The 4.1-like proteins of the bovine lens: spectrin-binding proteins closely related in structure to red blood cell protein 4.1.

The superficial cortical fiber cells of the bovine lens contain membrane-associated proteins of 150,000, 80,000, and 78,000 D that cross-react with antisera prepared against red blood cell (RBC) protein 4.1 (Aster, J. C., G. J. Brewer, S. M. Hanash, and H. Maisel, 1984, Biochem. J., 224:609-616). To further study their relationship to protein 4.1, these proteins were immunoprecipitated from detergent extracts of crude lens membranes with purified polyclonal and monoclonal anti-4.1 antibodies and resolved by SDS PAGE. The electrophoretic mobilities of the lens proteins of 80,000 and 78,000 D were found to be identical to bovine RBC protein 4.1a and protein 4.1b, respectively. One- and two-dimensional peptide mapping revealed that a high degree of structural homology exists among all three of the lens 4.1-like proteins and RBC protein 4.1a and protein 4.1b. Despite the large difference in apparent molecular mass, the 150,000-D lens protein showed only minor peptide map differences. A nitrocellulose filter overlay assay showed that all three of the lens 4.1-like proteins bind to RBC and lens spectrins. We conclude that the bovine lens contains proteins of 80,000 and 78,000 D that are highly similar to protein 4.1 in structure and functional capacity. Additionally, the lens also contains a 4.1 isomorph of 150 kD. Analogous to RBC protein 4.1, these proteins may function in the lens by promoting association of spectrin with actin and by playing a role in the coupling of lens cytoskeleton to plasma membrane.

Glial fibrillary acidic protein is localized in the lens epithelium.

The epithelium of the mouse lens stains intensely with antisera to glial fibrillary acidic protein (GFAP). A protein co-migrating with GFAP and immunoreactive with antisera to GFAP can be demonstrated in lens epithelium protein extracts by immunoblots. GFAP has previously been considered unique to cells of neural origin, but this study demonstrates that ectodermally derived cells express GFAP or a highly similar protein.

Calmodulin binds to chick lens gap junction protein in a calcium-independent manner.

A biochemically active conjugate of calmodulin and tetramethylrhodamine isothiocyanate (CaM-RITC) was synthesized. When incubated with sections of chick lens, this conjugate bound to the surface membranes of lens fiber cells in the presence of absence of calcium. Incubation of lens sections with antibodies to gap junction protein of lens completely blocked the binding of the conjugate to cell membranes, whereas serum from nonimmunized animals or antibodies to others lens proteins reduced the binding only slightly. By means of a gel overlay procedure, 125I-labeled calmodulin was found to bind to the gap junction protein of lens, also in a calcium-independent manner. These results support the concept that calmodulin may interact with and regulate gap junctions in living cells.

BASP1 in the lens.

BASP1 was detected in the embryonic and adult chicken lens, using immunological methods and by peptide sequence analysis. The protein was predominantly expressed in fiber cells and only faintly detected in annular pad cells. Localization of the protein was along the plasma membrane of fiber cells often in discrete areas. The role of BASP1 in the lens requires further study.

Subunit characterization of lens intermediate filaments.

The intermediate filaments from the bovine ocular lens can be fractionated into three 54000 dalton polypeptides (alpha, beta and gamma) which are present in equimolar quantities and differ from one another with respect to isoelectric point, two-dimensional peptide map pattern and total amino acid composition. The polypeptides, for which the name lentin is proposed, were purified from the 8 M urea extract of the water-insoluble residue of the cortical fiber cells by ion-exchange chromatography on DE- and CM-cellulose. The regeneration of native-appearance filaments from each purified fraction, or combinations thereof, definitively established the three polypeptides as the subunit species. Additionally, small diameter 'protofilaments' were regenerated when one or two fractions were utilized, but not when all three fractions were combined. This, together with the fact that the stoichiometric yield of the three species was invariably equimolar, suggests that alpha, beta, and gamma may be distinct entities not related by artifactual interconversion.

The lens epithelium contains glial fibrillary acidic protein (GFAP).

Both polyclonal and monoclonal antibodies to glial fibrillary acidic protein (GFAP) intensely immunostain the lens epithelium. Two-dimensional gels of mouse lens epithelium protein extracts reveal a protein which co-migrates with GFAP from spinal cord. A lens epithelial protein of approximately 50 000 MW on one-dimensional gels is immunoreactive with antiserum to GFAP (Hatfield et al. 1984). GFAP has been regarded as a protein unique to neuroglial cells but this study indicates that some cells of ectodermal origin (the lens epithelium) express GFAP.

