Hans Bloemendal

Structure and modifications of the junior chaperone α -crystallin

alpha-Crystallin is a high-molecular-mass protein that for many decades was thought to be one of the rare real organ-specific proteins. This protein exists as an aggregate of about 800 kDa, but its composition is simple. Only two closely related subunits termed alpha A- and alpha B-crystallin, with molecular masses of approximately 20 kDa, form the building blocks of the aggregate. The idea of organ-specificity had to be abandoned when it was discovered that alpha-crystallin occurs in a great variety of nonlenticular tissues, notably heart, kidney, striated muscle and several tumors. Moreover alpha B-crystallin is a major component of ubiquinated inclusion bodies in human degenerative diseases. An earlier excitement arose when it was found that alpha B-crystallin, due to its very similar structural and functional properties, belongs to the heat-shock protein family. Eventually the chaperone nature of alpha-crystallin could be demonstrated unequivocally. All these unexpected findings make alpha-crystallin a subject of great interest far beyond the lens research field. A survey of structural data about alpha-crystallin is presented here. Since alpha-crystallin has resisted crystallization, only theoretical models of its three-dimensional structure are available. Due to its long life in the eye lens, alpha-crystallin is one of the best studied proteins with respect to post-translational modifications, including age-induced alterations. Because of its similarities with the small heat-shock proteins, the findings about alpha-crystallin are illuminative for the latter proteins as well. This review deals with: structural aspects, post-translational modifications (including deamidation, racemization, phosphorylation, acetylation, glycation, age-dependent truncation), the occurrence outside of the eye lens, the heat-shock relation and the chaperone activity of alpha-crystallin.

Evolution of eye lens crystallins: the stress connection

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.

Identification of the cytoskeletal proteins in lens-forming cells, a special epithelioid cell type

Abstract Proteins of contractile and cytoskeletal elements have been studied in bovine lens-forming cells growing in culture as well as in bovine and murine lenses grown in situ by immunofluorescence microscopy using antibodies to the following proteins: actin, myosin, tropomyosin, α-actinin, tubulin, prekeratin, vimentin, and desmin. Lens-forming cells contain actin, myosin, tropomyosin, and α-actinin which in cells grown in culture are enriched in typical cable-like structures, i.e. microfilament bundles. Antibodies to tubulin stain normal, predominantly radial arrays of microtubules. In the epithelioid lens-forming cells of both monolayer cultures grown in vitro and lens tissue grown in situ intermediate-sized filaments of the vimentin type are abundant, whereas filaments containing prekeratin-like proteins (‘cytokeratins’) and desmin filaments have not been found. The absence of cytokeratin proteins observed by immunological methods is supported by gel electrophoretic analyses of cytoskeletal proteins, which show the prominence of vimentin and the absence of detectable amounts of cytokeratins and desmin. This also correlates with electron microscopic observations that typical desmosomes and tonofilament bundles are absent in lens-forming cells, as opposed to a high density of vimentin filaments. Our observations show that the epithelioid lens-forming cells have normal arrays of ( i ) microfilament bundles containing proteins of contractile structures; ( ii ) microtubules; and ( iii ) vimentin filaments, but differ from most true epithelial cells by the absence of cytokeratins, tonofilaments and typical desmosomes. The question of their relationship to other epithelial tissues is discussed in relation to lens differentiation during embryogenesis. We conclude that the lens-forming cells either represent an example of cell differentiation of non-epithelial cells to epithelioid morphology, or represent a special pathway of epithelial differentiation characterized by the absence of cytokeratin filaments and desmosomes. Thus two classes of tissue with epithelia-like morphology can be distinguished: those epithelia which contain desmosomes and cytokeratin filaments and those epithelioid tissues which do not contain these structures but are rich in vimentin filaments (lens cells, germ epithelium of testis, endothelium).

