Age and cataract-related changes in the heavy molecular weight proteins and gamma crystallin composition of the mouse lens.

Heavy molecular weight (HMW) proteins were detected in normal and cataractous mouse lenses. The HMW aggregates increased with the age of the lens in normal mouse. Alpha and β-crystallins were detected by immunodiffusion in the HMW fractions from normal and Nakano mice. No γ-crystallin could be detected in these aggregates by immunodiffusion; however, a slight amount of this crystallin was detected using the radioimmunoassay. The polypeptide composition of the HMW proteins was different in the Nakano mouse from the normal. By SDS polyacrylamide gel electrophoresis, a polypeptide of 27 000 mol. wt. was evident in the Nakano HMW material that was not present in the normal HMW protein, but a 15 000 mol. wt. band was absent in the Nakano.Two other differences were seen with the Nakano lens. First, the water insoluble lens protein was extremely high. By 90 days, about two thirds of the protein was insoluble in these lenses. Secondly, the sharp drop in γ-crystallin at the time of complete opacification of the lens was in part a result of the leakage of this protein into the anterior chamber of these mice. By radioimmunoassay, the level of γ-crystallin in the Nakano aqueous humor at the time of the cataract was greater than 100 ng per microliter. These data demonstrate that crystallins are converted to the insoluble proteins and some diffuse out of the lens during cataract formation.     

In vitro incubation paralleling changes occurring during mouse cataract formation

Certain biochemical and morphological changes involved in the formation of osmotic cataracts in the Nakano mouse and galactosemic rat lenses also occur in vitro. Incubation of young, normal, albino mouse lenses in glucose-free medium for 48 hr resulted in a gain in wet weight due to dramatic hydration. The glucose deprived lenses showed a marked decrease in the soluble protein content as is noted in cataract formation in vivo. Chromatography of the soluble protein demonstrated a substantial increase in heavy molecular weight material in the lenses incubated without glucose in comparison to the fresh lenses. A decrease in β_H-crystallin was also striking and might be correlated with the disappearance of a 31 000 mol. wt polypeptide band seen by SDS-PAGE gel. Interestingly, the membrane polypeptides in the in vitro incubated lenses mimicked changes observed during cataract formation in vivo. A 23 000 mol. wt polypeptide band became prominent in the membrane fraction of these lenses and there was a concomitant decrease in the 26 000 mol. wt component. Messenger RNAs were present in the glucose deprived lenses as shown by cell-free translation in vitro, although no [³⁵S]methionine incorporation into crystallin was noted. Both the hexokinase and glyceraldehyde phosphate dehydrogenase activities in the glucose deprived lenses fell rapidly and were virtually absent at the end of 48-hr incubation period. The data in this report suggest that the glucose deprived incubated mouse lens may serve as an in vitro model to the study of some biochemical and morphological changes occurring in the development of osmotic cataract in vivo.     

Biochemical alterations of the lens capsule in mice with hereditary cataract

Lens capsules from normal and cataractous mice (Nakano strain) were compared histologically and chemically. Histologically, the anterior capsule of the cataract lens was thicker than the normal, while the posterior capsule appeared almost the same. Amino acid analysis revealed some differences between normal and cataractous lens capsules: more glycine, hydroxylysine and arginine and less hydroxyproline and tyrosine were found in cataractous capsules. Hydroxyproline showed the largest difference, cataract lens being about 11% lower than normal.To see if the content of hydroxylysine-linked carbohydrates is different in normal and cataractous lenses, as has been shown for renal basement membranes from normal and diabetic animals, a procedure was developed to separate basic amino acids, including the hydroxylysine-linked carbohydrates, from other amino acids. It was found that the content of hydroxylysine-linked disaccharide (Glc-Gal-Hyl) was slightly but significantly higher in cataractous lenses.The possibility was suggested that the compositional alterations observed for the cataract lens might be correlated with functional changes in the lens capsule of the cataract, e.g. lowered elasticity, decreased permeability etc.     

Studies on primary cultures of adult lens cells from normal and hereditary cataractous mice.