Isoproterenol treatment causes cytoskeletal reorganization in chicken lens fiber cells.

Cytoskeletal organization in organ cultured embryonic chicken lens was affected by isoproterenol treatment. Immunofluorescent localization of a 49 kD fiber cell-specific cytoskeletal protein showed a cytoplasmic and membrane localization in control lenses. Following isoproterenol treatment, fluorescence was predominantly membrane-associated. Changes in 49 kD distribution as determined by immunofluorescence occurred within minutes of isoproterenol application and were completely blocked by propranolol pretreatment. These results suggest that beta-adrenergic stimulation influences the apparent localization of a fiber cell-specific cytoskeletal protein.

The plasma membrane of the rabbit lens cortical fiber. I. Isolation, characterization, and biosynthesis of two membrane intrinsic polypeptides.

Rabbit lens cortical fiber plasma membrane polypeptides were isolated and two membrane intrinsic proteins were characterized by SDS-PAGE. A polypeptide with a molecular weight of 26 kilodaltons is the major constituent of the plasma membrane. The molecular weight and antigenic properties of the other polypeptide studied are similar to polypeptides of cortical water-soluble alpha-crystalline. Biosynthesis of these two polypeptides was studied. After initially high rates, synthesis of both of these polypeptides decreased considerably and maintained a steady rate for the rest of the culture time.

Lens fodrin binds actin and calmodulin.

Fodrin was isolated from the chick lens and shown to bind to actin and calmodulin. Its localization to the cytoplasmic side of lens plasma membranes was demonstrated immunologically.

Cytoskeletal proteins of the aging human lens.

The cytoskeletal proteins of the human lens were studied by SDS-PAGE, 2-D electrophoresis and immunologically. Spectrin, vimentin and actin were identified in the superficial fiber cells of human lenses even to age 87 years. These proteins are lost from the deeper cortical and nuclear fiber cells, where a broad zone of acidic protein (36-42 Kd) emerges even in the transparent normal lens. Age-related changes in the water-soluble fraction include the increased prominence of proteins of 56 Kd and 36 Kd in the cortex, and of 38 Kd in the nucleus.

Evidence for a calcium activated protease specific for lens intermediate filaments.

The calcium mediated loss of intermediate filament protein from lens cytoskeletal preparations was examined by soft laser scanning densitometry of polyacrylamide gels. The time course of proteolysis by the lens Ca++ activated proteinase and inhibition by EGTA or PMSF and leupeptin were also determined. Proteolytic breakdown products were identified on electroblots with specific antiserum to vimentin.

Phosphorylation of chick lens proteins.

Phosphorylated proteins of the chick lens were identified following incubation of lenses in a medium containing 32P and subsequent analysis by gel electrophoresis. The acidic variant of the vimentin and both subunits of fodrin were phosphorylated, as were the 95 Kd and 49 Kd proteins associated with the beaded-chain filaments. Neither crystallins nor the main intrinsic membrane proteins were phosphorylated. Several low molecular weight phosphoproteins of the epithelial cell were not present in the fiber cells.

NCAM of the mammalian lens.

NCAM is present in the plasma membranes of human and rat lens epithelial cells and superficial fiber cells. The predominant isoform in epithelial cells is NCAM 140, while NCAM 120 appears only in the superficial fiber cells. The immunofluorescence patterns are consistent with a decreasing concentration of NCAM associated with fiber cell differentiation.

Adrenergic stimulation of lens cytoskeletal phosphorylation.

Stimulation of lens fiber cytoskeletal phosphorylation by adrenergic drugs is described. The effect of isoproterenol on phosphorylation of the 47 Kd beaded filament protein is dose-dependent, detectable as soon as one minute after treatment and blocked by propranolol. Epinephrine increases the phosphorylation of both 47 Kd and the intermediate filament protein, vimentin. 47 Kd phosphorylation is also increased by norepinephrine, dibutyryl-cAMP or forskolin. The results indicate that lens fiber cytoskeletal phosphorylation is regulated, at least in part, via a beta-adrenergic receptor coupled to cyclic AMP production.

Synthesis of cytoskeletal and membrane polypeptides by the organ cultured chicken lens.

