Role of albumin as a fatty acid carrier for biosynthesis of lens lipids.

Although serum albumin is the major protein component of the aqueous humor and vitreous humor, its possible physiological role(s) in lens metabolism has not yet been determined. BODIPY fatty acid, a fluorescent analogue of a C(12) long chain fatty acid, was bound to serum albumin, then incubated with lenses in culture. After various times of incubation, the lenses were homogenized, lipids extracted, and the extracted lipids resolved by thin layer chromatography. Fluorescence analyses demonstrated that the BODIPY fatty acid--albumin complex was translocated into the lens, where the BODIPY fatty acid was incorporated in a time dependent manner into numerous lipids, including phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin. Together, the results strongly suggest that serum albumin in the aqueous humor and/or vitreous humor facilitates the translocation of long chain fatty acids into the lens, where they are used for the biosynthesis of lens lipids.

Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice.

PURPOSE: Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals. METHODS: In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times. RESULTS: Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months. CONCLUSIONS: The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.

PKC-gamma phosphorylation of connexin 46 in the lens cortex.

PURPOSE: To identify the role of PKC-gamma in control of and phosphorylation of connexin 46 (Cx46) in the lens cortex. METHODS: The association between PKC-gamma and Cx46 was determined by co-immunoprecipitation from whole lens. Phosphorylation of Cx46 and activity of PKC-gamma were determined using Western blots, PKC activity assays, and inhibition of PKC activity by addition of isoform-specific PKC pseudosubstrate inhibitors. RESULTS: Co-localization of PKC-gamma and Cx46 was observed in the bow regions and cortical regions of rat lens. PKC-gamma was not observed in the nuclear region and Cx46 was not observed in the epithelial layer. PKC-alpha was not found in lens cortex or nuclear regions. PKC-gamma could be co-immunoprecipitated with Cx46 from lens cortical regions. Cx46 was phosphorylated on both serine and threonine. No tyrosine phosphorylation was observed. The PKC-gamma specific pseudosubstrate inhibitor caused a 73% inhibition of serine phosphorylation on Cx46 at 1 microM, and, 36% inhibition of threonine phosphorylation at the same concentration. Inhibition of phosphorylation of Cx46 with PKC-alpha pseudosubstrate inhibitor was not observed. CONCLUSIONS: PKC-gamma may phosphorylate Cx46, primarily on serine in whole lens. A role for PKC-gamma in control of lens cortical gap junctions is suggested.

Comparison of d-aspartic acid contents in alpha A-crystallin from normal and age-matched cataractous human lenses.

We have previously shown that biologically uncommon d-beta-aspartic acids (Asp) were localized with very high contents at Asp-151 and Asp-58 of alpha A-crystallin from aged human lenses. The amounts increased with age, and we have proposed the mechanism of this reaction. In the present study, in order to elucidate the possible relationship between the formation of d-beta-aspartic acids in alpha A-crystallin and cataract formation, we measured the d/l ratio of beta-Asp-151 of alpha A-crystallin from both cataractous and age-matched normal human lenses. alpha A-crystallin from total proteins of cataractous and age-matched normal lenses was prepared, followed by tryptic digestion and quantification of d/l ratios for tryptic fragments containing the alpha- and beta-aspartate forms of Asp-151 residues. The results demonstrate that the d/l ratio of beta-Asp-151 of alpha A-crystallin from normal lenses is not statistically significant from that of alpha A-crystallin from cataractous lenses, suggesting that formation of this biologically uncommon amino acid may not play a role in human cataractogenesis.

Formation of four isomers at the asp-151 residue of aged human alphaA-crystallin by natural aging.