Lens epithelial cells from normal and congenital cataractous mice strains were cultured under similar conditions. Both normal and cataractous cells actively propagated and reached confluency on the eleventh day. These cells, thereafter, underwent morphological changes characterized by cell elongation, aggregation and formation of lentoid bodies at about 15 days.Electron microscopy revealed these lentoid bodies to consist of immature lens cells. These structures derived from cataractous cells had numerous vacuoles in the cytoplasm much more so than in the normal lens cells. In addition, some lentoid bodies closely resembled mature fibers of the intact lens. It was also demonstrated that these lentoid bodies showed positive immunofluorescence when reacted with fluorescent antiserum to γ-crystallin.There were certain differences observed between the cultured cells derived from normal lens and Nakano cataract. The disappearance of organelles and denucleation process were delayed in the lentoid bodies found in cultured Nakano cells when compared to normal cell culture. In addition a second type of lentoid body, although present as a minor population, was observed in the Nakano cell culture. Other subtle differences were observed during the course of culturing normal and cataractous lens cells.     

Origin of urea-soluble protein in the selenite cataract. Role of beta-crystallin proteolysis and calpain II

Nuclear cataract resulting from an overdose of selenite was characterized by a five-fold increase in nuclear urea-soluble protein. The origin of this urea-soluble protein was examined by two-dimensional electrophoresis, immunoblotting with monospecific antisera against rat lens crystallins, and tryptic mapping. Cataractous urea-soluble protein was primarily composed of insolubilized beta- and gamma-crystallin polypeptides. Polypeptides from cataractous urea-soluble protein, and normal beta L-crystallin aggregates were compared by tryptic mapping. Approximately 19% of the urea-soluble protein from opaque nuclei was composed of 24.7 and 24.0 K polypeptides derived by limited proteolysis of 26.5 K beta L-crystallin polypeptide. Incubation of 26.5 K beta-crystallin polypeptide with purified rat lens calpain II in vitro caused production of fragments with similar molecular weights to polypeptides found in cataractous lenses. These results support the hypothesis that proteolysis may contribute to formation of urea-soluble protein in selenite cataract.     

Calcium-activated proteolysis in the lens nucleus during selenite cataractogenesis

A single injection of 20 mumol sodium selenite/kg body weight in 10-day-old rats caused severe nuclear cataract within 4 days. By 4 days postselenite injection, nuclear calcium levels increased from 0.4 to 6.8 mmol/kg lens dry weight. The purpose of these experiments was to determine if this calcium increase was associated with proteolysis specifically in the lens nuclear region. Sodium dodecyl sulfate polyacrylamide electrophoresis of lens nuclear proteins following selenite injection showed: loss of 30, 27, and 26 K molecular weight polypeptides in the soluble fraction, loss of 83, 52, 30, 27, and 26 K polypeptides in the insoluble fraction, and loss of the major 26 K membrane protein. Gel chromatography of nuclear soluble proteins indicated a decrease in beta H and beta L crystallins following selenite injection. Two-hour in vitro incubation of nuclear lens homogenates with calcium duplicated many of the proteolytic changes occurring in lenses in vivo following selenite injection. Calcium induced proteolysis in vitro was inhibited by EGTA, leupeptin, and iodoacetate but was not inhibited by phenylmethylsulfonyl fluoride. These properties are similar to calcium activated protease (CAP) from other tissues. Activation of CAP, and subsequent degradation of nuclear proteins, may be causes of selenite cataract.     

Characterization of water-insoluble proteins in normal and cataractous human lens

The mechanism of lens protein aggregation with age and/or cataract formation was investigated using the peptides resolubilized from the insoluble protein fraction of normal and cataractous human lenses. The insoluble fraction was treated with reductants for cleaving disulfide bonds, or with chelating agents for removing calcium ions from the aggregates. This study demonstrates that the insoluble protein aggregates consist of an approximately 400 Kd complex, which is formed by the peptides with lower molecular weight. Protein aggregation in the cataractous lens might be caused by disulfide bonds whereas, in aging, the aggregate might be preferentially formed by calcium ion bridges rather than by disulfide bonds. It was observed that the aggregate from the cataractous lenses involved a peptide with a molecular mass lower by 1 Kd or 2 Kd than the peptides found in the normal lens. The composition of crystallins in aggregating proteins and their secondary structures were also different in the normal and the cataractous lenses. Such changes of molecular weight, conformation, and/or crystallin species in the lens may lead to the disintegration of the orderly arrangement of crystallins, resulting in the diffused reflection and lens opacities which are seen in senile cataract.     

