Susceptibility of the bovine lens 115kDa beaded filament protein to degradation by calcium and calpain.

The 115kDa cytoskeletal beaded filament protein of bovine lens fibres is degraded during opacification induced by increased internal calcium. The monoclonal antibody R2D2 to this protein has been used in whole lenses and native homogenates to follow the process of degradation and the production of break-down products. In the opaque outer cortex of whole bovine lenses with an internal Ca2+ of 2.0mM, both the 115kDa parent protein and the main degradation product (57kDa) were reduced in amount by almost 60%. No additional products were detected by the antibody. When native homogenates were incubated overnight with 10mM Ca2+ the protein could no longer be detected in SDS gels, but faintly reactive bands were detected by the antibody. Since these changes were dependent on the presence of increased calcium they were compared with changes induced by incubating freshly isolated cytoskeletal proteins with Ca2+ and the Ca(2+)-activated protease, calpain. The 115kDa protein was shown to be susceptible to degradation by calpain, with the formation of a number of breakdown products. These results indicate that degradation of the beaded filament protein can be brought about by the activation of calpain. Since the enzyme is present in lens cortex it is likely to have a role in the protein degradation observed during Ca(2+)-induced opacification, and may also be involved in the changes occurring as the lens fibres mature.     

The 53kDa polypeptide component of the bovine fibre cell cytoskeleton is derived from the 115kDa beaded filament protein: evidence for a fibre cell specific intermediate filament protein.

The 115kDa protein found enriched in the PMCC (plasma membrane-cytoskeleton complex) fraction of the cortex in bovine lens fibre cells is proteolytically processed to a stable 53kDa product. The 115 kDa protein and the 53kDa polypeptide have been purified by a combination of ion exchange and hydroxyapatite chromatography. Tryptic peptide mapping using reverse phase HPLC and subsequent peptide sequencing confirmed that the 53kDa polypeptide is derived from the 115kDa protein. The 53kDa fragment is also a component of the PMCC as well as being a major component of the urea soluble fraction of lens plasma membranes which have been extracted with buffers containing 1M KC1. The 53kDa polypeptide has escaped identification as a breakdown product of the 115kDa protein because it is not recognised by a commonly used monoclonal antibody, R2D2, specific for the bovine 115kDa protein. This result suggests that proteolysis is important in determining the function(s) of the 115kDa protein, and that part of this function is satisfied by the 53kDa protein core. Both the purified 115kDa protein and the 53kDa polypeptide were unable to form either beaded or intermediate filaments on their own but they were able to form short 10nm rods indicative of an intermediate stage in intermediate filament assembly. Comparison ot the assembly properties of the 53 and 115kDa proteins indicate that there are sequences in the 115kDa protein which inhibit in vitro assembly. This is similar to the situation with neurofilament proteins. We suggest that the 115kDa protein is a lens-specific intermediate filament protein.     

Human lens membrane proteinase: purification and age-related distributional changes in the water-soluble and insoluble protein fractions

A proteinase from human lens membrane was purified by a procedure previously developed for a similar proteinase from bovine lenses (Srivastava, 1988a). The purification of the human proteinase was achieved by solubilization of the enzyme from membranes with 2% sodium deoxycholate followed by two consecutive passages through an Agarose A-1.5 m column. The purified proteinase exhibited molecular weight of 38 kDa on a SDS-polyacrylamide gel. A polyclonal antiserum was raised against bovine lens membrane proteinase, and used as a probe to examine distribution of the enzyme among water-soluble and insoluble proteins of human lenses of different ages. The antiserum had strict specificity to the human membrane proteinase as it showed immunoreaction to only the proteinase among human membrane proteins and crystallins. In addition, the antiserum also inhibited the proteinase activity on incubation. The Western blot of water-soluble proteins from 2-yr-old lens showed a 22 kDa immunoreactive protein, but an additional protein of 43 kD in lenses older than 19 years was observed. A similar Western blot analysis of the water-insoluble proteins from these lenses showed a single protein of 18 kDa that was identified as the subunit of the bovine lens membrane proteinase (Srivastava, 1988a). Furthermore, the immunoreactive 18 kD protein of the water-insoluble protein fractions could be solubilized with urea. The proteinase activity was found to increase with aging, as judged by the extraction of the enzyme with 2% deoxycholate from membranes of lenses of different ages and proteinase activity determination. Similarly, an age-related increase in the immunoreaction was also observed on measuring radio-iodinated protein A bound to an immunoreactive 18 kD protein of the water-insoluble protein fractions.     

