Lipids and the ocular lens

The unusually high levels of saturation and thus order contribute to the uniqueness of human lens membranes. In addition, and unlike in most biomembranes, most of the lens lipids are associated with proteins, thus reducing their mobility. The major phospholipid of the human lens is dihydrosphingomyelin. Found in significant quantities only in primate lenses, particularly human ones, this lipid is so extremely stable that it was reported to be the only lipid remaining in a frozen mammoth 40,000 years after its death. Unusually high levels of cholesterol add peculiarity to the composition of lens membranes. Beyond the lateral segregation of lipids into dynamic domains known as rafts, the high abundance of cholesterol in the human lens leads to the formation of patches of pure cholesterol. Changes in human lens lipid composition with age and disease as well as differences among species are greater than those observed for any other biomembrane. The relationships among lens membrane composition, structure, and lipid conformation reviewed in this article are unique to the mammalian lens and offer exciting insights into lens membrane function. This review focuses on findings reported over the last two decades that demonstrate the uniqueness of mammalian lens membranes regarding their morphology and composition. Becaue the membranes of human lenses do undergo the most dramatic changes with age and cataractogenesis, the final sections of this review address our current knowledge of the unusual composition and organization of adult human lens membranes with and without opacification. Finally, the questions that still remain to be answered are presented.     

The heterogeneity of the plasma membrane H+-ATPase response to auxin

The sensitivity of the plasma membrane H⁺-ATPase in tobacco was investigated in vitro, both at the proton translocation level and the ATPase level, according to plant development and leaf location. Both activities are stimulated by auxin in all leaves, whatever the plant age and the leaf age. However, the sensitivity to auxin was heterogeneous with respect to plant development and leaf location. In parallel experiments using the same plasma membrane samples, polypepides patterns were investigated by two-dimensional gel electrophoresis and image analysis was used to quantify the relative abundance of 110 peptides. Systematic analysis of the two kinds of data identified 8 polypeptides, the abundance of which changed in a consistent way with the sensitivity, whatever the plant developmental state and leaf location. These unknown polypeptides are proposed as potential markers of the membrane response to auxin.     

Effect of iron on the relative abundance of two large polypeptides of the Escherichiacoli outer membrane

The relative abundance of two polypeptides of the $\text{Escherichia}\text{coli}$ outer membrane is affected by the growth medium. The polypeptides have molecular weights of 85,000 and 95,000 and, in cells grown in medium containing low concentrations of iron, are dominant outer membrane proteins.     

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.     

Lens crystallin changes associated with amphibian metamorphosis: involvement of a beta-crystallin polypeptide

Lens crystallins isolated from the tadpole and frog lenses were compared with regard to the developmental changes of crystallin compositions. The major changes during the process of metamorphosis were (1) the total contents of alpha- and gamma-crystallins decrease from more than 70% to less than 60% and (2) one of the major beta-crystallin polypeptides increases from less than 1% to about 6% and (3) an amphibian-specific rho-crystallin also increases from about 6% to more than 10% of total soluble proteins of the lens. We have characterized the metamorphosis-dependent beta-crystallin polypeptide by peptide mapping and sequence determination of the protease-digested fragments. This polypeptide showed very high sequence homology to that of the major beta Bp-crystallin chain reported for the mammalian lenses. The changes of the relative abundance of various crystallins and the gradually-elevated levels of the expression of this beta Bp-like crystallin in the developing lens during metamorphosis may also have some bearing on the maintenance of lens stability in the adult frog lenses.     

Rate of formation of AGEs during ascorbate glycation and during aging in human lens tissue