Although proteins are generally composed entirely of l-amino acids, we have previously shown that Asp-151 in alphaA-crystallin from aged human lens is converted to the biologically uncommon d-isomer to a high degree. The formation of d-isomer was not simple racemization, but stereoinvertion. The reaction was also accompanied with isomerization to form beta-Asp (isoaspartate) residue simultaneously; therefore, four isomers of Asp-151, normal l-alpha-Asp and biologically uncommon l-beta-Asp, d-alpha-Asp, and d-beta-Asp, are formed in alphaA-crystallins. In the present study, we measured the ratio of the four isomers of Asp-151 in alphaA-crystallins obtained from total lens proteins of human lenses of newborn and 30-, 60-, and 80-year-olds. The isomers increased with age, and the total amount of three isomers was more than that of normal l-alpha-Asp in the alphaA-crystallin of the human lenses of the 80-year-olds. These drastic changes started at birth, with about 45% of normal l-alpha-Asp lost by 30 years. These modifications of the Asp residue likely affect the three-dimensional packing array of the lens proteins.

Localization of MIP 26 in nuclear fiber cells from aged normal and age-related nuclear cataractous human lenses.

The purpose of the current study was to localize the lens membrane protein, MIP 26, in nuclear fiber cells from different regions of aged normal and age-related cataractous human lenses. Adult, juvenile, fetal and embryonic nuclear regions in aged normal and age-related nuclear cataractous lenses were morphologically and biochemically characterized using the technologies of immuno-gold (5 nm) labeling, semi-thin sections (200-500 nm), serial sections, DiI staining following by photobleaching, transmission electron microscopy and spot-blot analysis. Numbers of gold particles per micron length of plasma membrane and numbers of gold particles per square micron of cytosol in the embryonic-fetal and juvenile-adult nuclear regions were quantified. Results showed that the labeling pattern of MIP 26 localized to the cytosol was unique to senescent fiber cells from age-related cataractous lenses. Numbers of gold particles per square micron of cytosol in the embryonic-fetal nucleus of age-related cataractous lenses were significantly elevated (P<0.001) above numbers from fiber cells located within the adult or juvenile nuclei of the same lens or senescent fiber cells from aged normal lenses. Some of the cytosolic labeling in cataracts was localized to lipid vesicles, while the remaining labeling was negative for the lipid specific stain DiI. Spot blot analysis demonstrated that binding of the ant-MIP 26 serum was exclusive to large molecular weight components greater than 10 kDa, and not to small molecular weight fragments of the protein. The results of the current study supply further evidence that damage to membranes occurs in senescent fiber cells during age-related nuclear cataracts, resulting in the internalization of structures containing the membrane protein MIP 26.

Quantitation of asparagine-101 deamidation from alpha-A crystallin during aging of the human lens.

PURPOSE: To quantitate deamidation of asparagine-101 from the alpha-A crystallin protein of human lenses of different ages. METHODS: Alpha-A crystallin was purified from total proteins of human lenses of different ages, followed by tryptic digestion and resolution of the peptides, using reverse phase chromatography. Known amounts of synthetic peptide standards, corresponding to the amidated and deamidated forms of the expected tryptic peptide containing asparagine-101, were used to identify and quantitate the amount of deamidation. RESULTS: From 0-30 yrs of age, approximately 45% of asparagine-101 was deamidated, while only approximately 5% additional deamidation occurred during 30-68 yrs of age. CONCLUSIONS: In the normal human lens, most deamidation of asparagine-101 occurs during the first approximately 30 years of age, followed by a small additional amount of deamidation (approximately 5%) during the next approximately 38 years, resulting in a maximum of approximately 50% deamidation during the lifetime of the individual.

Finger-like projections of plasma membrane in the most senescent fiber cells of human lenses.

PURPOSE: To corroborate the findings of finger-like membrane projections in monkey and baboon lenses, in human lens fiber cells. METHODS: Normal human lenses, two months to 76 years old, as well as age-related nuclear cataracts, were immersion fixed in 2% paraformaldehyde-0.2% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 for 24 h at room temperature, cut into 200-500 microm thick sections along the equatorial axis, fixed for an additional 12-18 h at room temperature, dehydrated in an ascending ethanol series and embedded in Lowicryl K4M. Semi-thick sections (0.25-1.0 microm) were cut, absorbed onto 75 or 100 nickel slotted grids, labeled with anti-MIP 26 or phalloidin, stained with 2% uranyl acetate and viewed by transmission electron microscopy at 100 kV. RESULTS: Transmission electron microscopy micrographs demonstrated the presence of finger-like plasma membrane projections measuring 0.16-0.25 microm in diameter and 1.0-6.5 microm in length with bulbous terminal tips in the most senescent fiber cells in two-month and older normal lenses, as well as, in nuclear cataracts. These projections appeared to overlie furrowed membrane domains in the extracellular space, as well as project into the cytosol along the cytosolic leaflet of plasma membrane. CONCLUSIONS: The results extend the findings in monkey and baboon lenses, to the human lens, and demonstrate that these projections, which sparsely label with antiserum against MIP 26, but not filamentous actin, not only extend into the extracellular space, but also project inward into the cytosol.