The Mr 115 kd fiber cell-specific protein is a component of the lens cytoskeleton

Electron microscope level immunocytochemistry was used to localize a lens fiber cell-specific protein with an Mr of 115 kd. Affinity-purified polyclonal antibodies were utilized on sections of detergent-extracted, acrylic-embedded lens cortical fiber cells. Monoclonal antibodies were utilized for pre-embeddment labelling of a subcellular fraction of lens fiber cells generated by homogenization, and high-speed centrifugation. The results indicate that the Mr 115 kd antigen is a component of the lens fiber cell cytoskeleton, specifically the beaded filament (BF), a cytoskeletal element thought to be unique to the differentiated lens fiber cell.     

Immunochemical characterization of a Mr 115 lens fiber cell-specific extrinsic membrane protein

Monoclonal and polyclonal antibodies have been produced against a lens fiber cell extrinsic membrane protein, with a relative molecular weight of approximately 115 kd. Enzyme Linked Immunosorbent Assays (ELISA) of retina, ciliary body-iris, liver, and skeletal muscle, utilizing these antibodies, suggest that the antigen is unique to the lens. Immunocytochemistry indicates that the antigen is present only in the differentiated fiber cell, and is absent from the lens epithelium. Further, immunocytochemical reactivity is predominantly associated with the fiber cell plasma membrane. However, sequential extraction of fiber cell homogenate, followed by quantitative, competitive ELISA analysis, indicates that most of the antigen is recovered in the neutral buffer extract. ELISA analysis using monoclonal antibodies indicates that an analogous antigen is present in human and rabbit lenses. On the basis of these results we characterize this antigen as a conserved extrinsic membrane protein, which is unique to the differentiated lens fiber cell. The relationship of this antigen to a previously described Mr 95 beaded filament-associated protein is discussed.     

Discrimination between the lens fiber cell 115 kd cytoskeletal protein and alpha-actinin.

The lens fiber cell cytoskeleton includes a protein with a relative molecular weight, by SDS PAGE, of 115 kD. This protein has been purported to be, or be related to alpha-actinin, a highly-conserved family of actin-binding cytoskeletal proteins common to many tissues across a wide phylogenetic range. In this report we assess the relationship between the 115 kd lens fiber cell protein and alpha-actinin. Assessment of relative molecular weight, immunologic cross-reactivity, and partial sequence analysis suggest that the 115 kD lens fiber cell cytoskeletal protein and alpha-actinin are either unrelated, or, at best, that the lens protein represents an unusually divergent isoform of the alpha-actin family of proteins. Immunochemical analysis of homogenates of bovine heart and red blood cells indicate that these tissues express a protein which is weakly cross-reactive with the lens 115 kD protein, but that this cross-reactive protein is not alpha-actinin.     

Kinetics of cyclic AMP-dependent accumulation of novel intermediate filament proteins in cultured chick lens cells.

PURPOSE: To refine the parameters affecting the accumulation of cytoskeletal markers of lens fiber terminal differentiation. METHODS: Primary cultures of chick lens annular pad cells were treated with a lipid soluble cyclic AMP analog under various culture conditions. The accumulation of beaded filament proteins, unique markers of lens fiber terminal differentiation, was quantified with an ELISA assay. The incorporation of beaded filament proteins into macromolecular structures was followed with immunofluorescence microscopy. RESULTS: In a time- and dose-dependent manner, beaded filament protein levels were increased in cyclic nucleotide treated cells. The addition of serum to treated cells caused a further dose-dependent increase in beaded filament protein levels. The continuous presence of cyclic nucleotides for maximal beaded filament protein accumulation was also established. At the light microscopic level, cyclic nucleotide treatment produced much more extensive multilayering of cells and lentoid formation. Macromolecular structures containing beaded filament proteins also increased in both abundance and complexity after cyclic nucleotide treatment and were restricted to the multilayers/lentoids. CONCLUSIONS: These results indicate that multiple mechanisms (including cyclic AMP, serum factors, and the degree of cell-cell interactions) affect the accumulation of beaded filament proteins during the normal differentiation of lens fibers.     

