Herman J. Hoenders

The interrelationship between monomeric, oligomeric and polymeric α-crystallin in the calf lens nucleus

Calf lens nuclear α-crystallin was separated into the three known molecular weight populations on Ultrogel AcA22. Electron microscopy illustrates that the two higher molecular weight classes, which are formed upon aging of the lens, are chain-like oligomers and polymers of the spherical monomeric α-crystallin molecules of 15 nm average diameter and 1·1 × 106 average molecular weight which form the lowest molecular weight class. Our combined sedimentation and electron microscopy data make the alleged non-equilibrium state within these populations questionable. From the polypeptide chain composition, determined by isoelectric focusing, it can be inferred that the total amount of C-terminal shortened chains increases progressively from 33 to 53% from monomeric to polymeric α-crystallin. Reassociation of different types of isolated shortened A-chains in various mixtures indicates a tendency for formation of higher molecular weight reaggregates with increasing relative amount of degraded chains. The involvement of degraded chains in polymerization is postulated although it is not likely to be the only factor causing aggregation.

The distribution of the soluble proteins in the calf lens

Abstract Lenses of 3-month-old calves were divided into nine concentric layers. The protein extracts of these fractions were submitted to gel chromatography on agarose A-5 m. From the epithelium to the nucleus, the content of α-crystallin decreased significantly, whereas the content of a high molecular weight fraction, resembling α-crystallin in u.v. spectrum as well as polypeptide composition, increased markedly. An analogous phenomenon was observed in the group of the β-crystallins. The proportion of the lower molecular weight fraction decreased and that of the higher molecular weight fraction increased from epithelium to nucleus, i.e. according to the age of the fibre cells. The highest content of γ-crystallin was found in the nucleus. The activity of lens leucine aminopeptidase was found to be highest in the outer cortex fractions, i.e. in the synthetically active area of the lens.

A Model for the Architecture of α-Crystallin

Knowledge of the quaternary structure of α-crystallin is required to understand the age-dependent superaggregation processes, eventually leading to lens opacification. Different approaches, e.g. sulfhydryl modification, chemical cross-linking, limited proteolysis and dissociation studies revealed new information, providing a basis for further studies of aging and higher-order structures. A model for the architecture of native α-crystallin from calf lens cortex, featuring subunit arrangement and surface exposure, will be presented.

Structural aspects of bovine β-crystallins: Physical characterization including dissociation-association behavior*

Bovine cortical lens extracts were fractionated on Bio-Gel A-5m or Sephacryl S-200 and revealed three and four size classes for the β -crystallins, respectively. The greatest aggregates, β H , turned out to be subject to reversible, concentration-dependent dissociation into smaller aggregates. It was inferred that the association-dissociation equilibrium is mainly affected by hydrophilic interactions. On Sephacryl S-200 a β -crystallin size class with mol. wt 71 000, determined at sedimentation equilibrium, was isolated. Moreover, a β L -crystallin fraction (mol. wt 48000) and β S -crystallin (mol. wt 28 500) could be isolated. The polypeptide compositions of all β -crystallin aggregates have been determined by sodium dodecylsulfate (SDS) gel electrophoresis and isoelectric focusing in urea. On SDS gels β S -crystallin revealed a molecular weight of 22 000 contrasting sharply with the value determined at sedimentation equilibrium.

Protein changes in the human lens during development of sentile nuclear cataract

Senile nuclear cataractous lenses were divided into three groups of increasing nuclear color. These groups were considered as successive stages in the development of senile nuclear cataract. The cortex and the nucleus of normal and cataractous lenses were separated into water-soluble, urea-soluble and urea-insoluble fractions. Fractionation on a Sephadex G-200 column of the water-soluble components revealed five protein fractions for both cortex and nucleus. Only minor quantitative differences in polypeptide chain composition were found by isoelectric focusing between corresponding protein fractions isolated from normal and cataractous lenses. The weight percentages of the water-soluble, urea-soluble, and urea-insoluble fractions of cortex and nucleus from the normal and cataractous lenses were determined. A decrease of the amounts of the water-soluble and urea-soluble fractions and a concomitant increase of the urea-insoluble fraction were observed in the nucleus as a function of cataract development. Lens wet weight and protein content did not change significantly. The carbohydrate content of the urea-soluble fractions increased, that of the urea-insoluble fraction decreased. A striking decrease of the phospholipid content in the urea-insoluble fraction was found.

