Lenticular Proteins Research Articles

Differences in Susceptibility Between Crystallins and Non-Lenticular Proteins to Copper and H2O2-Mediated Peptide Bond Cleavage

The relative susceptibilities of lenticular proteins (alpha, beta and gamma-crystallins) and a number of proteins of non-lenticular origin, to hydroxyl radical-mediated peptide bond cleavage were compared. The non-lenticular proteins (bovine serum albumin, ovalbumin, alcohol dehydrogenase, lysozyme, thyroglobulin, beta-amylase, haemoglobin and carbonic anhydrase) were readily cleaved into acid-soluble fragments following 5 hours treatment with copper ions and hydrogen peroxide. In contrast the crystallins were almost totally unaffected by similar treatment. When alpha-crystallin was pre-treated with acid or cleaved into large fragments with cyanogen bromide it became susceptible to hydroxyl radical attack, yet heating the protein did not diminish its resistance. It is suggested that the resistance of alpha-crystallin to the copper/peroxide-mediated fragmentation may be dependent on the conformation of the protein.

Histological and biochemical characterization of the murine cataract mutant Nop

Nop, a spontaneous murine dominant cataract mutation, was detected by slit lamp investigations and preliminary characterized as a nuclear opacity. Histological investigations confirmed these findings and revealed additionally polar cataracts with vacuolization. In contrast to wild-type lenses, the nuclei of the cortical cells could also be detected in the area of the lens nucleus in Nop lenses. No other pathological alterations were found in the eyes. Lens wet and dry weights, as well as the content of water-soluble lens proteins, were reduced in heterozygous and homozygous mutants. The body weight was only slightly altered, indicating a rather lens-specific growth retardation. Some parameters concerning the osmotic state of the lens were changed, however, only in the homozygous mutants. Electrophoresis of the water-soluble lens proteins of the mutants revealed either additional bands, not present in the wild types, or bands of overrepresented proteins only slightly present in wild-type lenses. The changes might be related to the reduced amount of γ-crystallins, which alters the composition of lenticular proteins in the mutants. Northern blots probed with cDNA specific for α-, β- or γ-crystallin genes suggested a reduced transcription of the γ-crystallin genes. In contrast, the transcription of α- and β-crystallins appeared to be similar in wild type and the mutants. The selective reduced amount of γ-crystallin specific RNA can be discussed as a biochemical indicator for the histologically observed changes of differentiation in the cataractous Nop lenses.

Characterization of autoantigens relevant to autoimmune ophthalmitis and thyroiditis in mice immunized with the syngeneic tissue extracts and Klebsiella O3 lipopolysaccharide.

The histological localization and biochemical properties of the autoantigens relevant to experimental autoimmune ophthalmitis and thyroiditis were studied using sera from mice hyperimmunized with the corresponding tissue extract of syngeneic mice and Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. Specific antigens were detected in the lens of the eyeball by immunofluorescence test with sera from mice in which ophthalmitis had been induced and the antigens were lenticular proteins with molecular weights (MW) of 15,000 (15K) to 25K, and 45K. The lenticular proteins with MW of 15K to 25K correspond to the subunits of crystalline. These findings clearly demonstrated that our experimental model for autoimmune ophthalmitis was classified as the lens-induced uveitis. The colloids of the thyroid follicles and the follicular cells were markedly stained by sera from mice in which thyroiditis had been induced. One of the autoantigens detected in the thyroid gland was biochemically consistent with a thyroglobulin subunit. It was also shown that these autoantigens detected in the present study were organ-specific but not species-specific. The nature of autoantigens in the eye and the thyroid gland is discussed.

Lanthionine, a protein cross-link in cataractous human lenses

Oxidative processes are suggested to be a main cause of the covalent cross-linking of lenticular proteins. For a major part these cross-links are disulfides (cystine). With progression of nuclear cataract, cystine may be oxidized further to cysteic acid. Another oxidative degradation product of cystine is the symmetric thioether lanthionine. In this study, we clearly demonstrate that lanthionine is a protein cross-link of cataractous human lenses. Possible mechanisms of its formation are discussed.

Hereditary cataract. Perspective for prenatal screening.