Multi-crystallin complexes exist in the water-soluble high molecular weight protein fractions of aging normal and cataractous human lenses

The purpose of the study was to identify non-covalently held complexes that exist in the water-soluble high molecular weight (WS-HMW) protein fractions of normal human lenses of 20-year-old and 60- to 70-year-old, and in the age-matched 60- to 70-year-old cataractous lenses. The WS protein fractions were prepared from five pooled normal lenses of 20-year-old donors or five pooled lenses of 60- to 70-year-old donors or four pooled cataractous lenses (with nuclear opacity) of 60- to 70-year-old donors. Each WS protein fraction was subjected to size-exclusion chromatography using an Agarose A 5m column to recover the void volume WS-HMW protein fraction. A method known as blue-native polyacrylamide gel electrophoresis (BN-PAGE), which allows the isolation of large multi-protein complexes (MPCs) in their native state for further characterization, was used to separate such complexes from individual WS-HMW protein fractions. The protein species that existed as a complex were excised from a gel and trypsin-digested, and the amino acid sequences of the tryptic fragments analyzed by electrospray tandem mass spectrometry (ES-MS/MS). After the second-dimensional sodium dodecyl sulfate-PAGE during BN-PAGE, protein complexes containing a total of 16, 12, and 24 species with M_r between 10 and 90 kDa were identified in the HMW protein fractions of normal lenses of 20-year-old, 60- to 70-year-old and cataractous lenses of 60- to 70-year-old donors, respectively. Based on the amino acid sequences of tryptic peptides of individual protein species in the complexes by the ES-MS/MS method, the presence of α-, β-, and γ-crystallin species along with beaded filament proteins (filensin and phakinin) was observed in the 20-year-old normal lenses. The 60- to 70-year-old normal lenses contained filensin and aldehyde dehydrogenase in addition to the above crystallins. Similarly, the age-matched cataractous lenses also contained the above crystallins and aldehyde dehydrogenase but lacked beaded filament proteins. Protein complexes, held mostly via non-covalent bonding, were seen in the WS-HMW proteins of 20-year-old normal, 60- to 70-year-old normal, and 60- to 70-year-old cataractous lenses. The complexes in the normal lenses were made of α-, β-, and γ-crystallin species, beaded filament proteins (filensin and/or phakinin), and aldehyde dehydrogenase. The complexes in the age-matched cataractous lenses also contained these crystallins, and aldehyde dehydrogenase, but not the beaded filament proteins. Further, the crystallin fragments were greater in number in the cataractous lenses compared to the age-matched normal lenses. During multi-angle light scattering (MALS), the HMW proteins from cataractous lenses exhibited species with lower molecular weight range than age-matched normal lenses. The HMW protein preparations from both normal and cataractous lenses showed spherical structures on electron microscopic analysis.     

Reconstitution of the filamentous backbone of lens beaded-chain filaments from a purified 49kD polypeptide.

The beaded-chain filaments unique to the fiber cells of the crystalline lens are composed of a linear array of spheroidal particles which appear to be connected by a filamentous backbone. In order to determine the existence of the putative backbone and to characterize its constituents, one of the major proteins associated with beaded-chains in the chicken lens was investigated. 49kD was isolated in an enriched fraction derived from the 8M urea extract of the lens cell water-insoluble residue. The polypeptide (which exists in several charge isoforms, the major at pI 5.2) was purified sequentially by gel filtration on Sephacryl S-200, hydrophobic interaction chromatography on phenyl-Sepharose, and anionic exchange chromatography on Mono Q, all under denaturing conditions. Immunoblot analyses established that 49kD was immunologically distinct from vimentin, actin, and tubulin/MAPs (representing the three classes of cytoplasmic filaments), as well as from the crystallins. Amino acid analyses demonstrated compositional differences for 49kD compared with lens actin and vimentin, and one- and two-dimensional peptide mapping of 49kD and vimentin revealed no homology. Electron microscopy demonstrated that short, contorted filaments were produced upon removal of purified 49kD from urea to low-salt buffers. In the presence of physiological salt concentrations 49kD assembled into extensive 4-6nm diameter, straight filaments similar to the backbone seen in native beaded-chain filaments, but morphologically distinct from the other cytoplasmic filament classes.     

