Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.

The in vivo phosphorylation sites of bovine alpha B-crystallin.

Phosphate content determinations established that in alpha B-crystallin two phosphate groups can be present in vivo in bovine lenses. Comparison of tryptic digests of phosphorylated and unphosphorylated alpha B chains, revealed the location of the two phosphorylation sites in tryptic peptides T2 and T3. Thermolytic digestion and gas-phase sequencing demonstrated that Ser-19 and Ser-45 are the in vivo phosphorylation sites of bovine alpha B-crystallin. This pattern of phosphorylation differs from the previously reported in vitro obtained results.

MP17, a fiber-specific intrinsic membrane protein from mammalian eye lens.

A major protein with a molecular weight of 17,000, designated as MP17, has been identified in mammalian eye lens plasma membranes. Hydrophobic photolabeling experiments revealed that MP17 is a genuine intrinsic membrane protein. By using monoclonal antibodies we demonstrated that MP17 is not detectable in liver, heart, muscle, spleen and kidney, and thus can be considered, like MP26, as a lens-specific membrane protein. Furthermore, we showed that MP17 is a substrate for cAMP-dependent protein kinase and that it is a calmodulin-binding protein.

Differential synthesis of crystallins in the developing rat eye lens.

The patterns of protein synthesis in rat lenses ranging in age from newborn to 4 months were compared. After incubation of lenses in [35S]methionine-containing medium it was possible to identify the de novo synthesized crystallins by two-dimensional gel electrophoresis and fluorography, in combination with peptide mapping and immunoblotting. It was found that the relative synthesis of alpha A and beta A3 stays fairly constant in rat lenses of all investigated ages. The relative synthesis of beta B2 and gamma s shows a pronounced increase with age in these post-natal lenses. A differential decrease can be observed in the relative synthesis of the other six gamma-crystallins (gamma A-gamma F). There appears to be a good correlation between the changes in relative synthesis of the various crystallins and previously reported alterations in mRNA levels, although certain mRNAs exhibit marked differences in translational efficiency.

Characterization of anti-crystallin autoantibodies in patients with cataract.

Anti-crystallin autoantibodies have often been demonstrated in the serum of healthy persons and, especially, patients with cataract. In no case, however, have the specific crystallin subunits been identified against which such antibodies are directed. This information would be of particular interest in view of the recent finding that several crystallin subunits occur constitutively outside the lens. To fill this gap, we analysed the sera of 15 patients with mature cataract by means of 1- and 2-dimensional immunoblotting. The circulating antibodies turned out to be directed against several beta- and gamma-crystallin subunits. The types of subunits and the intensities of the responses varied considerably between patients. No or only occasional and very weak reactions were observed against the alpha A-, alpha B- and beta B2-crystallin subunits. These are in fact the only crystallins at present known to occur outside the lens in mammals. Our findings thus indicate that anti-crystallin autoantibodies are specifically directed against those crystallins that appear to be lens-restricted, while immunological tolerance would exist for the extra-lenticularly occurring crystallins.

Spontaneous peptide bond cleavage in aging alpha-crystallin through a succinimide intermediate.

Cleavage of specific peptide bonds occurs with aging in the alpha A subunit of bovine alpha-crystallin. One of the breaks occurs at residue Asn-101. This same residue undergoes in vivo deamidation, isomerization, and racemization. Deamidation and isomerization are known to occur via succinimide ring formation of labile asparagine residues. Model studies on peptides have shown that imide formation can also lead to peptide bond cleavage (Geiger, T., and Clarke, S. (1987) J. Biol. Chem. 262, 785-794). In that case, both asparagine and aspartic acid amide would be expected as C termini of the truncated polypeptide, and this is indeed the case in the alpha A-(1-101)-chain. This thus represents a first example of nonenzymatic in vivo peptide bond cleavage in an aging protein through the formation of a succinimide intermediate. In addition, we found that in bovine lens no detectable conversion (through the action of protein-carboxyl methyltransferase) of isoaspartyl to normal aspartyl residues occurs in vivo after deamidation of Asn-101.

Structural and functional similarities of bovine alpha-crystallin and mouse small heat-shock protein. A family of chaperones.

alpha-Crystallin, composed of the subunits alpha A and alpha B, is a major vertebrate eye lens protein, accomplishing a structural role in maintaining lens stability and transparency. Both subunits also occur in low amounts outside the lens, where their precise function is unknown. They are structurally related to the small heat-shock proteins (HSPs), and increasing evidence indicates that they have also functional similarities with the small HSPs. To extend our insight into these structural and functional relationships, the mouse small HSP (HSP25) was compared with bovine alpha-crystallin, with respect to several known properties of the latter. We show that alpha-crystallin and HSP25 resemble each other in secondary structure and have similar stability toward urea dissociation at pH 7.0. Mixed polymers can be formed from any combination of alpha A-crystallin, alpha B-crystallin, and HSP25 subunits. Furthermore, we demonstrate that HSP25, like alpha-crystallin, can function as a molecular chaperone, by suppressing heat-induced aggregation of other proteins, and is an efficient inhibitor of elastase. Finally, HSP25 is found to be a substrate for protein cross-linking by tissue-type transglutaminase, like alpha B-crystallin. Our results thus corroborate that alpha-crystallin and the small HSPs have comparable functions, probably being involved in the protection of other proteins under conditions of stress.