding properties of ␥-crystallin, and we show that it binds 4 mol eq of calcium with a dissociation constant of 90 M. It also binds the calcium-mimic spectral probes, terbium and Stains-all. Calcium binding does not significantly influence protein secondary and tertiary structures. We present evidence that the Greek key crystallin fold is the site for calcium ion binding in ␥-crystallin. Peptides corresponding to Greek key motif of ␥-crystallin (42 residues) and their mutants were synthesized and studied for calcium binding. These peptides adopt ␤-sheet conformation and form aggregates producing ␤-sandwich. Our results with peptides show that, in Greek key motif, the amino acid adjacent to the conserved aromatic corner in the "a" strand and three amino acids of the "d" strand participate in calcium binding. We suggest that the ␤␥ superfamily represents a novel class of calcium-binding proteins with the Greek key ␤␥-crystallin fold as potential calcium-binding sites. These results are of significance in understanding the mechanism of calcium homeostasis in the lens.Calcium homeostasis plays an important role in lens transparency, opacification, and cataractogenesis. Cataracts can occur both under hypocalcemic and hypercalcemic conditions, so the actual amount of available calcium in the lens is an important parameter for the health of the lens (1, 2). The normal mammalian lens has around 0.2 mM total calcium, of which the amount of free Ca 2ϩ is only of the order of a few micromolars. Thus there must exist calcium regulation systems in the lens, and it is of interest to identify what they are and how they change in health and in disease. Vrensen et al. (3) have done an ultrastructural analysis of calcium distribution in the rat lens and have found calcium precipitates in the intermediate cortex fiber membranes, cytoplasm, and the nuclear envelope and very low levels of calcium in gap junctions, epithelial cells, and superficial fibers (3-5). The question of what the calcium-binding and -storing agents are in the lens is open; phospholipids and crystallins have been thought of as candidates. The major components of the lens are cytosolic proteins, crystallins, which account for about 40% of the wet weight of the lens. It is worth investigating whether any of the crystallins could act as a calcium sponge or storage depot in the tissue, particularly since the ultrastructural analysis shows calcium distribution in the cytoplasm. We have earlier shown that the ␤-and avian core protein ␦-crystallins show significant calcium-binding ability (6, 7). Thus, the possibility of crystallins acting as lenticular calcium-sequestering and -storing systems exists. However, the calcium binding properties of ␥-crystallin have not yet been...

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