The calcium sensor protein caldendrin is abundantly expressed in neurons and is thought to play an important role in different aspects of synapto-dendritic Ca2+ signaling. Caldendrin is highly abundant in the postsynaptic density of a subset of excitatory synapses in brain and its distinct localization raises several decisive questions about its function. Previous work suggests that caldendrin is tightly associated with Ca2+ - and Ca2+ release channels and might be involved in different aspects of the organization of the postsynaptic scaffold as well as with synapse-to-nucleus communication. In this report we introduce two new EF-hand calcium sensor proteins termed calneurons that apart from calmodulin represent the closest homologues of caldendrin in brain. Calneurons have a different EF-hand organization than other calcium sensor proteins, are prominently expressed in neurons and will presumably bind Ca2+ with higher affinity than caldendrin. Despite some significant structural differences it is conceivable that they are involved in similar Ca2+ regulated processes like caldendrin and neuronal calcium sensor proteins.

AIM1 (absent in melanoma), a candidate suppressor of malignancy in melanoma, is a nonlens member of the betagamma-crystallin superfamily, which contains six predicted betagamma domains. The first betagamma-crystallin domain of AIM1 (AIM1-g1) diverges most in sequence from the superfamily consensus. To examine its ability to fold and behave like a normal betagamma domain, we cloned AIM1-g1 and overexpressed it in Escherichia coli as a recombinant protein. The recombinant domain was found to be a stable, soluble protein, similar to lens protein gammaBeta-crystallin in secondary structure. The tertiary structure of AIM1-g1 is dominated by the contribution of aromatic amino acids and cysteine. AIM1-g1 undergoes concentration-independent, noncovalent homodimerization with no trace of monomer, similar to a one-domain protein spherulin 3a. Since many betagamma domain proteins bind calcium, we have also investigated the calcium-binding properties of AIM1-g1 by various methods. AIM1-g1 binds the calcium-mimic dye Stains-all, the calcium probe terbium (with K(D) 170 microM), and (45)Ca when blotted on a membrane. AIM1-g1 binds calcium (K(D) 30 microM) with a comparatively higher affinity than bovine lens gamma-crystallin (90 microM). However, calcium binding does not induce significant change in the protein conformation in the near- and far-UV CD and in fluorescence. The AIM1-g1 domain is as stable as domains of betagamma-crystallins (betaB2- or gammaS-crystallins) as monitored by guanidinium chloride unfolding (midpoint of unfolding transition is 1.8 M GdmCl), and the stability of the protein is not altered upon binding calcium as evaluated by equilibrium unfolding. These results show that, despite the sequence variation, AIM1-g1 folds such as a betagamma domain, binds calcium and undergoes dimerization.

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...

Corneal epithelium is known to have high levels of some metabolic enzymes such as aldehyde dehydrogenase in mammals, gelsolin in zebrafish, and ␣-enolase in several species. Analogous to lens crystallins, these enzymes and proteins are referred to as corneal crystallins, although their precise function is not established in any species. Although it is known that after lentectomy, the outer cornea undergoes transdifferentiation to regenerate a lens only in anuran amphibians, major proteins expressed in an anuran cornea have not been identified. This study therefore aimed to identify the major corneal proteins in the Indian toad (Bufo melanostictus) and the Indian frog (Rana tigrina). Soluble proteins of toad and frog corneas were resolved on two-dimensional gels and identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight and electrospray ionization quadrupole time-of-flight. We report that anuran cornea is made up of the full complement of ubiquitous lens ␣-, ␤-, and ␥-crystallins, mainly localized in the corneal epithelium. In addition, some taxon-specific lens crystallins and novel proteins, such as ␣-or ␤-enolase/-crystallin, were also identified. Our data present a unique case of the anuran cornea where the same crystallins are used in the lens and in the cornea, thus supporting the earlier idea that crystallins are essential for the visual functions of the cornea as they perform for the lens. High levels of lens ␣-, ␤-, and ␥-crystallins have not been reported in the cornea of any species studied so far and may offer a possible explanation for their inability to regenerate a lens after lentectomy. Our data that anuran cornea has an abundant quantity of almost all the lens crystallins are consistent with its ability to form a lens, and this connection is worthy of further studies.

Posttranslational modification of proteins, such as glycosylation, can impact cell signaling and function. ST6Gal I, a glycosyltransferase expressed by B cells, catalyzes the addition of α-2,6 sialic acid to galactose, a modification found on N-linked glycoproteins such as CD22, a negative regulator of B cell activation. We show that SNA lectin, which binds α-2,6 sialic acid linked to galactose, shows high binding on plasma blasts and germinal center B cells following viral infection, suggesting ST6Gal I expression remains high on activated B cells in vivo. To understand the relevance of this modification on the antiviral B cell immune response, we infected ST6Gal I−/− mice with influenza A/HKx31. We demonstrate that the loss of ST6Gal I expression results in similar influenza infectivity in the lung, but significantly reduced early influenza-specific IgM and IgG levels in the serum, as well as significantly reduced numbers of early viral-specific Ab-secreting cells. At later memory time points, ST6Gal I−/− mice show comparable numbers of IgG influenza-specific memory B cells and long-lived plasma cells, with similarly high antiviral IgG titers, with the exception of IgG2c. Finally, we adoptively transfer purified B cells from wild-type or ST6Gal I−/− mice into B cell-deficient (μMT−/−) mice. Recipient mice that received ST6Gal I−/− B cells demonstrated reduced influenza-specific IgM levels, but similar levels of influenza-specific IgG, compared with mice that received wild-type B cells. These data suggest that a B cell intrinsic defect partially contributes to the impaired antiviral humoral response.