The similarity of the yellow chromophores isolated from human cataracts with those from ascorbic acid modified calf lens proteins was recently published [Biochim. Biophys. Acta 1537 (2001) 14]. The data presented here additionally quantify age-dependent increases in individual yellow chromophores and fluorophores in the water-insoluble fraction of normal human lens. The water-insoluble fraction of individual normal human lens was isolated, solubilized by sonication and digested with a battery of proteolytic enzymes under argon to prevent oxidation. The level of A(330)-absorbing yellow chromophores, 350/450 nm fluorophores and total water-insoluble (WI) protein were quantified in each lens. The total yellow chromophores and fluorophores accumulated in parallel with the increase in the water-insoluble protein fraction during aging. The digest from each single human lens was then subjected to Bio-Gel P-2 size-exclusion chromatography. The fractions obtained were further separated by a semi-preparative prodigy C-18 high-performance liquid chromatography (RP-HPLC). Bio-Gel P-2 chromatography showed four major fractions, each of which increased with age. RP-HPLC of the amino acid peak resolved five major A(330)-absorbing peaks and eight fluorescent peaks, and each peak increased coordinately with age. A late-eluting peak, which contained hydrophobic amino acids increased significantly after age 60.Aliquots from an in vitro glycation of calf lens proteins by ascorbic acid were removed and subjected to the same enzymatic digestion. Ascorbic acid-modified calf lens protein digests showed an almost identical profile of chromophores, which also increased in a time-dependent manner. The late-eluting peak, however, did not increase with the time of glycation and may not be an advanced glycation endproduct (AGE) product. The data indicate that the total water-insoluble proteins, individual yellow chromophores and fluorophores increased equally both with aging in normal human lens and during ascorbate glycation in vitro. The major protein modifications, which accumulate during aging, therefore, appear to be AGEs. Whereas the late-eluting peak, which showed poor correlation to ascorbylation, may represent UV filter compounds bound to lens proteins.     

Comparative analysis of the major polypeptides from liver gap junctions and lens fiber junctions

Gap junctions from rat liver and fiber junctions from bovine lens have similar septilaminar profiles when examined by thin-section electron microscopy and differ only slightly with respect to the packing of intramembrane particles in freeze-fracture images. These similarities have often led to lens fiber junctions being referred to as gap junctions. Junctions from both sources were isolated as enriched subcellular fractions and their major polypeptide components compared biochemically and immunochemically. The major liver gap junction polypeptide has an apparent molecular weight of 27,000, while a 25,000-dalton polypeptide is the major component of lens fiber junctions. The two polypeptides are not homologous when compared by partial peptide mapping in SDS. In addition, there is not detectable antigenic similarity between the two polypeptides by immunochemical criteria using antibodies to the 25,000-dalton lens fiber junction polypeptide. Thus, in spite of the ultrastructural similarities, the gap junction and the lens fiber junction are comprised of distinctly different polypeptides, suggesting that the lens fiber junction contains a unique gene product and potentially different physiological properties.     

AGEs in human lens capsule promote the TGFβ2‐mediated EMT of lens epithelial cells: implications for age‐associated fibrosis

Proteins in basement membrane (BM) are long‐lived and accumulate chemical modifications during aging; advanced glycation endproduct (AGE) formation is one such modification. The human lens capsule is a BM secreted by lens epithelial cells. In this study, we have investigated the effect of aging and cataracts on the AGE levels in the human lens capsule and determined their role in the epithelial‐to‐mesenchymal transition (EMT) of lens epithelial cells. EMT occurs during posterior capsule opacification (PCO), also known as secondary cataract formation. We found age‐dependent increases in several AGEs and significantly higher levels in cataractous lens capsules than in normal lens capsules measured by LC‐MS/MS. The TGFβ2‐mediated upregulation of the mRNA levels (by qPCR) of EMT‐associated proteins was significantly enhanced in cells cultured on AGE‐modified BM and human lens capsule compared with those on unmodified proteins. Such responses were also observed for TGFβ1. In the human capsular bag model of PCO, the AGE content of the capsule proteins was correlated with the synthesis of TGFβ2‐mediated α‐smooth muscle actin (αSMA). Taken together, our data imply that AGEs in the lens capsule promote the TGFβ2‐mediated fibrosis of lens epithelial cells during PCO and suggest that AGEs in BMs could have a broader role in aging and diabetes‐associated fibrosis.     