Solubilization of the lens water-insoluble fraction by sonication

A method is reported whereby the solubilization of the bulk of the lens water-insoluble fraction is accomplished by a short sonication of the suspended proteins in low salt buffers. This procedure solubilized greater than 90% of a bovine lens water-insoluble fraction and 80% of the normal human lens water-insoluble fraction. Decreased protein was solubilized from cataractous lenses, but in every case sonication was at least equivalent to extraction with 6.0 M urea. Fractionation of the solubilized proteins by Agarose A-1.5 m gel filtration chromatography showed native molecular weights for bovine lens, but only partial disaggregation with human lens extracts. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) comparison of the proteins solubilized by sonication and 6.0 M urea extraction showed no major differences except that sonication solubilized more of the highly cross-linked protein which remained at the top of the gel.     

The EDTA extractable protein is glycosylated in the bovine lens

The EDTA extractable protein (EEP) can be prepared from the bovine lens using sonication; however, urea washing of the membrane fraction was essential in decreasing the non-specific binding of the crystallins to the EEP fraction. Both the 32 and the 35 kilodalton (kD) proteins from EEP were shown to be glycoconjugates using periodate labelling. The EEP from the lens bound a variety of lectins at both 32 and 35 kD. The binding of the lectins suggested that EEP is composed of proteins having galactose or N-acetylgalactosamine groups as well as fucose residues. The glycosylation of EEP adds another possible regulatory step for the control of these calcium binding proteins in the lens.     

Further studies on low molecular weight crystallins: relationship between the bovine beta s, the human 24kD protein and the gamma-crystallins

Three classes of monomeric crystallins separable by SDS gel electrophoresis or by gel filtration have been demonstrated in human lens. In previous studies we have concluded from physico-chemical and immunological data that all three polypeptides are related and should be classified as gamma-crystallins. The present paper presents further evidence supporting this conclusion, but also demonstrates that the 24,000 dalton (24kD) polypeptide corresponds to the beta s-crystallin. beta s-crystallin was purified by classical techniques from bovine lens and was shown to cross-react with a monoclonal antibody specific for the human 24kD polypeptide. This antibody exhibited no reactivity to other crystallin fractions from either bovine or human lenses. The identification of the 24kD polypeptide as beta s was further supported by analysis of the tertiary structures of the molecules by near UV-circular dichroism and by the finding of a blocked amino terminus on the 24kD polypeptide. Our finding that the human beta s (24kD polypeptide) should actually be classified as a gamma-crystallin is fully consistent with recently reported sequence data on bovine beta s-crystallin.     

Characterization of a mutation in the lens-specific CP49 in the 129 strain of mouse

PURPOSE: The 129 strain of mouse carries a mutation in the gene for CP49 (phakinin), an intermediate filament protein thus far demonstrated only in the lens fiber cell. As such, these mice represent naturally occurring mutants of interest in the study of the lens cytoskeleton. However, this strain of mouse is also widely used as a source of embryonic stem cells in gene-targeting studies. The presence of a mutation in a lens-specific gene can confound interpretation of studies in which lens genes have been knocked out. In the present study, both the genotype and phenotype of these mice were characterized, to permit an evaluation of the biological impact of this mutation and to facilitate the discrimination between wild-type and mutant animals that have been derived from this strain in gene-targeting studies. METHODS: The CP49 cDNA and, when relevant, the genomic DNA sequences were determined for the 129/SvJ and C57BL/6J mice and from a commercially available 129/OLa P1 genomic clone. PCR primers were screened for their capacity to discriminate between the mutant and wild-type CP49 genes. Northern blot analysis was used to assess mRNA levels for CP49, filensin, and gammaS-crystallin (control). Western blot analysis was used to identify changes in protein size and abundance. The impact of the mutation on lens architecture was evaluated at the light-microscope level. Lens fiber cell ghosts from mutant and wild-type mice were examined in the electron microscope for the presence of beaded filaments. Lens clarity was assessed by slit lamp. RESULTS: The 129 strain of mice exhibited a 6303-bp deletion from the end of intron B, and the beginning of exon 2. This deletion results in the loss of the exon 2 splice acceptor site, absence of exon 2 from the CP49 mRNA, and dramatically reduced levels of CP49 mRNA. The CP49 protein was undetectable by Western blot analysis. Messenger RNA levels for filensin, CP49's assembly partner, were normal, but protein levels were sharply reduced. Light microscopy established that the initial differentiation and elongation of the fiber cells proceeded normally. Electron microscopy showed the absence of beaded filaments, whereas slit lamp microscopy showed a slowly emerging and progressive loss of optical clarity. CONCLUSIONS: The 129/SvJ and 129/OLa strains of mice harbor a mutation that sharply reduces CP49 mRNA levels and essentially eliminates both CP49 and the beaded filament. These lenses exhibited a slow but progressive loss of optical clarity with age. Thus, the 129 strain of mouse behaves as a functional CP49 knockout. The loss of clarity in the lenses of these animals and the absence of beaded filaments (and any attendant interactions that may exist between beaded filaments and other lens proteins/structures) suggest that gene-targeting studies of lens proteins in which the 129 strain was used as a source of embryonic stem cells may need reevaluation.     