The possibility that defects in lenticular proteins are one cause of hereditary cataract is discussed. Possible mutant loci for such proteins can be detected by linkage to restriction fragment length polymorphisms within or around these loci. In a family in which a Coppock cataract occurs, close linkage between the locus for this cataract and the gamma-crystallin gene cluster was found. The restriction fragment length polymorphism within the gamma-crystallin gene family is sufficiently informative to allow prenatal diagnosis of this disease within this family.

Activity of glutathione peroxidase and glutathione reductase in the human lens related to age.

Lenses from 42 eye bank eyes were assayed for glutathione peroxidase and glutathione reductase activities. The activity of glutathione peroxidase, when considered as a function of age, was lowest in the neonate lens, increasing with age to reach maximal values in young adult lenses, and thereafter progressively decreasing with ages greater than 40 years. Glutathione reductase activity was little affected by age when expressed as activity per lens, per gram lens or per mg soluble protein, indicating that activity of this enzyme did not increase with lens size as would a representative lenticular protein. However, the activity of this enzyme per gram lens was among the highest of any species yet examined.

The role of ascorbic acid in senile cataract.

The reductone ascorbic acid, present in the crystalline lens in concentrations higher than those of glucose, is capable of undergoing nonenzymatic "browning" in the presence of lenticular proteins. We studied the nonenzymatic browning with ascorbate in model systems employing bovine serum albumin and lens crystallins. When bovine serum albumin, alpha-crystallin, or gamma-crystallin was incubated with [14C]ascorbic acid, the formation of yellow and then brown condensation products appeared to correlate with increasing protein-associated radioactivity. The fluorescence spectrum of these products was similar to that of homogenates of human cataractous lenses. We suggest that the nonenzymatic reaction of lens crystallins with ascorbic acid may contribute, at least in part, to the color changes of aging lenses and to the physical lenticular deterioration leading to senile cataract. High dietary intake of ascorbic acid did not affect the fluorescence spectrum of murine lenses; thus, we assume that the speed and extent of the lenticular browning reactions must depend on a deterioration of other factors of the multicomponent antioxidant system of the eye.

Alterations in the lenticular proteins of rats on riboflavin deficient diet.

Effect of feeding riboflavin deficient diet to rats on lens protein composition was investigated. Total proteins and profile of soluble and insoluble proteins in lenses from rats fed on a riboflavin deficient diet for seven weeks were found to be similar to that of the vitamin supplemented diet. Distribution of high molecular weight protein (above 4 X 10(6) daltons) isolated from the 9,900 g supernatant fraction was found to be significantly higher and gamma crystallin was significantly lower in riboflavin deficient group as compared to the normal lenses. However, the distribution of alpha and beta crystallins was not affected. Gel electrophoresis in sodium dodecyl sulfate of soluble lens proteins from riboflavin deficient animals had lower proportions of polypeptide species with molecular weight above 40,000 daltons while insoluble protein fraction had higher proportions of these polypeptide species as compared to control rats. These data suggest that the composition of lens proteins is altered in riboflavin deficiency.

The state of sulfhydryl groups in normal and cataractous human lens proteins. II. Cortical and nuclear regions

The effects of aging and development of senile cataract on the distribution of lenticular protein and total sulfhydryl groups (SH + SS) in the water-soluble and insoluble fractions of the cortex and nucleus of the human lens have been examined. In normal lenses between 33 and 80 years of age, protein and total SH + SS levels of both regions remain fairly constant. With the possible exception of the cortex of the most advanced cataracts, values for these parameters in senile cataracts of various stages show no significant change from those for normal 63-year-old lenses. In normal lenses between 18 and 80 years of age amounts of water-insoluble proteins increase from 20 to 44% of the total cortical protein and from 20 to 55% of the total nuclear protein. In mature cataracts as much as 60% of the cortical and more than 90% of the nuclear proteins can be water-insoluble. Protein disulfides, not detectable in the normal 18-year-old cortex, accumulate by 63 years of age to a level of about 9% of the total cortical SH + SS and 17% of the total nuclear SH + SS groups. The percentages of total SH + SS represented by protein disulfides in either the soluble or insoluble cataractous proteins of Groups II through IV show large increases beyond those in the normal 63-year-old lens. Absolute amounts of soluble protein disulfides, however, remain within the range of normal values. Nonprotein disulfides, i.e. TCA-soluble sulfhydryl groups released by borohydride, are present at low levels in the normal aging lens. The nuclear insoluble fraction of most cataractous lenses is characterized by marked increases in these groups. Losses of non-protein sulfhydryl groups during aging and cataract development are confined largely, if not entirely, to the soluble fraction of the cortical region.