Protein complement of rod outer segments of frog retina

Rod outer segments (ROS) from frog retina have been purified by Percoll density gradient centrifugation, a procedure that preserves their form and intactness. One- and two-dimensional electrophoretic analysis reveals a smaller number of proteins than is observed in many cell organelles and permits quantitation of the 20 most abundant polypeptides. Rhodopsin accounts for 70% of the total protein (3 X 10(9) copies/outer segment), and approximately 70 other polypeptides are present at more than 6 X 10(4) copies/outer segment. Another 17% of the total protein is accounted for by the G-protein (3 X 10(8) copies/outer segment) that links rhodopsin bleaching and the activation of cyclic GMP phosphodiesterase (PDE). The phosphodiesterase accounts for 1.5% of the protein (1.5 X 10(7) copies/outer segment), and a 48,000-dalton component that binds to the membrane in the light accounts for a further 2.6%. The function of approximately 90% of the total protein in the outer segment is known, and two-thirds of the non-rhodopsin protein is accounted for by enzyme activities associated with cyclic GMP metabolism. The relative molar abundance of rhodopsin, G-protein, and PDE is 100:10:1. Apart from these major membrane-associated proteins, most of the other proteins are cytosolic. Thirteen other polypeptides are found at an abundance of one or more copies per 1000 rhodopsins, nine soluble and four membrane-bound, and their abundance relative to rhodopsin has been quantitated. ROS have been separated into subcellular fractions which resolve three classes of soluble, extrinsic membrane, and integral membrane proteins. A listing of the proteins that are phosphorylated and their subcellular localization is given. Approximately 25 phosphopeptides are detected, and most are in the soluble fraction. Fewer phosphorylated proteins are associated with the purified outer segments than with crude ROS. Distinct patterns of phosphorylation are associated with intact rods incubated with [32P]Pi and broken rods incubated with [gamma-32P]ATP.     

Fatty acid acylation of lens fiber plasma membrane proteins. MP26 and alpha-crystallin are palmitoylated

We describe in this report the fatty acylation of some of the main polypeptides from the eye lens fibers. MP26, the major lens fiber plasma membrane protein, and probably MP22, its natural degradation product, are palmitoylated in a post-translational process. This is also the case for alpha-crystallin, a major cytoplasmic structural protein shown to interact directly with the plasma membrane. Furthermore, a 65 kDa non-identified polypeptide and a high molecular weight component are also modified in the same way.     

Post-translational modifications of aquaporin 0 (AQP0) in the normal human lens: spatial and temporal occurrence

Because of the lack of protein turnover in fiber cells of the ocular lens, Aquaporin 0 (AQP0), the most abundant membrane protein in the lens, undergoes extensive post-translational modification with fiber cell age. To map the distribution of modified forms of AQP0 within the lens, normal human lenses ranging in age from 34 to 38 were concentrically dissected into several cortical and nuclear sections. Membrane proteins still embedded in the membranes were digested with trypsin, and the resulting C-terminal peptides of AQP0 were analyzed by HPLC tandem mass spectrometry, permitting the identification of modifications and estimation of their abundance. Consistent with earlier reports, the major phosphorylation site was Ser 235, and the major sites of backbone cleavage occurred at residues 246 and 259. New findings suggest that cleavage at these sites may be a result of nonenzymatic truncation at asparagine residues. In addition, this approach revealed previously undetected sites of truncation at residues 249, 260, 261, and 262; phosphorylation at Ser 231 and to a lower extent at Ser 229; and racemization/isomerization of l-Asp 243 to d-Asp and d-iso-Asp. The spatial distribution of C-terminally modified AQP0 within the lens indicated an increase in truncation and racemization/isomerization with fiber cell age, whereas the level of Ser 235 phosphorylation increased from the outer to inner cortex but decreased in the nucleus. Furthermore, the remarkably similar pattern and distribution of truncation products from lenses from three donors suggest specific temporal mechanisms for the modification of AQP0.     

Chemical composition and solubility of human glomerular and tubular basement membranes of adult and senescent men

The effect of aging on the composition of human renal basement membranes was studied in persons aged 22-90 years. The relative proportion of cortex in kidney appears to decrease with aging. Twenty pairs of GBM and TBM preparations were isolated using the detergent method. Protein content of the basement membrane preparations amounts to about 66% and is independent of type of membrane or age. Amino acid and carbohydrate analyses of both GBM and TBM revealed that the extents of hydroxylation of proline at the C4 position and of lysine decrease with aging. In the case of lysine this occurred from the seventh of life onwards. The decreases are probably not caused by a change of the collagen content. The extent of glycosylation of hydroxylysine is similar for all basement membrane preparations. An adapted protein assay is presented for solutions containing SDS and dithiothreitol. In the presence of these two compounds, solubility of GBM and TBM from adult and aged persons is similar and amounts to about 55 and 85% after 10 and 60 min, respectively, of heating at 95 degrees C. In SDS-polyacrylamide gels, no differences were observed for the major peptide bands between the preparations, irrespective of basement membrane type or age.     