Expression and distribution of cytoskeletal IFAP-300kD as an index of lens cell differentiation.

By their implication in the organization of the intermediate filament (IF) cytoskeleton, IF-associated proteins (IFAPs) can delineate subsets of the same IF type within a cell; moreover, they are proving useful as markers of the differentiation states of certain cells. For these reasons the expression of the vimentin-associated IFAP-300kD was investigated in the constantly differentiating cell lineage of the adult bovine lens. Immunofluorescence microscopy and immunoblot analysis were employed using a monoclonal anti-IFAP-300kD and a rabbit anti-lens vimentin. Cultures of adult lens epithelial cells were immunopositive for the IFAP. By double-label studies the IFAP-300kD pattern co-localized with that of the vimentin-type IF; moreover, the IFAP pattern co-distributed with that of both colchicine-sensitive and -insensitive IF systems. IFAP-300kD was also present in a co-distributing pattern with vimentin IF in fresh lens epithelial cells on whole mounts. There was a differential expression of the IFAP in the lens fiber cells in that those of the cortex exhibited the IFAP and vimentin IF, while both proteins were absent from the nuclear fiber cells. Furthermore, there was a differential distribution of the IFAP within the cortical fiber cells in that the IFAP localized only with a paramembranal subset of IF. Immunoblot analysis supported the presence of IFAP-300kD in the lens cytoskeletal fraction. IFAP-300kD thus identified a subset of vimentin IF whose location may have functional significance for the cortical fiber cell. The changes in the IFAP's expression and distribution pattern throughout lens cell differentiation in the adult organ suggest the usefulness of IFAP-300kD as a potential marker in studying lens cell differentiation in vitro.     

Development- and differentiation-dependent reorganization of intermediate filaments in fiber cells

PURPOSE. To define the remodeling of lens fiber cell intermediate filaments (IF) that occurs with both development and differentiation. METHODS. Prenatal and postnatal mice were probed for the IF proteins phakosin, filensin, and vimentin, using light microscope immunocytochemical methodology. RESULTS. The pattern of vimentin accumulation in elongating fiber cells changed with development. Early in development vimentin first emerged predominantly as focal accumulations in the basal region of both epithelial and primary fiber cells. A light diffuse cytoplasmic staining was also noted. Later in embryonic development, and through maturity, vimentin in fiber cells was predominantly associated with the plasma membrane with no anterior-posterior polarity. Phakosin and filensin were first detected in the very latest stages of primary fiber elongation and continued to accumulate well after cells had completed elongation. Initially, these proteins accumulated in the anterior half of the fiber cells and were cytoplasmic in distribution. After P13, the pattern of initial distribution in differentiating fiber cells changed to a predominantly plasma membrane localization. Neither beaded filament protein showed focal basal accumulations. In mature lenses, all three proteins ultimately disappeared from the nuclear fiber cells. CONCLUSIONS. Beaded filament protein accumulation lags significantly behind both primary and secondary fiber cell elongation, suggesting a functional role subsequent to elongation. The subcellular distribution of vimentin and the beaded filament proteins showed marked differences within the cell, with differentiation, and with development. The differences in time of initial synthesis and in distribution of these IF proteins may bear on hypotheses about the role of IFs in fiber cell elongation and in structural-functional polarity of the fiber cell.     