Differential synthesis and degradation of protein in the hereditary Philly mouse cataract

Lens protein metabolism was investigated in the Philly mouse between the third and eighth postnatal week. As demonstrated in an accompanying article, the Philly mouse develops a hereditary, osmotic, cataract associated with influx of Na⁺ and loss of K⁺ during this time interval. The contents of β- and γ-crystallin were strikingly reduced in the Philly lens, as judged by sodium dodecyl sulfate (SDS)-urea-polyacrylamide gel electrophoresis and by immunodiffusion. This appeared to be due to proteolysis, since there were negligible amounts of crystallins found in the medium of cultured Philly lenses. α-Crystallin remained in the Philly lens but apparently accumulated discrete polypeptide cleavage products. The incorporation of [³⁵S]methionine into β- and γ-crystallin polypeptides was markedly reduced in the Philly lens. By contrast, the incorporation of [³⁵S]methionine into the α-crystallin and the higher molecular weight non-crystallin polypeptides was as great if not greater, in the Philly lens than in the normal lens. The non-crystallin polypeptides were associated with the 10 000 × g pellet of the homogenate. The present data extend the correlation between alterations in protein metabolism and electrolyte concentrations to this hereditary cataract, and support the idea that selective degradation of crystallins and differential reduction in the synthesis of crystallins are primary causes for the lowered amounts of soluble protein—especially β- and γ-crystallin—found in cataracts associated with ionic imbalances.     

Biosynthesis of proteoglycans by lens epithelial cells of cataractous mouse (Nakano strain)

The capsules (with epithelial cells attached) of lenses from normal and cataractous mice (Nakano strain) were biosynthetically labeled in vitro with radioactive precursors. The labeled macromolecules were chromatographed on a Sepharose CL-4B column and analyzed by specific enzyme digestion. The incorporation of [3H]-proline and [3H]-glucosamine into macromolecules was comparable in the cataract and normal capsules, while that of [35S]-sulfate was reduced by 60% in the cataract capsules, indicating that the proteoglycan synthesis was specifically decreased in the cataract lens. Glycosaminoglycan analyses showed an increased synthesis of hyaluronic acid and decreased synthesis of heparan sulfate in the cataract capsules. It is possible that the alterations in the synthetic level and glycosaminoglycan components of proteoglycan affect the permeabilities of macromolecules to lens capsule and lead to cataract in Nakano mouse lens.     

Lens growth in the Nakano mouse.

The growth curve of the lens of the Nakano mouse was compared to that of the normal mouse. There was no obvious difference for the first two weeks of age. After this period the growth of the normal lens continued while that of Nakano mouse lens slowed abruptly and eventually ceased. Studies with labeled leucine, however, showed that even after the appearance of the "pin-head" opacity the protein synthesis, although depressed, continued. These findings combined with previous histological observations suggest that new fiber formation is probably unaffected in the early stages of the Nakano cataract. The apparent cessation of lens growth is probably associated with the extensive liquefaction that is observed to occur at the posterior nuclear region.     

Changes in lens proteins induced at the early stage of cataractogenesis in cac (Nakano) mice

Changes in lens proteins induced at the early stages of cataractogenesis in cac (Nakano) mice were investigated by two-dimensional gel electrophoresis (2D-PAGE). The 2D-PAGE profile of lens proteins in 21-day-old cac mice differed from that in 27-day-old normal mice, even though the appearance of 'pin-head' nuclear opacity (26-day-old) had not yet been observed in the lenses. Especially noticeable were great differences in the polypeptides associated with the alpha- and beta- crystallin subfractions, the appearances of which corresponded to an increase in a ratio of the amounts of Na+ to that of K+ in the lenses of defective mice. No dramatic decrease in the gamma-crystallin fraction was observed until the mature cataract stage.     