Genetic and physiological regulation of intrinsic proteins of the outer membrane of Salmonella typhimurium.

Four major outer membrane polypeptides, accounting for approximately 20% of the total protein of the outer membrane of Salmonella typhimurium, were induced by growth in minimal medium. The polypeptides were tightly bound membrane components. Physiological and genetic evidence indicates that the four polypeptides fall in two separate regulation groups. Synthesis of one of these groups was coordinately regulated by the concentration of iron in the medium, and a mutant strain has been identified in which there is constitutive synthesis of this group of major outer membrane proteins.     

Proteome Remodeling of the Eye Lens at 50 Years Identified With Data-Independent Acquisition

The eye lens is responsible for focusing and transmitting light to the retina. The lens does this in the absence of organelles, yet maintains transparency for at least 5 decades before onset of age-related nuclear cataract (ARNC). It is hypothesized that oxidative stress contributes significantly to ARNC formation. It is in addition hypothesized that transparency is maintained by a microcirculation system that delivers antioxidants to the lens nucleus and exports small molecule waste. Common data-dependent acquisition methods are hindered by dynamic range of lens protein expression and provide limited context to age-related changes in the lens. In this study, we utilized data-independent acquisition mass spectrometry to analyze the urea-insoluble membrane protein fractions of 16 human lenses subdivided into three spatially distinct lens regions to characterize age-related changes, particularly concerning the lens microcirculation system and oxidative stress response. In this pilot cohort, we measured 4788 distinct protein groups, 46,681 peptides, and 7592 deamidated sequences, more than in any previous human lens data-dependent acquisition approach. Principally, we demonstrate that a significant proteome remodeling event occurs at approximately 50 years of age, resulting in metabolic preference for anaerobic glycolysis established with organelle degradation, decreased abundance of protein networks involved in calcium-dependent cell–cell contacts while retaining networks related to oxidative stress response. Furthermore, we identified multiple antioxidant transporter proteins not previously detected in the human lens and describe their spatiotemporal and age-related abundance changes. Finally, we demonstrate that aquaporin-5, among other proteins, is modified with age by post-translational modifications including deamidation and truncation. We suggest that the continued accumulation of each of these age-related outcomes in proteome remodeling contribute to decreased fiber cell permeability and result in ARNC formation.     

Changes in proteins of the human lens in development and aging.

A number of proteins have been isolated from the human lens at different stages of development, from before birth to old age. These proteins have been characterized and compared with each other and with corresponding proteins from bovine lens. Many similarities were found between human and bovine crystallins, but alpha-crystallin isolated from old human lenses using DEAE-cellulose, unlike bovine alpha-crystallin similarly isolated, is not found as large soluble aggregates. The amide contents of various lens protein fractions were determined. No extensive changes were found during adult life, but there was evidence that significant deamidation of alpha-crystallin had occurred before birth and possibly during infancy. The results are related to the unique development and aging of the lens.     

Conformational stability of the polypeptide components of human high density serum lipoprotein.

An investigation of the denaturation of the major polypeptides from human serum high density lipoprotein by guanidine hydrochloride reveals that the lipid-free, water-soluble states are minimally stable relative to the random coil states. These findings are in direct contrast to the resistance of intrinsic membrane proteins (in the absence of ligand) to complete unfolding by the same denaturant. Consideration of the denaturation data for the apolipoproteins together with previously published data from this laboratory on ligand-induced conformational changes indicate that these polypeptides possess several similar conformational states which are readily interconvertible.     

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.     

Mitochondria induce oxidative stress, generation of reactive oxygen species and redox state unbalance of the eye lens leading to human cataract formation: disruption of redox lens organization by phospholipid hydroperoxides as a common basis for cataract disease

