Martinus A. M. van Boekel

Conversion from Oligomers to Tetramers Enhances Autophosphorylation by Lens A-Crystallin SPECIFICITY BETWEEN αA- AND αB-CRYSTALLIN SUBUNITS

Previously we showed that α-crystallins are autophosphorylated (Kantorow, M., and Piatigorsky, J.(1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3112-3116). Here we report that addition of 1% deoxycholate converted αA-crystallin aggregates into 80-kDa tetramers which were 10-fold more active for autophosphorylation. Circular dichroism (CD) spectra of α-crystallin revealed little or no change in secondary and tertiary structures in 1% deoxycholate. αA2D, a truncated form of bovine αA that exists as a tetramer, was as active for autophosphorylation in the absence of deoxycholate as intact αA was in the presence of deoxycholate. At least one serine between amino acids 131 and 145 of bovine αA was autophosphorylated in peptide mapping experiments. Chicken αA-crystallin, which lacks the Ser-122 cAMP-dependent kinase site of bovine αA, was also autophosphorylated in the presence of deoxycholate. In contrast to αA-crystallin, autophosphorylation by αB-crystallin was not activated by deoxycholate despite its conversion to a tetrameric form, and αB was also more efficiently phosphorylated by cAMP-dependent kinase than αA. These data suggest metabolic differences between the α-crystallin subunits that may be related to specific expression of αA in the lens and ubiquitous expression of αB in numerous normal and diseased tissues.

Eye lens αA- and αB-crystallin: complex stability versus chaperone-like activity

Abstract The major lens protein α-crystallin is composed of two related types of subunits, αA- and αB-crystallin, of which the former is essentially lens-restricted, while the latter also occurs in various other tissues. With regard to their respective chaperone capacities, it has been reported that homomultimeric αA-crystallin complexes perform better in preventing thermal aggregation of proteins, while αB-crystallin complexes protect more efficiently against reduction-induced aggregation of proteins. Here, we demonstrate that this seeming discrepancy is solved when the reduction assay is performed at increasing temperatures: above 50°C αA- performs better than αB-crystallin also in this assay. This inversion in protective capacity might relate to the greater resistance of αA-crystallin to heat denaturation. Infrared spectroscopy, however, revealed that this is not due to a higher thermostability of αA-crystallin’s secondary structure. Also the accessible hydrophobic surfaces do not account for the chaperoning differences of αA- and αB-crystallin, since regardless of the experimental temperature αB-crystallin displays a higher hydrophobicity. It is argued that the greater complex stability of αA-crystallin, as evident upon urea denaturation, and the higher chaperone capacity of αB-crystallin at physiological temperatures reflect the evolutionary compromise to obtain an optimal functioning of heteromeric α-crystallin as a lens protein.

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.

In vivo glycation of bovine lens crystallins

To investigate the possible role of glycation in the aging of lens proteins, we used bovine lenses as a model. We studied crystallins isolated from prenatal bovine lenses, calf lenses and lenses from mature animals (up to 20 years old). The experiments show an increase in glycation levels with age in all crystallin fractions. Regarding the lysine content of the different crystallins, gamma-crystallin showed relatively high levels of early glycation products. The results also revealed high levels of early glycation products for the HM material (containing mainly alpha-crystallin). In alpha-crystallin, alpha A-subunits were glycated to a higher extent compared with the alpha B-subunits. There is an age-related increase in advanced glycation products, measured as specific fluorescence (excitation/emission wavelengths 370/440 nm), mainly present in the HM and alpha-crystallin fraction.

Evolution of the Aldose Reductase-Related Gecko Eye Lens Protein ρB-Crystallin: A Sheep in Wolf's Clothing

Abstract.ρB-crystallin (AJ245805) is a major protein component (20%) in the eye lens of the gecko Lepidodactylus lugubris. Limited peptide sequence analysis earlier revealed that it belongs to the aldo-keto reductase superfamily, as does the frog lens ρ-crystallin. We have now determined the complete cDNA sequence of ρB-crystallin and established that it is more closely related to the aldose reductase branch of the superfamily than to frog ρ-crystallin. These gecko and frog lens proteins have thus independently been recruited from the same enzyme superfamily. Aldose reductase is implicated in the development of diabetic cataract in mammals, and, if active, ρB-crystallin might be a potential risk for the gecko lens. Apart from a replacement 298 Cys → Tyr, ρB-crystallin possesses all amino acid residues thought to be required for catalytic activity of the aldose reductases. However, modeling studies of the ρB-crystallin structure indicate that substrate specificity and nicotinamide cofactor affinity might be affected. Indeed, neither recombinant ρB-crystallin nor the reverse mutant 298 Tyr → Cys showed noticeable activity toward aliphatic and aromatic substrates, although cofactor binding was retained. Various other oxidoreductases are known to be recruited as abundant lens proteins in many vertebrate species; ρB-crystallin demonstrates that an aldose reductase-related enzyme also can be modified to this end.

Detection of lens proteins in phaco tips after phacoemulsification

PURPOSE To determine the efficacy of cleaning procedures to remove water-soluble and water-insoluble lens proteins from phaco tips after phacoemulsification. SETTING Academic Hospital Maastricht, Department of Ophthalmology, University Eye Clinic, and Rotterdam Hospital, Rotterdam, The Netherlands. METHODS Detection of alpha A-crystallins, cytoskeletal lens proteins (vimentin), and lens-cell membranes (MP26) was performed on new and reused phaco tips with specific markers. To detect antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques were used. RESULTS Threshold detection levels for alpha A-crystallins, vimentin, and MP26 were 100, 43, and 108 ng, respectively. No detectable residues of water-soluble or water-insoluble lens proteins were found in outflow samples of routinely cleaned phaco tips or in outflow samples of phaco tips that were not cleaned after phacoemulsification. Urea effectively eliminated water-insoluble lens proteins from contaminated phaco tips. CONCLUSION After regular cleaning of phaco tips, no detectable lens protein residues were found in the outflow samples. Moreover, omitting the cleaning of phaco tips after phacoemulsification did not lead to detectable lens proteins in the outflow samples.

Diabetic Cataract: Glycation of the Molecular Chaperone α-Crystallin and its Binding to the Membrane Protein MIP26

Summary We investigated the influence of glycation on the chaperone-like activity of the eye lens protein α-crystallin. Formation of early glycation products in vitro had no consequences for the protecting properties of the protein. Late (cross-linking) glycation products had a marked negative influence on the chaperone-like activity of α-crystallin. Similar results were found for homopolymers of the α-crystallin subunits αA2 and αB2. Of all crystallins, only α-crystallin specifically binds to lens membranes. The presence of early glycation products on α-crystallins does not seem to alter their affinity for membranes in vitro . These processes are possibly involved in the development of diabetic cataract.