Calcium-binding to lens βB2- and βA3-crystallins suggests that all β-crystallins are calcium-binding proteins

Abstract

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, β- and γ-crystallins, are the prototype and founding members of the βγ-crystallin superfamily. βγ-Crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of βγ-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in βB2-crystallin and other βγ-crystallins from fish and mammalian genomes, it was argued that lens βγ-crystallins should not bind calcium. In order to probe whether β-crystallins can bind calcium, we selected one basic (βB2) and one acidic (βA3) β-crystallin for calcium-binding studies. Using calcium-binding assays such as ⁴⁵Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both βB2- and βA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, ¹⁵N-¹H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to βB2- and βA3-crystallins, we suggest that all β-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.