Beta A3/A1 crystallin from human cataractous lens contains an intramolecular disulfide bond.

PURPOSE: To develop an experimental approach that can identify amino acid sequences containing cysteine residues involved in disulfide bonding during cataractogenesis of the human lens. METHODS: Total proteins from cataractous and normal human lenses were solubilized anaerobically, followed by carboxy-methylation of free sulfhydryl groups with iodoacetate. Carboxymethylated proteins were partially purified by reverse phase chromatography, then subjected to lys-C endoprotease digestion. Using reverse phase chromatography, each digest was resolved in the presence and absence of dithiothreitol (DTT), to identify peptides that were linked by disulfide bonds. These peptides were further characterized using a combination of Edman degradation and mass spectrometry. RESULTS: The reverse phase chromatography profiles of lys-C peptides from proteins of normal lenses were very similar in the presence and absence of dithiothreitol, while identical analysis of proteins from cataractous lenses demonstrated the presence of a lys-C peptide that corresponded to residues 163-193 of human beta A3/A1, with cysteine 170 and cysteine 185 linked via an intramolecular disulfide bond. CONCLUSIONS: Reverse phase chromatography of complex mixtures of lens protein digests, in the presence and absence of dithiothreitol, provides a rapid method of identifying sequences involved in disulfide bonding. The results of this analysis using the endoprotease lys-C have demonstrated that beta A3/A1 crystallin from cataractous lenses contains an intermolecular disulfide bond involving cysteine residues 170 and 185.

Confocal microscopy of cataracts from animal model systems: relevance to human nuclear cataract.

A recent study demonstrated that cytosolic lipid membrane structures, independent of the plasma membrane, preferentially occurred in human cataractous lenses. Animal model systems of cataractogenesis (selenite treated rats: galactose fed rats; buthionine-sulfoxime treated mice; Emory mice) were screened for possible relevant structures using the lipid membrane probe DiI and confocal microscopy. Well delineated plasma membranes of lens fiber cells with independent cytosolic staining structures were only observed in the selenite model system. These cytosolic structures were not observed in aged matched control lenses or within the transparent cortical regions of selenite treated animals with intense nuclear opacification. These results suggested that the morphological changes in DiI staining structures seen in the nucleus of the human cataractous lens were best approximated by those seen in the selenite model system.

Disulfide bond formation of cysteine-37 and cysteine-66 of beta B2 crystallin during cataractogenesis of the human lens.

Beta B2 crystallin has been prepared from total protein of normal (transparent) and cataractous human lenses. After digestion with endoprotease lys-C, the crude digests were resolved on a C18 reverse phase column. The lys-C digests of beta B2 crystallin from cataractous lenses consistently showed the presence of a major peptide that was not present in the lys-C digests from normal lenses. Treatment of this peptide with dithiothreitol resulted in the production of two peptides, corresponding to beta B2 crystallin sequences 18-41 and 48-67, containing cysteine-37 and cysteine-66, respectively. The results demonstrated that intramolecular disulfide bonding of these two residues had occurred in vivo. Based upon previous knowledge of the three dimensional structure of beta B2 crystallin, formation of this disulfide bond suggests that significant denaturation of the beta B2 crystallin molecule has occurred during the process of human lens opacification.

Changes in the C-terminal region of alpha-A crystallin during human cataractogenesis.