Urea formation in rat,bovine, and human lens

Lenses produce both ammonia and urea, and a previous report suggested that bovine lenses contain a complete urea cycle capable of synthesizing urea from bicarbonate and ammonia. To determine whether lenses produce urea by a complete urea cycle or by arginase alone, intact lenses were cultured with [guanido-¹⁴C]-arginine or [¹⁴C]-bicarbonate. The [¹⁴C]-urea was volatilized to [¹⁴C]-CO₂ by urease and collected in KOH. The cultured rat, bovine and human lenses produced [¹⁴C]-urea from [¹⁴C]-arginine; therefore lens arginase activity was also examined in homogenates of rat and human lenses. Rat lens homogenates had constant arginase activity for at least 2 hr at 37°C, and activity increased linearly with the concentration of lens homogenate. Rat lens arginase had an apparent V_max of approximately 13 nmol/hr/mg lens wet weight in lens homogenates and produced 4–6 nmol urea/hr/mg at 25 mm arginine. Human lens homogenates produced 1–5 nmol/hr/mg. In contrast, neither bovine nor rat lenses cultured with [¹⁴C]-bicarbonate produced detectable [¹⁴C]-urea, although label was incorporated into unidentified nonvolatile products. These products were shown by ion exchange chromatography and enzymatic assay to contain no detectable arginine or urea. It was concluded that although arginase activity is present, neither rat nor bovine lenses contain significant urea cycle activity. However, it is possible that arginase serves as a source of lens ornithine.     

Lens membrane fraction associated intermediate filaments of different aged rats

PURPOSE: To describe the intermediate filament proteins vimentin, filensin and phakinin associated with different fractions isolated from neonatal, 10 day old and 20 day old rat lenses. METHODS: Fractions were isolated by differential and density gradient centrifugation of lens homogenates from neonatal, 10 day old and 20 day old rats. Aliquots of the 8 M urea soluble proteins of each fraction were separated by SDS PAGE, transferred to PVDF membranes, the membranes were probed with antibodies to vimentin, filensin or phakinin, and analyzed by computer. RESULTS: Over the 20 day growth period, the water soluble fraction increased and the most abundant membrane fraction was characterized by a significant increase in its urea insoluble protein and a significant decrease in its urea soluble protein. There were no significant quantitative changes in any of the other fractions. The concentration of each intermediate filament protein was greatest in the cytoskeletal fraction and over the 20 day period, the amount of vimentin associated with this fraction dramatically decreased, and the amounts of filensin and phakinin dramatically increased. Among the membrane fractions, the greatest concentration of each intermediate filament protein was found in the non sedimenting membrane fraction (NSMF) which was the least abundant fraction recovered. Filensin and phakinin associated with the other three major membrane fractions increased over the 20 day growth period, but the level of vimentin did not significantly change. CONCLUSIONS: The NSMF may represent a domain of the lens plasma membrane particularly important in interaction between plasma membrane and cytoskeleton and as the membrane-cytoskeleton protein architecture of rat lens changes over the first 20 days of life, the changes are readily detected in the different membrane fractions.     

Comparative studies of beta s-crystallins from human, bovine, rat and rabbit lenses

Soluble extracts from young bovine, human, rat and rabbit lenses were fractionated by high resolution size-exclusion chromatography to demonstrate the existence of three discrete size-classes of monomeric crystallins in each species. These were identified by ion exchange chromatography, amino acid analysis, SDS electrophoresis and isoelectric focusing as the beta s-, gamma A- and gamma B-crystallins. Conventional SDS electrophoretic analysis of these proteins revealed apparent Mr values of about 23kD, 22kD and 19kD, respectively. Similar analysis in the presence of 6 M urea showed the proteins all co-migrated with an apparent Mr of about 20,500, which is far more consistent with the molecular weights calculated from beta s- and gamma-crystallin sequence data. Amino acid compositions of all the beta s samples indicate a high degree of homology to the bovine protein, whose sequence is known. The different species beta s-crystallins showed other general similarities in size, charge, thiol content and secondary structural properties. On the other hand, near UV CD and fluorescence emission and energy transfer measurements indicate that these proteins have subtle yet significant differences in their tertiary structures. Unlike the gamma-crystallins, the secondary structure of all of the beta s samples is completely denatured in the presence of 8 M urea at 20 degrees C.