Disulfide-linked membrane proteins in X-ray-induced cataract

The formation of membrane protein disulfide was investigated at various stages of development of X-ray-induced cataract in the rabbit. Intermolecular disulfide bonding of lens membrane proteins was detected not only in the mature cataract (occurring 8-9 weeks after the X-ray dose) but also at 1 week prior to maturation, in which no significant increase in lens hydration occurs and where opacification is confined mainly to the posterior subcapsular region. Two-dimensional diagonal electrophoresis revealed that polypeptides with apparent molecular weights of 21, 23, 25, 31, 35, 46 and 53 kilodaltons were involved in cross-linking. The MP26 membrane polypeptide was not significantly involved in the disulfide bonding. The oxidation of membrane proteins in stages other than mature was evident only in the lens nucleus (which remained clear) and not in the cortex. The results of this study indicate that an intermolecular disulfide linkage of cytosolic proteins to membranes occurs prior to formation of mature cataract, and may be a precursor to protein aggregation and insolubilization in the mature nuclear cataract.     

The development and application of a radioimmunoassay to lens crystallins

A radioimmunoassay was developed for mouse α- and γ-crystallins. A standard inhibition curve against known quantities of the crystallins served as a basis for quantitating unknown samples. The sensitivity of the assay was 2 ng for α-crystallin and 4 ng for γ-crystallin. It was possible to quantitate the amount of γ-crystallin present in the cultures from normal mouse and Nakano mouse lenses. The results were consistent with the previous immunofluorescent observation of γ-crystallin localization in the lentoid bodies. In addition, changes in the lens crystallins in normal and Nakano mouse lenses were followed. A dramatic decrease of γ-crystallin occurred during cataract development, however the level of α-crystallin in the soluble lens protein remained unchanged during this period.     

Human alpha-crystallin-III isolation and characterization of protein from normal infant lenses and old lens peripheries.

Alpha-crystallin isolated from the peripheries of old normal or cataractous lenses appears to be identical, consisting of eleven polypeptides, five B, and six A chains. In contrast, alpha-crystallin isolated from normal six-week-old human lenses has only three major polypeptides, corresponding to B1, A1, and A2 of the old human lens protein as well as small amounts of some of the other components. Comparisons with bovine alpha-crystallin are also reported. Based on gel filtration experiments with Bio-Gel A-1.5m, two distinct populations of alpha-crystallin were found in old lens periphery, one containing species greater than 1.5 X 10(6) daltons and another of approximately 9 X 10(5) daltons. In the cataract preparations, the higher molecular weight fraction is predominant. This fraction is not present in young lenses.     

Selective association of crystallins with lens 'native' membrane during dynamic cataractogenesis.

Plasma membrane with its associated extrinsic proteins was isolated from normal and cataractous rat lenses by centrifugation of the total water insoluble fraction from homogenized lenses on a discontinuous sucrose gradient. Membrane, which we call "native" membrane, was recovered mainly from the 25/45% sucrose interface. Development of the experimental U18666A cataract resulted in plasma membrane shifting to higher density (the 50/55% sucrose fraction) and great increases in the urea soluble protein content of the lens. At early stages of cataract development, most of the increased urea soluble protein was membrane associated, presumably as extrinsic protein. With advancing cataract, most of the urea soluble protein appeared in an essentially membrane-free pellet fraction. The urea soluble protein associated with the cataract membrane was shown by combined IEF, SDS-PAGE, Western blotting, amino acid compositional analysis and protein sequence determinations to be mainly composed of modified alpha- and beta-crystallins. Alpha A-crystallin truncated by not more than 27 residues from the carboxyl terminus plus beta b1 crystallin truncated by 49 residues from the amino terminus were conclusively identified. In addition to beta b1, a population of six alpha-crystallin derived polypeptides were specifically enriched in the cataract membrane fraction. Four of these six alpha-crystallins appear to be truncated from their carboxyl terminus, a modification which should have increased their hydrophobicity. The pellet fraction, which accumulated in the lens nucleus as the cataract advanced, was enriched in urea soluble gamma-crystallin derived polypeptides. We suggest that protein insolubilization in this experimental cataract involves the selective and tight association of principally modified alpha-crystallins to the fiber cell plasma membrane.     