Looking at lens proteins

The eye lens is highly suitable for studies on differentiation and aging since it is derived from a single pure cell line and does not show the phenomenon of cell death. Yet the major lenticular proteins (crystallins) undergo changes in their structure upon aging. The evolution of these proteins has been very conservative.

Investigations on Normal and Cataractous Human Lenses by EPR

Normal and senile cataractous human lenses were studied by spin probe electron paramagnetic resonance (EPR) technique analyzing the reduction of added stable organic-free radicals. It has been concluded that the radical reduction kinetics were different at the different states of the senile cataract, and the conformational changes in lenticular proteins induced by the urea had no influence on the kinetics of the radical reduction. It is supposed that the decrease of the reduced glutathione is an exceedingly sensitive indicator of the change in the lens metabolism in the course of cataractous progression.

Immunopathology of the lens. II. 86Rb efflux and protein leakage from normal lenses exposed to antilens and antiuveoretina antibodies.

Normal rabbit lenses exposed in vitro to heterologous antilens and antiuveoretina antibodies lose control of the intracellular 86Rb and this effect is followed by a late leakage of lenticular proteins; both phenomena take place only in the presence of complement. This antibody-induced complement-dependent membrane damage might be involved in the pathogenesis of complicated cataracts in uveitis and in the self-maintenance of recurrent uveal inflammations.

Immunopathology of the lens. I 86Rb efflux and protein leakage from normal lenses exposed to unrelated antigen-antibody interactions.

Normal rabbit lenses exposed in vitro to heterologous antibody (Ab)--antigen (Ag) interactions lose control of the intracellular 86Rb; this membrane damage, which seems to be related to the quantitative ratio Ab/Ag, is relatively complement independent and is followed by a subsequent leakage of lenticular proteins. A possible implication of these findings in the development of complicated cataracts in uveitis (as 'permeability cataracts') and in the self-maintenance of recurrent uveal inflammation is shortly discussed.

Cellular hypersensitivity to lenticular protein in lens-induced uveitis: by H Hammer and M Olah. Albrecht von Graefes Arch Klin Ophthalmol 192:339–344, 1974

Cellular hypersensitivity to lenticular protein in lens-induced uveitis: by H Hammer and M Olah. Albrecht von Graefes Arch Klin Ophthalmol 192:339–344, 1974

Physical and chemical changes in alpha-crystallin during maturation of lens fibers

A mechanical separation of calf lens into three fractions composed of outer cortex, inner cortex and nucleus provided material representing three phases of fiber maturation. Separation of the lenticular proteins in each fraction was achieved by gel filtration on agarose 15 m. The amount of high mol. wt. alpha-crystallin aggregates was found to increase appreciably as the fibers age. The amount of low mol. wt. alpha-crystallin, however, decreases while the average size gradually increases as the fibers are displaced toward the center of the lens. An increase in sedimentation coefficient corresponding to an increase of 50% in mol. wt. was found when the low mol. wt. alpha-crystallin from the outer cortex was compared to that from the nuclear region. No detectable change in the amino acid or the subunit composition accompanied this increase in size. The circular dichroism (CD) of these crystallins also revealed no conformation changes of the polypeptide backbone. Unique to the CD spectrum for the nuclear alpha-crystallin are minima at 235, 214 and 206 nm. This observation was corroborated by far u.v. spectroscopy to 195 nm. The loss of these CD minima as well as changes in the u.v. profile that can be effected by 7 m-urea suggests the presence of chromophore(s) attached non-covalently to the nuclear protein. One or more other chromophore(s) common to all low mol. wt. alpha-crystallins were revealed by a 40% reduction in the extinction at 195 nm after urea treatment of the cortical alpha-crystallins with no associated change in the CD spectrum. The 20% reduction in extinction for the nuclear protein suggests the presence of similar chromophore(s). Deaggregation followed by reaggregation not only markedly diminished the size of the low mol. wt. alpha-crystallins but also drastically reduced the disparities in size of this protein among the three fiber fractions. These results suggest that chromophore(s) removed by urea treatment may play a decisive role in determining the size of the alpha-crystallin marcromolecule during the maturation of the lens fibers.

Cellular hypersensitivity to lenticular protein in lens-induced uveitis.

