Calcium Binding Properties of Beta-Crystallins

Sharma, Yogendra; Balasubramanian, D.

doi:10.1159/000267942

Cited by 13 publications

(16 citation statements)

References 11 publications

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“…Its colocalization with synaptotagmin 1, which is a leading calcium sensor within the retina and likely triggers exocytosis (52), may point to a similar mechanism of secretion into the culture medium. Colocalization of crybb2 with calmodulin, which is the major calcium-binding protein in the retinal ganglion cell layer (53) and in the RGC-5 cell line (54), provides further evidence that crybb2 also operates through calcium binding by its Greek key motifs (55). The double staining with crybb2 and tau-1 protein, which is a specific marker for axons, showed that crybb2 was translocated into the processes, whereas tau-1 remained confined to the cell body (32).…”

Section: Discussionmentioning

confidence: 99%

Elongation of Axons during Regeneration Involves Retinal Crystallin β b2 (crybb2)

Liedtke

Schwamborn

Schröer

et al. 2007

Molecular & Cellular Proteomics

103

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Adult retinal ganglion cells (RGCs) can regenerate their axons in vitro. Using proteomics, we discovered that the supernatants of cultured retinas contain isoforms of crystallins with crystallin ␤ b2 (crybb2) being clearly up-regulated in the regenerating retina. Immunohistochemistry revealed the expression of crybb within the retina, including in filopodial protrusions and axons of RGCs. Cloning and overexpression of crybb2 in RGCs and hippocampal neurons increased axonogenesis, which in turn could be blocked with antibodies against ␤-crystallin. Conditioned medium from crybb2-transfected cell cultures also supported the growth of axons. Finally real time imaging of the uptake of green fluorescent protein-tagged crybb2 fusion protein showed that this protein becomes internalized. These data are the first to show that axonal regeneration is related to crybb2 movement. The results suggest that neuronal crystallins constitute a novel class of neurite-promoting factors that likely operate through an autocrine mechanism and that they could be used in neurodegenerative diseases. Molecular & Cellular Proteomics 6:895-907, 2007. Adult retinal ganglion cells (RGCs)1 exhibit only a short and transient sprouting reaction after injury, and they fail to extend axons throughout the interior of the optic nerve (1). The failure of regeneration is commonly attributed to inhibitory factors associated with myelin components and/or the glial scar that includes cells and extracellular matrix proteins (2-7). The inhibitory myelin proteins have been shown to include NogoA, myelin-associated glycoprotein, and oligodendrocyte-myelin glycoprotein, all of which act through the Nogo receptor, NgR (8 -11). Expanding on this pathway of inhibition, blockage of signaling through NgR, applying antibodies against NogoA, and inactivation of RhoA (a downstream effector of NgR) signaling result in only modest axonal regeneration (12-14).There are several experimental conditions that permit the regrowth of RGC axons, including (i) replacing the distal segment of the cut optic nerve with a sciatic nerve segment (15), (ii) injuring the optic nerve and delayed culturing of retinal explants in vitro (16) or dissociation of RGCs and culturing of primary cells mainly in the postnatal retina (17), and (iii) injuring the lens (18 -21) or implanting a peripheral nerve fragment directly into the vitreous (22). Expanding on the mutual inflammatory mechanism of lens injury, stimulation of ocular macrophages with intravitreal injections of zymosan (19, 21, 23) also supports axonal growth. Explorations of alternative noninflammatory mechanisms have revealed that coculturing dissociated RGCs with injured lens tissue devoid of macrophages also improves the growth of axons (24). In an attempt to localize the growth factors within the lens, a coculture of retinal stripes with intact lens epithelium cells was shown to also support axonal growth in vitro (25), suggesting that independent mechanisms can result in the successful growth of axons. Consequently axon regene...

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Section: Discussionmentioning

confidence: 99%

Elongation of Axons during Regeneration Involves Retinal Crystallin β b2 (crybb2)

Liedtke

Schwamborn

Schröer

et al. 2007

Molecular & Cellular Proteomics

103

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“…This leaves us to explain the tight binding of calcium in these deeper zones. In a scries of studies Sharma and Balasubramanian [13] have shown that (3-crystallin has calcium binding properties and additionally is present in large amounts in lens fibers. Although (3- The possible consequences of these local variations in specific calcium storage sites are not yet understood.…”

Section: Discussionmentioning

confidence: 99%

Calcium Distribution in the Human Eye Lens

Vrensen¹,

Wolf²

1996

Ophthalmic Res

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The distribution of calcium in the human eye lens was investigated using the oxalate-pyroantimonate technique. In epithelial cells and superficial cortical fibers calcium is sequestered and tightly bound to proteins in the endoplasmic reticulum, nuclear envelope, Golgi cisterns and mitochondria. In intermediate fibers these cell organelles are broken down and the liberated calcium is ionically bound to the phospholipids at the extracellular side of the fiber-limiting membrane. It is argued that calcium is probably additionally bound to β-crystallin. This site-specific variation in calcium storage might explain the deeper cortical localization of early focal dot opacities and retrodots.

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“…An interesting feature of some of these proteins is their calciumbinding ability, e.g. ␤-crystallin (6,7,26), protein S (14,27), and spherulin 3a (17). Putative calcium-binding sites have been shown in the EP37 proteins (20).…”

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confidence: 99%

Calcium Binding Properties of γ-Crystallin

Rajini

Shridas

Sundari

et al. 2001

Journal of Biological Chemistry

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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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Calcium Binding Properties of Beta-Crystallins

Cited by 13 publications

References 11 publications

Elongation of Axons during Regeneration Involves Retinal Crystallin β b2 (crybb2)

Elongation of Axons during Regeneration Involves Retinal Crystallin β b2 (crybb2)

Calcium Distribution in the Human Eye Lens

Calcium Binding Properties of γ-Crystallin

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