The aging eye appears to be at considerable risk from oxidative stress. Lipid peroxidation (LPO) is one of the mechanisms of cataractogenesis, initiated by enhanced promotion of oxygen free radicals in the eye fluids and tissues and impaired enzymatic and non-enzymatic antioxidant defenses of the crystalline lens. The present study proposes that mitochondria are one of the major sources of reactive oxygen species (ROS) in mammalian and human lens epithelial cells and that therapies that protect mitochondria in lens epithelial cells from damage and reduce damaging ROS generation may potentially ameliorate the effects of free radical-induced oxidation that occur in aging ocular tissues and in human cataract diseases. It has been found that rather than complete removal of oxidants by the high levels of protective enzyme activities such as superoxide dismutase (SOD), catalase, lipid peroxidases in transparent lenses, the lens conversely, possess a balance between peroxidants and antioxidants in a way that normal lens tends to generate oxidants diffusing from lenticular tissues, shifting the redox status of the lens to become more oxidizing during both morphogenesis and aging. Release of the oxidants (O(2)(-)·, H(2)O(2) , OH·, and lipid hydroperoxides) by the intact lenses in the absence of respiratory inhibitors indicates that these metabolites are normal physiological products inversely related to the lens life-span potential (maturity of cataract) generated through the metal-ion catalyzed redox-coupled pro-oxidant activation of the lens reductants (ascorbic acid, glutathione). The membrane-bound phospholipid (PL) hydroperoxides escape detoxification by the lens enzymatic reduction. The lens cells containing these species would be vulnerable to peroxidative attack which trigger the PL hydroperoxide-dependent chain propagation of LPO and other damages in membrane (lipid and protein alterations). The increased concentrations of primary LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moiety of the aqueous humor samples obtained from patients with cataract as compared to normal donors. Since LPO is clinically important in many of the pathological effects and aging, new therapeutic modalities, such as patented N-acetylcarnosine prodrug lubricant eye drops, should treat the incessant infliction of damage to the lens cells and biomolecules by reactive lipid peroxides and oxygen species and "refashion" the affected lens membranes in the lack of important metabolic detoxification of PL peroxides. Combined in ophthalmic formulations with N-acetylcarnosine, mitochondria-targeted antioxidants are promising to become investigated as a potential tool for treating a number of ROS-related ocular diseases, including human cataracts.     

An investigation into the ATP requirement for phosphorylation of thylakoid proteins and for the ATP-induced decrease in the yield of chlorophyll fluorescence in chloroplasts at different stages of development

The phosphorylation of thylakoid membrane polypeptides has been investigated in chloroplasts prepared from peas that had been grown under intermittent light and then exposed to between 4 and 48 h of continuous light. At 4 h, when the ratio of the total amount of labelling of a 9 kDa-polypeptide relative to light-harvesting chlorophyll protein (LHCP) polypeptides was much greater than 1, the affinity for ATP was found to be the same (S_0.5, approx. 100 μM) for both polypeptides. In contrast, in fully greened chloroplasts, when labelling of LHCP was much greater than that of the 9 kDa-polypeptide, the S_0.5 for ATP was 40 μM for LHCP and 500 μM for the 9 kDa-polypeptide. A correlation was observed during development between the affinity for ATP of the 9 kDa-species and its abundance relative to LHCP. It is suggested that these polypeptides compete for phosphorylation by the same protein kinase. Simultaneous assay of the ATP-induced fluorescence decrease at different ATP concentrations revealed a close correlation with LHCP labelling but not with labelling of the 9 kDa-polypeptide. This correlation held irrespective of which polypeptide was the major phosphoprotein.     

Actin filament patterns in mouse lens epithelium: a study of the effects of aging, injury, and genetics

Using mainly fluorescence microscopy after rhodamine-phalloidin staining, the F-actin distribution in the mouse lens epithelium was studied with regard to the effects of age, genetic strain, and mechanical injury. These studies have revealed that aside from its association with the plasma membrane the structural organization of F-actin in the mouse lens epithelium in situ is characterized by two major configurations: (1) a filamentous arrangement in such patterns as stress fibers, polygonal arrays (PAs), and meshworks, and (2) a highly concentrated structure called a sequestered actin bundle (SAB). The aging study indicated that the SAB is a consistent character in C57BL/6 mice from the age of 5 wk on, but not in CF1 mice. The size and shape of the SAB change gradually with age as inferred from two-dimensional measurements. The genetic study on the SAB character using hybrids and congenic strains showed that it is inherited as a Mendelian dominant, probably multigenic mode. Finally, the injury study revealed a structural modification in cells around the wound, including flattening of cells at the edge and extension of processes into the wound space. In the rest of the epithelium, injury amplified membrane infolding and fluorescence of polygonal arrays but diminished the size and fluorescence intensity of SABs. These changes are thought to be correlated with wound repair involving cell division and migration. These studies illustrate the variability in F-actin expression in situ in lens epithelial cells that can be induced by intrinsic and extrinsic factors.