Effect of Calpain on Hereditary Cataractous Rat, ICR/f.

Abstract

The ICR/f rat is a recessive-type hereditary cataractous strain. We previously reported that this mutant differs from normal in the composition of lens crystallins.1) Analysis of the cataractous lens crystallins showed remarkable decreases in g-, bB1-and bA3-crystallin contents. Also, the calcium content in the cataractous lenses was about 10-fold higher than in the normal lenses. Two-dimensional electrophoresis of the cataractous lens proteins revealed the loss of b-and acrystallins and the appearance of new polypeptides.The major classes of crystallins are the a-crystallins which are molecular chaperones related to the small heatshock proteins, 2) and the b-and g-crystallins.3) The b-and gcrystallin superfamilies are characterized by four Greek key motifs in their amino acid sequences. The b-crystallins also have amino-and carboxy-terminal extensions designated as "arms". All b-crystallins have N-terminal extensions of 10 to 58 amino acids in length, and the subtype of bB-crystallin has C-terminal extensions of approximately 15 amino acids.3,4) g-Crystallins lack these N-and C-terminals. While b-crystallins form mixtures of homodimers, heterodimers, and higher order oligomers, 5) the native g-crystallins are characterized as monomers.3) It has been hypothesized that the extensions function to stabilize the intermolecular interaction between b-crystallins and to prevent their random aggregation.3) Thus some research has examined the possible function of b-crystallin N-terminal extensions in stabilizing the structure of b-crystallin oligomers. 6,7)Calpain (EC 3.4.22.17) is a non-lysosomal cysteine-protease activated by calcium. The ubiquitous m-and m-calpains, by far the best characterized calpains, are heterodimers comprising distinct but quite homologous 80-kDa "large" Lsubunits and a common 30-kDa "small" S-subunit. On exposure to calcium at concentrations of 5-50 mM (for m-calpain) and 200-1000 mM (for m-calpain), these calpains are activated and partially autolyzed.8) m-Calpain has been found in lenses of all species examined so far. David et al. have reported that the amino acid sequence of the N-terminal of bcrystallins in the insoluble protein fraction of lens from rodent cataractous lenses is very similar to the N-terminal of bcrystallin polypeptides degraded partially by the purified mcalpain. 11,12) This result suggests that endogenous calpain cleaves the N-terminal extension of b-crystallin, and the resulting N-terminal-missing b-crystallins aggregate randomly. Cysteine protease inhibitors suppressed this proteolysis and temporarily prevented the progression of cataract formation caused by various inducers in vitro.13) In addition, incubation of total soluble proteins from young mouse and rat lenses with the purified m-calpain, [11][12][13] or activation of endogenous lens calpain in a soluble fraction by calcium, caused the insolubilization of crystallins and an increase in the turbidity of the fraction. 9) On the other hand, David et al. reported that 120 mM KCl (the concentration in normal lenses)...

Phosphoproteome Analysis of Hereditary Cataractous Rat Lens .ALPHA.-Crystallin

“…3) In previous studies, we have reported that oxidative stress occurred in cataractous lens (ICR85). 16,21) In this study, we have found an increase in the phosphorylation of serine residues of truncated of a-crystallin in cataractous lens (ICR85). In conclusion, C-terminal truncation and phosphorylation of a-crystallin could both reduce its chaperone-like activity and lead to cataract formation.…”

“…Isolation and purification of lens crystallins were performed according to the method of previous paper. 16) Briefly, 4 ml of the supernatant (lens water-soluble fraction) was applied to a Superose 12 column (1.6ϫ50 cm, Amersham Biotech) and eluted with 50 mM phosphate (pH 7.0) containing 150 mM NaCl, 1 mM EDTA, and 0.02% NaN 3 at a flow rate of 0.5 ml/min at room temperature. The eluates from the column were monitored by measuring absorbance at 280 nm.…”

Pharmacokinetic and Pharmacodynamic Evaluation of the Anti-cataract Effect of Eye Drops Containing Disulfiram and Low-Substituted Methylcellulose Using ICR/f Rats as a Hereditary Cataract Model

“…31,32) The Ca 2+ content in the cataractous lenses of ICR/f rats is about 10-fold higher than in the lenses of Wistar rats, and autolytic products formed by calpain can also be detected in cataractous lenses. 33,34) We also reported that excessive nitric oxide produces an increase in lipid peroxide, and that the elevated lipid peroxide levels cause an decrease in Ca 2+ -ATPase activity and increase in Ca 2+ content in ICR/f rat lenses during cataract development. 18,19) It is noteworthy that ICR/f rat cataracts are not related to diabetic cataracts, and that the mechanism of cataract development is similar to that of senile cataracts.…”

Comparison of the Mechanisms of Cataract Development Involving Differences in Ca2+ Regulation in Lenses among Three Hereditary Cataract Model Rats

“…[10][11][12][13][14] The Ca 2ϩ content in the cataractous lenses of UPLR, SCR and ICR are higher than in the lenses of Wistar rats, and autolytic products formed by calpain can also be detected in cataractous lenses. 11,12,15,16) It is noteworthy that the cataracts formed in these rat models are not diabetic cataracts. Therefore, these rats have been used for studies to develop anti-cataract drugs.…”