Age-related changes of calpain II and alpha-crystallin in the lens of hereditary cataract (Nakano) mouse

The age-related changes of calpain II (high-Ca2+-requiring form of Ca2+-dependent cysteine proteinase; EC 3.4.22.17) and alpha-crystallin in the lens of hereditary cataract (Nakano; cac/cac) mouse were studied. Before the onset of the cataract formation, i.e., at the end of the 2nd week after birth, the calpain activity in Nakano mice was as high as that in the control ICR mice, but it decreased rapidly as the cataract progressed to completion during the 4th and the 12th week. Marked degradation of lens proteins ensued between the 2nd and the 4th weeks, and one of these proteins was identified, using monospecific antibodies, as B chain of alpha-crystallin. A chain of alpha-crystallin was not degraded in vivo, in contrast to its known susceptibility to calpain in vitro. The present data suggest that in Nakano mice, calpain may be involved in the onset or early stage of the cataract formation.     

Retarded and distinct progress of lens opacification in congenic hereditary cataract mice, Balb/c-nct/nct

The Nakano cataract gene, nct, was introduced into Balb/c mice by repeated backcrosses to elucidate the possible effects of background genes on its expression. The resulting congenic Balb/c-nct/nct mice were characterized by retarded and sporadic cataract formation with a tendency of further retardation in males and by the different disease process of cataract as compared with Nakano mice. The age of 50% cataract incidence was 60 days in females and 90 days in males compared with 22 days in Nakano mice, and lens opacification commenced in a diffuse, mild form at the cortex in congenic but in a pin-head, intense form at the core in Nakano mice. Sex hormones seemed to be involved in the difference in cateractogenesis between male and female mice. Microphthalmia was slighter in degree in Balb/c background mice. The results indicated that the nct-dependent cataractogenesis may be influenced by background genes and some non-hereditary factors. Balb/c-nct/nct mice will provide a new type of hereditary cataract model.     

A possible cataractogenic factor in the Nakano mouse lens.

A factor present in the Nakano mouse lens is an inhibitor of NaK ATPase. This inhibitor is not found in the normal mouse lens. It is not associated with any of the major crystallins but is found with the low molecular weight components. It is resistant to high temperatures and to acid and alkaline pHs. It appears to be a polypeptide since its activity is abolished by treatment with carboxy-peptidase A and leucine amino peptidase. Other properties of the inhibitor indicate that this factor appears quite different from other substances which inhibit NaK ATPase.     

The comparison of human lens crystallins using three monoclonal antibodies

Three monoclonal antibodies against lens crystallin have been used to study the accumulation of specific polypeptides during development of the human lens. One antibody which recognizes an antigen common to three polypeptides with molecular weights close to 31,000 reacted equally well to the human lens cortex and nucleus and had a similar binding activity to proteins isolated from embryonic and older human lenses. This suggests that the antigen accumulates to the same degree in human lenses during development. The second monoclonal antibody to the 24,000 molecular weight gamma-crystallin showed increased binding at the older ages indicating the increased accumulation of this protein during lens development. The third monoclonal antibody to a beta-crystallin of 27,000 molecular weight showed little if any reactivity at the embryonic age and revealed a clear difference between the binding with cortical and nuclear protein at older ages. It appears that the 27,000 molecular weight beta-crystallin may not be synthesized in the embryonic human lens.     

Biochemical evidence for conversion to milder form of hereditary mouse cataract by different genetic background

Congenic hereditary cataract mice, BALB/c-nct/nct, were established by introducing the nct gene from Nakano into BALB/c mice. These mice developed a milder cortical form of cataract which developed sporadically and later in life than in Nakano mice. Combined use of BALB/c and BALB/c-nct/nct mice enables biochemical comparison of normal clear lenses, congenic clear lenses which are destined to be opacified some time later, and opacified lenses in the same genetic and aging statuses. We compared the age-related changes in water content and water-soluble and -insoluble fractions among these three types of lenses. Congenic clear lenses and opaque lenses were more similar to BALB/c normal clear lenses and Nakano opaque ones, respectively, in these parameters. These results suggest, in addition to formation of aggregated crystallins and their accumulation in water-insoluble fractions, that decreased protein synthesis, increased protein degradation and augmented leakage of crystallin might have a significant role in the nct-induced lens opacification.