Christina E.M. Voorter

The in vivo phosphorylation sites of bovine αB-crystallin

Phosphate content determinations established that in αB‐crystallin two phosphate groups can be present in vivo in bovine lenses. Comparison of tryptic digests of phosphorylated and unphosphorylated αB 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 αB‐crystallin. This pattern of phosphorylation differs from the previously reported in vitro obtained results.

Site-specific racemization in aging α-crystallin

Of all aspartyl residues in bovine αA‐crystallin, only Asp‐151 exhibits pronounced racemization. Asp‐151 is also one of the sites where peptide bond cleavage occurs in in vivo aging αA‐crystallin. This aspartyl residue is followed by an alanyl residue and resides in a flexible carboxyl terminal extension of α‐crystallin. Both in vivo and in vitro racemization studies indicate that the pronounced and site‐specific racemization of Asp‐151 proceeds via formation of a succinimide intermediate. The in vivo racemization of aspartyl residues in αA‐crystallin is discussed with regard to the proposed tertiary structure of α‐crystallin.

Age‐dependent deamidation of αB‐crystallin

Bovine and human αB‐crystallin undergo deamidation upon aging in the lens. In bovine αB‐crystallin, the specific site of dearnidation has been identified by peptide mapping after tryptic digestion. Asn‐146 was found to be subject to deamidation, whereas the only other asparagine residue, at position 78, is not affected. Asn‐146 is flanked at the carboxylic side by a glyeyl residue. Yet, the rate of in vivo deamidation is low. In vitro studies reveal that the deamidation is accompanied by significant racemization, indicating that the deamidation proceeds via formation of a succinimide intermediate.

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.

Relocalization of αB-crystallin by heat shock in ovarian carcinoma cells

αB‐Crystallin, a major lens protein, is present in clearly detectable amounts in cultured ovarian carcinoma cells. After heat‐shock treatment of these cells at 45°C αB‐crystallin relocalizes from the detergent‐soluble, cytosolic fraction to the non‐ionic detergent‐insoluble nuclear/cytoskeletal fraction. Colchicine treatment of the cells, alhough giving rise to a vimentin collapse on the nucleus, does not result in redistribution of ß‐cyrstallin. When this colchicine treatment is followed by heat shock, αB‐crystallin relocalizes again to the insoluble fraction, indicating that this relocalization is independent of the collapse of the vimentin network.

Age-dependent deamidation of chicken αA-crystallin

The major posttranslational modification product of αA‐crystallin from chicken eye lenses has one more negative charge than the corresponding primary gene product. These polypeptides were compared by peptide mapping after tryptic digestion and cyanogen bromide cleavage, and the charge difference could be located in a peptide, comprising residues 146–150 of the amino acid sequence of αA‐crystallin. Subsequent enzymatic hydrolysis with aminopeptidase showed that asparagine at position 149 of the primary gene product is replaced by aspartic acid. Two‐dimensional gel electrophoresis of total lens homogenates from chickens of different ages revealed an age‐dependent increase of the deamidated αA‐subunit.

Distribution of αB-crystallin in the anterior segment of primate and bovine eyes

The presence and distribution of αB-crystallin in the anterior segment of human, monkey and bovine eyes was investigated immunocytochemically. In all three species the most intense staining was seen in the lens and in the nonpigmented and pigmented epithelial cells covering the tips of the pars plicata of the ciliary body. The staining intensity of the ciliary epithelial cells was comparable to that seen in the lens fibers. Strong labeling was also found in the corneal endothelium.In bovine eyes the presence of αB-crystallin in lens, ciliary epithelium of the pars plicata and corneal endothelium was also shown by biochemical analysis using SDS-PAGE and immunoblotting. Cum. Eye Res. 12: 871-876, 1993.

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.

cAMP-dependent protein kinase phosphorylates gap junction protein in lens cortex but not in lens nucleus

MP70 (a 70 kDa membrane protein) is a component of the gap junctions of the young fibre cells in the lens outer cortex. In the older fibres deeper in the mammalian lens (lens nucleus), MP70 is processed to MP38 by cleavage and removal of the carboxy terminal half. It is shown here that cortical MP70, and its derivative MP64, can be phosphorylated with cAMP-dependent protein kinase. In contrast, MP38 from the lens nucleus is not phosphorylated by the enzyme. Proteolytic processing and this lens region specific phosphorylation are relevant for the future development of functional assays for lens gap junctions.

The alternative splicing product αAins-crystallin is structurally equivalent to αA and αB subunits in the rat α-crystallin aggregate

Abstract In rodents and some other unrelated mammals, alternative splicing of the αA-crystallin gene transcript results in the synthesis of the elongated αA ins -crystallin chain. This polypeptide is identical to the normal αA-crystallin chain of 173 residues, but contains an additional sequence of 23 amino acid residues inserted between positions 63 and 64. To determine the effects of this insert peptide, the structure of the rat α-crystallin aggregate and its subunits αA-, αA ins and αB-crystallin was studied using fluorescence spectra, partial urea dissociation, and lactoperoxidase-catalysed iodination of surface residues. The data suggest that all α-crystallin subunits occupy equivalent positions in the protein aggregate, and that the insert peptide merely elongates the connecting peptide between the putative amino- and carboxyl-terminal domain of the αA-crystallin subunit.