Effect of aging on the water-soluble and water-insoluble protein pattern in normal human lens

Total extracts of both water-soluble and water-insoluble portions are obtained from fetal and adult human lenses. In the water-soluble extracts of lenses older than 14 years of age little difference is seen between the cortical and nuclear preparations. In lenses older than 14 years a polypeptide chain of approximately 10 k appears. In fetal and newborn lenses a 43 k polypeptide is detected which becomes more diffuse upon aging. Concomitant with a decrease of a 29·5 k polypeptide, there is a relative increase of a 29 k chain, suggesting a situation comparable with the transition from calf βB1a to βB1b. In young lenses a major β-polypeptide co-migrating with calf βBp is seen. As for the water-insoluble portions, no difference between cortical and nuclear preparations is detectable after 14 years of age. In fetal lens there is a high amount of the 29·5 k polypeptide, which decreases with aging and finally disappears. Till 6 months four bands with molecular weights of 200 k, 100 k, 57 k and 43 k are detected, co-migrating with calf myosin, α-actinin, vimentin and actin, respectively.

The polypeptide chains of α-crystallin from old human eye lenses☆

Abstract The polypeptide chains of α-crystallin from 60- to 80-year-old human eye lenses were studied to determine whether they are subject to similar post-synthetic alterations as known from bovine α-crystallin. Isoelectric focusing in the presence of 6 M urea revealed that old human α-crystallin consists of five major polypeptide chains, designated as Ax, A2, Bx, By and B2. By dodecyl sulphate gel electrophoresis mainly chains with apparent molecular weights of 22 000 and 20 000 were observed. Ion-exchange chromatography in the presence of urea was applied to obtain enriched preparations of individual polypeptides. Additional data concerning the primary structure of some chains were gathered by tryptic peptide mapping in combination with amino acid analysis. The presence of two cysteine residues in the A-chains was demonstrated. The Ax component turned out to be composed of two chains: deamidated A2 of normal length (172 residues) and, presumably, a shortened A2-chain: A21–151. The Bx- and By-chain with isoelectric points slightly lower and higher, respectively, than that of bovine B1, appeared to be B21–170 and deamidated B2 of normal length (175 residues). A group of polypeptides with very acidic isoelectric points, present in low proportion, showed a heterogeneous molecular weight distribution below 18 000 and a high degree of similarity with the A-chains as judged by tryptic peptide mapping. In spite of several differences in subunit composition, it can be concluded that in human α-crystallin, as in bovine α-crystallin, deamidation and degradation processes occur.

Characteristics of α-crystallin related to fiber cell development in calf eye lenses

Abstract α-Crystallin was isolated from extracts of the epithelium, from seven successive concentric layers of the cortex and the nucleus of calf lenses. In this order the α-crystallin content of the extracts decreased strongly whereas a very high-molecular component with α-crystallin like subunit composition became more and more prominent. Polyacrylamide gel electrophoresis in 6 M urea revealed significant changes in polypeptide composition. In the epithelial cells only trace amounts of the α A 1 and no α B 1 polypeptides were present. The latter ones appeared at a relative late stage of fiber cell development. The sedimentation coefficients of the α-crystallin preparations obtained from the successive lens layers were also determined. Going from epithelium to nucleus the values obtained ranged from 17 to 21 S and passed through a minimum. A clear cut relationship between the polypeptide chain composition and sedimentation coefficient could not be found.

Glycation of human serum albumin: inhibition by Diclofenac.

Glycation is a non-enzymatic modification of proteins by sugars, probably responsible for the initiation of complications in diabetes patients and aging individuals. Our in vitro experiments show an inhibition of sugar attachment in the presence of Diclofenac. The levels of advanced glycation products, measured as specific fluorescent groups, were also lowered due to Diclofenac. These results were compared with inhibition by Aspirin (acetylsalicylic acid), a known inhibitor of the glycation process. The protection by Diclofenac is based on a non-covalent interaction of the drug with serum albumin. There is evidence that Diclofenac specifically blocks at least one of the major glycation sites of human serum albumin.

Glycation of crystalline in lenses from aging and diabetic individuals

Water‐soluble crystallins were obtained from clear human lenses of different age(4–81‐year‐olds) and lenses of individuals showing senile and diabetic cataracts. Levels of early glycation products were high in the high molecular weight material (HM) and the α‐crystallin fractions, compared with β and γ‐crystallins. This difference becomes more prominent upon aging. The content of total early glycation products in HM and α‐crystallin increases clearly with age, whereas levels remain relatively constant in the β‐ and α‐crystallins. There is an elevation of early products in cataractous lenses from diabetic individuals compared with those suffering from senile cataract. Specific non‐tryptophan fluorescence (excitation/emission wavelengths 370/440 nm), used as an indicator for late glycation products, increased dramatically with age and was 2‐fold higher in the diabetic subjects. Levels of fluorescence decreased in the order HM > α‐ > b > γ‐crystallins. The results suggest an increase in glycation rate in α‐crystallin as a result of aging and diabetes, while the rate of glycation of β‐ and γ‐crystallins remains almost constant.