Reverse phase chromatography was used to purify alpha-A crystallin from total protein of human cataractous and normal lenses, followed by tryptic digestion and quantitation of the peptides corresponding to the intact C-terminal region of the protein. Relative to alpha-A crystallin from normal lenses, alpha-A crystallin from cataractous lenses contained decreased amounts of the expected C-terminal tryptic peptides. In an alternative approach, antiserum specific for the C-terminal region of the protein was used to quantitatively probe Western blots of total proteins from cataractous and normal lenses. The results demonstrated a decrease in the amount of this antiserum binding to the C-terminal region of alpha-A crystallin from human cataracts. Together, these studies show that relative to alpha-A crystallin from normal lenses, there is a decrease in the amount of the intact C-terminal region during the process of human cataractogenesis.

Confocal microscopy of human lens membranes in aged normal and nuclear cataracts.

PURPOSE: To visualize the structure and determine the continuity of lipid membranes in lens fiber cells (LFCs) from human aged normal and cataractous lenses. METHODS: Thick sections from human nuclear cataracts and aged normal lenses were stained with the lipophilic probe DiI, and then analyzed by confocal microscopy. Staining patterns of membranes were observed in individual optical sections or three-dimensional projections of z-series taken in longitudinal section and cross-section of LFCs from different regions within the lens nucleus. RESULTS: DiI bound to and delineated the plasma membrane of LFCs from all regions of the lens nucleus. Three-dimensional projections of z-series from aged normal and cataractous lenses suggested that some of the stained lipid membranes were not continuous with LFC plasma membrane of cataractous lenses. CONCLUSIONS: The results obtained using these methods demonstrated that lipid membranes, discontinuous with the plasma membrane of LFCs, were indicative of a novel process occurring predominately in cataractous human lenses.

Oxidation of cysteine residues from alpha-A crystallin during cataractogenesis of the human lens.

Although it has been known for many years that opacification of the human lens is accompanied by oxidation of cysteine sulfhydryl groups to half-cystine residues, nothing is known concerning the exact amino acid sequences involved in this oxidative process. Since alpha-A crystallin is one of the major proteins of the lens, and since a decrease in its molecular chaperone activity has been implicated in possible mechanisms of cataract formation, alpha-A crystallin was purified from total lens proteins by reverse phase chromatography, followed by digestion with lys-C endoprotease. Mass spectral analysis of the digest indicated that in normal transparent lenses, cys-131 and cys-142 from alph-A crystallin are present as a mixture of cysteine sulfhydryl and half-cysteine disulfide groups, while identical analysis from cataractous lenses demonstrated undetectable levels of the cysteine sulfhydryl group. Together, these results demonstrate for the first time, the involvement of specific cysteine residues in the oxidative mechanism of human lens opacification.

Differential phosphorylation of alpha-A crystallin in human lens of different age.

Previous studies have demonstrated that a major site of in vivo phosphorylation of alpha-A crystallin from human lens is serine-122. To determine the relative degree of this phosphorylation in alpha-A crystallin from human lenses of different age, alpha-A crystallin was purified from total lens proteins, followed by sequential digestion with lys-C and asp-N endoproteases. Mass spectral analysis of the asp-N peptide fragments that contained serine-122 demonstrated undetectable levels of phosphorylation from infant human lenses (41 days, 2 months and 4 months of age). Identical analysis of alpha-A crystallin from older lenses (12, 15, 40 and 73 years of age) indicated significant phosphorylation of serine-122, demonstrating that phosphorylation of the serine-122 residue of alpha-A crystallin does not occur during the aging process, but is rather a developmentally regulated event in the human lens.

Gap junction structures and distribution patterns of immunoreactive connexins 46 and 50 in lens regrowths of Rhesus monkeys.