THE PLASMA MEMBRANES OF EYE LENS FIBRES. BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION

Abstract Plasma membranes have been isolated from calf eye lens fibre cells. The purified fraction is characterized by the occurrence of a large number of junctional complexes. The cholesterol phospholipid ratio is approximately 0.7, a value in between the values reported for erythrocyte ghosts and liver plasma membranes. Specific membrane protein components have been separated electrophoretically from structural lens proteins.

Immobilization of the C-terminal extension of bovine alpha A-crystallin reduces chaperone-like activity

α-Crystallins occur as multimeric complexes, which are able to suppress precipitation of unfolding proteins. Although the mechanism of this chaperone-like activity is unknown, the affinity of α-crystallin for aggregation-prone proteins is probably based on hydrophobic interactions. α-Crystallins expose a considerable hydrophobic surface to solution, but nevertheless they are very stable and highly soluble. An explanation for this paradox may be that α-crystallin subunits have a polar and unstructured C-terminal extension that functions as a sort of solubilizer. In this paper we have described five αA-crystallins in which charged and hydrophobic residues were inserted in the C-terminal extension. Introduction of lysine, arginine, and aspartate does not substantially influence chaperone-like activity. In contrast, introduction of a hydrophobic tryptophan greatly diminishes functional activity. CD experiments indicate that this mutant has a normal secondary structure and fluorescence measurements show that the inserted tryptophan is located in a polar environment. However, NMR spectroscopy clearly demonstrates that the presence of the tryptophan residue dramatically reduces the flexibility of the C-terminal extension. Furthermore, the introduction of this tryptophan results in a considerably decreased thermostability of the protein. We conclude that changing the polarity of the C-terminal extension of αA-crystallin by insertion of a highly hydrophobic residue can seriously disturb structural and functional integrity.

Cyanate formation in solutions of urea: I. Calculation of cyanate concentrations at different temperature and pH

Abstract Data from the literature on the equilibrium between urea and cyanate which were difficult to combine, have been correlated by computer calculation. Accumulation of cyanate in urea solutions was quantitatively studied at different temperature and pH.

Expression and aggregation of recombinant αA-crystallin and its two domains

The 20 kDa αA and αB subunits of α-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 αA-crystallin, as well as the intact αA-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20–60% of total protein). Recombinant αA-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant αA-crystallin forms aggregates comparable with α-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 αA-crystallin subunits are mainly located in the N-terminal half of the chain.

Eye-lens proteins: The three-dimensional structure of β-crystallin predicted from monomeric γ-crystallin

Recent sequence determinations [l-4] have demonstrated that two of the three classes of mammalian lens-specific proteins, the pand y-crystallins, form a single superfamily of proteins, accounting for >50% of the protein in the mammalian lens. show such a strong internal homology except for some residues at particular features in the 711 fold which do display a 4-fold repeat [ 121. There is, however, a general conservation of functional types of residue between the 4 folding motifs of the structure.

The genes coding for the cytoskeletal proteins actin and vimentin in warm-blooded vertebrates.

Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta‐ or gamma‐) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2‐3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm‐blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non‐coding sequences of both vimentin and actin genes are conserved in length.

Lens Proteins and Their Genes

Publisher Summary Recent developments made in lens research have demonstrated that certain enzyme proteins occurring in nonlenticular tissues in minor quantities exist in the lens in high concentration and seem, therefore, to be recruited as structural elements in evolution. In addition, it appears that at least, one of the typical structural proteins of the vertebrate lens—αB-crystallin—is by no means lens-specific. For example, heart, brain, skeletal muscle, kidney, and possibly other tissues express this characteristic component of the αcrystallin aggregate. In addition, research community is beginning to understand the way genes are expressed and regulated in the eye lens. Studies using gene constructs containing crystallin 5’-regulating sequences have enabled the mapping of positive and negative control elements responsible for tissue-specific expression. However, many features, such as post-transcriptional modifications, still have to be clarified at this level.