Partial amino acid sequences of six major subunits of bovine P-crystallin have been determined by automatic liquid-phase Edman degradation and the dansyl-Edman procedure, complemented by amino acid analyses of peptides. The results show that, including the previously established PBp sequence [H. P. Eur. J. Biochem. 121,, there exist at least seven primary gene products in bovine P-crystallin, which exhibit 40 % or more sequence homology. Two of the gene products are completely identical except for the presence in one of them of 17 additional residues at the N terminus, possibly caused by differential splicing of the same primary RNA transcript. The rate of evolutionary change of the fi chains (4 % sequence change per 100 x lo6 years) is about equally slow as that of a-crystallin, and the gene duplications giving rise to the different chains must have occurred very early in vertebrate evolution. The P chains can be divided into two groups, according to sequence homology and presence of deletions/insertions and C-terminal extension, on which basis a new, rational nomenclature for the /? subunits is introduced. The N-terminal extensions of all P chains are very different in length and sequence, even between homologous P chains in different species. Possible explanations for this finding are discussed.The evolutionarily highly conserved structural eye lens proteins, the crystallins, can be divided into four classes a, P, y and h-crystallin, of which P-crystallin is the most heterogeneous [I]. In bovine P-crystallin six or more chains are primary gene products, whereas some ten others arise by post-translational modification [2]. They can associate to oligomers, varying from dimers and trimers (filow) to octamers (fihigh) [2,3]. This diversity in P-crystallin subunits has also been found at the mRNA level in the chicken lens [4]. The primary structure of the predominant bovine P-crystallin chain, PBp, has been elucidated [5], while the protein structure of a major murine P-crystallin polypeptide (P 23) has been deduced from cDNA and genomic DNA analysis [6, 71. Several mRNAs of the rat lens crystallins have been cloned [8], and recently the nucleotide sequence of one of these has revealed the primary structure of the PBlchain (J. T. den Dunnen and J. G. G. Schoenmakers, unpublished data).Amino acid sequence determination of bovine y-crystallins [9, 101 and nucleotide sequence analyses of several y-crystallin cDNAs from rat [ll] and frog [12] have shown a considerable homology between P and y-crystallins. The three-dimensional structure of the bovine y-I1 chain has been determined [13] and a similar tertiary fold has been predicted for two / 3 chains, BBp [I41 and 823 [7]. The P and y-crystallins are built up of two domains, which show considerable sequence similarity, suggesting an intragenic duplication in the ancestral gene of these proteins. Each of the domains is again folded into two similar 'Greek key' motifs. A relationship between these structural motifs and the exons of the murine P23 gene has been suggeste...
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.
The amino acid sequence of the N-terminal region of the two basic bovine &crystallin B1 chains has been analyzed. The results reveal that @Bib is derived in vivo from the primary gene product @la by removal of a short N-terminal sequence. It appears that them1 chains have the same domain structure as observed in other /3-and y-crystallin chains. They have, however, a very long N-terminal extension in comparison with other &chains. This extension is mainly composed of a remarkable Pro-and Ala-rich sequence, which suggests an interaction of these structural proteins with the cytoskeleton and/or the plasma membranes of the lens cells.
A Ca2+-dependent transglutaminase (EC 2.3.2.13) has been demonstrated in the eye lenses of several mammalian species [Lorand, L., Hsu, L. K. M., Siefring, G. E., Jr., & Rafferty, N. S. (1981) Proc. Natl. Acad. Sci. USA 78, 1356USA 78, -1360. Using [3H]methylamine as a convenient probe for transglutaminase activity, we have explored the action of this enzyme in the bovine eye lens. We could characterize the glutamine residues acting as acyl-donor sites in three (3-crystallin chains, which are the only substrates for lens transglutaminase among the various lens-specific structural proteins, the crystallins. A single glutamine was found to bind[3H]methylamine in each of these three chains: glutamine -9 in (3Bp ((3B2), glutamine -21 in (3B3, and glutamine -23 or -24 in (3A3. The four glutamines are all located in the NH2-terminal regions, which presumably extend from the compact two-domain structure of the .8-crystallin chains. It was, moreover, established that several components of the lens cytoskeleton are substrates for transglutaminase. Intracellular transglutaminase (EC 2.3.2.13), which catalyzes the formation of e-(y-glutamyl)-lysine isopeptide crosslinks between polypeptides (1, 2), has recently been related to a variety of important biological processes. It may be involved in neuronal aging and in Alzheimer disease (3,4), in the interaction between membrane proteins and cytoskeleton (5), in receptor-mediated endocytosis (6), in erythrocyte membrane stiffening (7), in terminal differentiation of keratinocytes (8), in the regulation of cell proliferation (9), and in the development of senile cataract (10). The endogenous substrate glutamines of intracellular transglutaminase have in no case been identified. This prompts further studies to reveal the precise substrate sites and the modes of action of transglutaminase to better understand the physiological significance of the enzyme in intracellular processes.In the rabbit eye lens two /-crystallin subunits among the water-soluble proteins have been shown to act selectively as substrates for lens transglutaminase (10). In the bovine lens seven primary gene products are identified in B-crystallin (11), and their sequences have been completely (12) or partially (11) elucidated. We previously could identify three ,& crystallin chains which act as substrates, and we located the site of amine incorporation in the major BBp-chain, using [14C]putrescine and dansylcadaverine as probes (13) ,AM, respectively. After 2 hr of incubation at 37°C the reaction was stopped by the addition of 150 ,ul of 50 mM EDTA to chelate the Ca2, and the mixture was centrifuged at 13,000 x g for 30 min at 4°C. The supernatant was briefly dialyzed against two changes of 1% ammonium bicarbonate at 4°C and then lyophilized. The pellet was washed several times with buffer and subsequently extracted with 6 M urea. After centrifugation at 13,000 x g and 4°C, the supernatant was dialyzed and lyophilized. The pellet was washed several times with 6 M urea and buffer and subsequently l...
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