Limited proteolysis of bovine lens α-crystallin by calpain, A Ca2+-dependent cysteine proteinase, isolated from the same tissue

The purpose of this study was to examine changes in calcium-dependent proteolytic activity in the lens epithelium from whole rabbit lenses exposed to long-term oxidative stress at near physiological levels. Rabbit lenses, incubated in 50 µM H₂O₂ for 1 or 24 h, were checked for clarity and morphological changes in the epithelium. Proteolytic activity was measured in the epithelium using a fluorogenic synthetic substrate; N-succinyl-Leu-Tyr-7-amino-4-methylcoumarin, both in the presence and the absence of calcium (1 mM Ca²⁺ and 5 mM EDTA respectively). The effect on transparency and morphology of the epithelium following a 1-hour incubation in 100 µM H₂O₂ was also studied. Lenses incubated in 50 µM H₂O₂ were clear even after 24 h. After a 1-hour incubation in 50 µM H₂O₂ the epithelium of the exposed lens appeared normal. However, after 24 h the epithelium cells appeared swollen and microscopical examination showed extensive intracellular and subepithelial vacuolization. Incubation in 100 µM H₂O₂ for 1 h caused loss of transparency; vacuole formation, globulization of the superficial lens fibers and death of the epithelial cells. There was a 55% increase in calcium-dependent proteolytic activity after 1 h in 50 µM H₂O₂, implying a role for the calcium-activated protease calpain in oxidatively induced cataract.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Calcium-Dependent Proteolysis in Rabbit Lens Epithelium after Oxidative Stress

Andersson

Sjöstrand²,

Petersen³

et al. 1998

“…The possible physiological function of this protein might be to act as a calcium store or sequester through binding the available calcium, thus regulating the free calcium level in the lens. This is important since high level of free calcium might (1) trig ger calpain-like enzymatic activity for which a-and (3-crystallins are the possible substrates in lens [16,17]; (2) induce transglutaminase activity causing covalent crosslinking in (3-crystallins [18]; (3) induce aggregation of acrystallin and degenerate the lens fibre cells. The high content of p-crystallin in lens with its affinity and moderate capacity for calcium binding favours its possible role in the regula tion of free calcium level in the lens cyto plasm, specifically in the cortical region of the lens.…”

Section: Com Parison Of the Affinities Of P-crystallins From V Ariousmentioning

confidence: 99%

Calcium Binding Properties of Beta-Crystallins

Sharma

Balasubramanian²

1996

β-Crystallins, the oligomeric proteins of the eye lens, were found to bind calcium ions with low affinity, in the millimolar range. Calcium was found to induce conformational changes in the secondary and tertiary structure of β-crystallins. While these changes in conformation are seen in low ionic strength media, they are masked when the protein is dissolved in an intermediate ionic strength medium, where oligomers of lower molecular weight are formed. The calcium binding takes place in these conditions. The behaviour of this protein with respect to calcium binding is reviewed.

“…Activated calpain is known to proteolyze a number of lens crystallins [8][9][10], and we pre viously showed proteolysis of lens crystallins in xylose cataract [15], There is some evi dence that degraded crystallins contribute to cataract formation. Degraded protein in creased in cataractous and aging lenses [9,[17][18][19], This degraded protein was associated with formation of high-molecular-weight pro tein [29] which is one factor in light scattering in cataractous lenses [30], Figure 4 emphasizes the role of calpain in xylose nuclear cataract after the initial os motic insult by xylose to produce cortical cataract.…”

Section: mentioning

confidence: 99%

“…Calpain has been demonstrated in human [7], bovine [8], mouse [9] and rat [10] lenses. E64 is a small peptide inhibitor of cysteine proteases, such as calpain.…”

Section: Introductionmentioning

confidence: 99%

Hydration and Elevated Calcium Alone Do Not Produce Xylose Nuclear Cataract: Role of Proteolysis by Calpain

Azuma

David

Shearer³

1992

The purpose of this experiment was to determine the contribution of calpain proteolytic enzyme (EC 3.4.22.17) in the formation of nuclear cataract during lens culture in xylose. Increased lens calcium was found to be required for formation of xylose nuclear cataract in our culture system. Inhibition of calpain by the cysteine protease inhibitor E64 was effective in slowing the formation of nuclear cataract, even though lens calcium and hydration were markedly elevated. These results showed that hydration and elevated calcium alone do not produce xylose nuclear cataract, and they indicated that calpain proteolysis may be necessary for xylose nuclear cataract in the rat lens.