In 9 of 12 patients suffering from lens-induced uveitis lymphocyte transformation could be induced in vitro with alphacrystalline, an isolated organ-specific antigen of the lens. This antigen also inhibited leukocyte migration in 8 out of 11 patients. The available date strongly suggest the cellular hypersensitivity to alpha-crystalline to be primarily involved in the development of this form of uveitis.

The elastic constants of the human lens.

1. When the lens is spun around its antero-posterior polar axis in an apparatus designed for the purpose, high speed photography can be used to record its changing profile. By this method a variable radial centrifugal force can be applied to the lens which mimics the pull of the zonule.2. If the lens is not stressed at its centre beyond 100 Nm(-2) it behaves as a truly elastic body. When stressed beyond this limit visco-elastic strain is produced at its poles.3. The human lens has isotropic elastic properties at the extremes of life, but at the other times Young's Modulus of Elasticity varies with the direction in which it is measured.4. Young's Modulus of Elasticity of the lens varies with age, polar elasticity and equatorial elasticity, at birth being 0.75 x 10(3) and 0.85 x 10(3) Nm(-2) respectively, while at 63 years of age both are equal to 3 x 10(3) Nm(-2).5. A comparison of Young's Modulus of the young human lens with that of the rabbit and cat shows that the polar elasticity of the lenses of these animals was 5 times greater in the young rabbit, and 21 times greater in the adult cat. Equatorial elasticities of the rabbit and human lens were equal, while in the cat the equatorial elasticity was four times greater.6. A mathematical model showing the lens substance possessing a nucleus of lower isotropic elasticity than that of the isotropic elastic cortex surrounding it, accounts for the difference between polar and equatorial elasticity of the intact adult lens.7. The implications of these findings are discussed in relation to:(i) accommodation and the rheological properties of the lens;(ii) possible differences in the physical state of the lenticular proteins in the cortex and nucleus which may account for the senile variations in Young's Modulus of Elasticity in these regions of the lens;(iii) the loss of accommodation due solely to an increase in Young's Modulus of Elasticity of the lens between the ages of 15 and 60. This would amount to 44% of the total observed in vivo.

Agar microelectrophoresis at high tension of soluble lens proteins.

Hesselvik was the first to succeed, in 1939, in fractionating lenticular proteins by electrophoresis according to Tiselius. This method of investigation has since been used by numerous investigators studying the soluble proteins of the ocular lens, namely, Viollier, Labhart and Sullmann (1947), Smelzer and von Sallmann (1949), Lewis and Moses (1950), Bon (1955), Rao et al. (1955), Orekhovich (1957), and Bloemendal (1957). Fractionation has subsequently been perfected with the introduction of paper electrophoresis (d'Ermo, 1952; Croisy, 1952; Francois, Wieme, Rabaey, and Neetens, 1953; Stemmerman, 1953; Bon, 1955), and it soon became apparent that the protein composition of the lens is more complex than would be suggested by the classical schema with its three crystallins (α-crystallin, (β-crystallin, and albumin). A comparative study of results obtained by electrophoresis has shown, among other things, that in a great many animal species the number of protein constituents of the lens exceeds the three fractions

Some problems of protein chemistry of the eye.

Proteins are substances on which the organic composition and structure of the cell depends. The protein concentration of the crystallin lens is higher than that of any other organ of the body. This predominance of proteins in the lens' substance points clearly towards the cardinal role they play in the metabolism of this organ. The present review is concerned with the problem of protein chemistry of the lens and mainly with the determination of the amino acid composition of the lenticular proteins. Such determinations were made on the undivided total proteins or on the isolated individual proteins.

CHEMISTRY OF THE LENS

It has always been assumed, without actual evidence, that the various lenticular proteins were homogeneous in chemical composition. However, investigations on many other proteins have indicated that most proteins are heterogeneous in nature. On this basis, it is to be expected that the lenticular proteins are also composite. Technical difficulties prevent the fractionation of most of the naturally occurring proteins, but fortunately the nature of the anatomic growth of the lens aids in the isolation of the lenticular proteins, which are presumably of various ages. Since there is no reason to believe that the lenticular proteins of older cellular fibers are renewed during the life of the lens, the comparative quantitative analysis of the isolated proteins of the lamellae of the lens should give an insight into the process of growth of the lens. The chronological changes in the lens protein also have a bearing on the etiology