Gap junction structures and distribution patterns of immunoreactive connexin46 (Cx46) and connexin50 (Cx50) in normal lenses and lens regrowths of rhesus monkeys were studied using electron microscopy and immunofluorescence double-labeling. Lens regrowths were collected from aphakic eyes of young monkeys whose natural lenses had been surgically removed 11-34 months earlier to simulate monocular congenital cataract surgery in human infants. Approximately 90% of the lens regrowths examined was in the form of a doughnut-shaped Soemmerring's ring located behind the iris. The lens regrowth consisted of lens epithelium and lens fibers enclosed within hypertrophied capsular material. The superficial equatorial region usually contained nucleated young fibers of normal appearance. The other regions consisted of many swollen fibers. Gap junctions were readily observed between fiber cells of both normal and swollen configuration in the lens regrowth. In superficial fibers, gap junctions were not associated with cytoskeletal components. In the intermediate and the deeper cortical regions, actin filament bundles were found specifically associated with gap junctions along both of their cytoplasmic surfaces. An immunofluorescence double-labeling study showed that Cx46 and Cx50 were labeled in the same gap junctions in both superficial and deeper cortical fibers of the normal lens. In contrast, in the lens regrowth strong co-labeling of Cx46 and Cx50 was only observed in the superficial fibers. The labeling for Cx50 was very weak or absent in the deeper cortex, whereas the strong labeling for Cx46 persisted throughout the major portion of the deeper cortex. The labeling for Cx46 finally disappeared in the much deeper cortex. This study shows that (1) the same distribution pattern of actin bundle/gap junction association found in normal lenses is seen in the lens regrowth, and (2) the immunoreactive distribution of Cx46 and Cx50 differ in the lens regrowths as compared with those in the normal lenses of rhesus monkeys.

Identification of the in vivo truncation sites at the C-terminal region of alpha-A crystallin from aged bovine and human lens.

Total alpha-A crystallin was purified from young versus old lens, followed by digestion with cyanogen bromide. Laser desorption mass spectrometry of the C-terminal fragment demonstrated age-dependent loss of one and five amino acids from the C-terminus of alpha-A crystallin from both bovine and human lens. These results demonstrate specific peptide bonds of alpha-A crystallin are cleaved during the aging process of the normal lens. The C-terminal region is cleaved in two places between the two hydroxyl-containing amino acids present in the sequence -P-S(T)-S-.

Phakomatous choristoma (Zimmerman's tumor). Immunohistochemical confirmation of lens-specific proteins.

BACKGROUND: Phakomatous choristoma is a rare, congenital, ocular adnexal tumor that is presumed to be of lenticular anlage based on light and electron microscopy. METHODS: The authors performed immunohistochemistry using standard commercially available antibodies against vimentin, S-100 protein, and several cytokeratins on a phakomatous choristoma that was excised from the right lower eyelid of a 10-week-old white boy. In addition, a battery of antibodies against lens-specific proteins, including alpha, beta, and gamma crystallins, was used. RESULTS: The tumor cells showed intense immunoreactivity for all lens-specific proteins tested. The epithelial cells of the phakomatous choristoma stained positively for S-100 protein and vimentin, the intermediate filament normally found in lens epithelial cells. Keratin markers were negative. CONCLUSIONS: The results of immunohistochemistry indicate that the cells of phakomatous choristoma synthesize several types of lens-specific proteins. Complementing previous light and electron microscopic studies, these data strongly support Zimmerman's conclusion that this pediatric adnexal tumor is a choristoma of lenticular anlage.

Antipeptide antibodies that can distinguish specific subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.).

The amino acid sequences of the beta and gamma subunit polypeptides of glutamine synthetase from bean (Phaseolus vulgaris L.) root nodules are very similar. However, there are small regions within the sequences that are significantly different between the two polypeptides. The sequences between amino acids 2 and 9 and between 264 and 274 are examples. Three peptides (gamma 2-9, gamma 264-274, and beta 264-274) corresponding to these sequences were synthesized. Antibodies against these peptides were raised in rabbits and purified with corresponding peptide-Sepharose affinity chromatography. Western blot analysis of polyacrylamide gel electrophoresis of bean nodule proteins demonstrated that the anti-beta 264-274 antibodies reacted specifically with the beta polypeptide and the anti-gamma 264-274 and anti-gamma 2-9 antibodies reacted specifically with the gamma polypeptide of the native and denatured glutamine synthetase. These results showed the feasibility of using synthetic peptides in developing antibodies that are capable of distinguishing proteins with similar primary structures.