The synthesis of cytoskeletal and membrane polypeptides of the chick lens epithelium, superficial cortical fiber cells and deep cortical fiber cells was studied in the organ-cultured lens. The synthetic activity of the cytoskeletal polypeptides was restricted to the epithelium and superficial cortical fiber cells while membrane polypeptides were synthesized by deep cortical fiber cells as well. None of the polypeptides was observed to be synthesized by the lens nuclear fiber cells.

Lipid composition of chick lens fiber cell gap junctions.

Chick lens fiber cell gap junctions were isolated to homogeneity by the urea-deoxycholate method, characterized ultrastructurally and biochemically, and their lipid composition determined by quantitative thin layer chromatography (TLC). The junctions were estimated to comprise about 52% of the lens fiber plasma membrane. Unlike the junctions of other organs, the lens gap junctions were found to contain sphingomyelin. The cholesterol/phospholipid molar ratio was 2.1 for total fiber membranes but 3.1 for the fiber gap junctions. The levels of major phospholipids in decreasing order were SPH, PC, PE, PI for fiber junctions and PE, SPH, PC, PI for total fiber membranes. The gap junctions were found to contain about 57% of the total fiber cholesterol and 53% of the total fiber sphingomyelin. The high cholesterol and sphingomyelin content suggests that lens fiber gap junctions constitute highly rigid membrane regions conferring significant constraints to the movement of their intramembrane particles (connexons) in the plane of the membrane. The findings help to explain the resistance to the crystallization of their connexons, observed so far only in lens gap junctions.

Enolase in the avian and turtle lens.

Enolase is a dimeric enzyme of molecular weight of 100,000 daltons, which plays an important role in the glycolytic cycle. The aim of this study was to characterize the enzyme of the chicken lens epithelium and to compare its distribution in different regions of the chicken, duck and turtle lens. Enolase of the chicken lens epithelium was found to be an enzymatically active dimeric protein of molecular weight 100,000 daltons and representing alpha-enolase. It is a major component of the epithelium comprising 4%, 12% and 46% of the water-soluble protein of chicken, duck and turtle epithelium respectively. Enolase is found in trace amount in the fiber cells of the chicken and duck, but is retained in much greater concentration in the turtle fiber mass as a predominantly inactive enzyme.

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.

Human beta crystallins: regional and age related changes.

The composition of human beta-crystallins displayed specific changes with age and region of the lens. 27 kD and 29 kD human beta-crystallin subunits were singled out for study. The 29 kD beta-crystallin subunit constituted approximately 10% of the total lens crystallins at 8 months of fetal life. Its accumulation decreased steadily to 3.3% during postnatal year 1, to 0.5% by year 5 and to 0.3% thereafter. At all postnatal ages, however, it persisted mainly in the superficial fibers. Thus in a 17-years old lens it made up 1.3% of the superficial fiber soluble protein but was already absent from deep cortical and nuclear fibers. The 27 kD subunit increased steadily from 3.5% at 8 months fetal to 7% at year 5; it then decreased steadily to 1.2% in the 86-year old lens. It persisted in all regions of the lens but decreased markedly in the deep cortical and nuclear fibers with increasing age beginning at 5-17 years of age. Studies on the oligomeric structure of human beta-crystallin must take into account age-related changing quantitative patterns in the subunit polypeptide composition of this lens protein.

Electron microscopy supports a fibrous substructure for lens intermediate filaments.

The substructure of intermediate filaments from bovine lens cortical fiber cells was investigated by electron microscopy. Native filaments and synthetic ones regenerated from the total cytoskeletal extract and from the three purified subunits were examined. The morphologies from these various sources were essentially identical, with the exception that filaments reconstituted from one of the purified polypeptides were much shorter, very contorted and showed strings of aggregated protein. The solid cylindrical, unbranching filaments consisted of a helical arrangement of at least two, 5 nm diameter strands. The evidence indicated that each strand was composed of two, 2 nm diameter protofilaments which were also helically constructed (right-handed) with a periodicity of 11.6 nm. Intermediate filament diameter varied widely (8-14.8 nm, average 11.3 nm) and in a direct, linear manner relative to the apparent progression (helical) angle of the strands across the filaments face. These conclusions were obtained from observations on negatively stained intact filaments and reconstituted 4.4 nm fibrils and on positively stained transverse sections of fixed and embedded filaments.