cDNA Clones encoding bovine γ-crystallins

Oxidative damage of the lens causes disulfide bonds between cysteinyl residues of lens proteins and thiols such as glutathione and cysteine, which may lead to cataract. The effect of H 2 O 2 oxidation was determined by comparing bovine lenses incubated with and without 30 mM H 2 O 2 . The H 2 O 2 treatment decreased the glutathione and increased the protein-glutathione and proteincysteine disulfides in the lens. The molecular mass of the ␥B-crystallin isolated from lenses, not treated with H 2 O 2 , agreed with the published sequence (M r 20,966). Some lenses also had a less abundant ␥B-crystallin component 305 Da higher (M r 21,270), suggesting the presence of a glutathione adduct. The ␥B-crystallins from H 2 O 2 treated lenses had three components, the major one with one GSH adduct, another one with the mass of unmodified ␥B-crystallin, and a third with a mass consistent with addition of two GSH adducts. Mass spectrometric analysis of tryptic peptides of ␥B-crystallins from different lenses indicated that the ؉305 Da modifications were not at a specific cysteine. For the lenses incubated without H 2 O 2 , there was evidence of adducts at Cys-41 and in peptide 10 -31, which includes 3 cysteines. Analysis of modified peptide 10 -31 by tandem mass spectrometry showed GSH adducts at Cys-15, Cys-18, and Cys-22. In addition, ␥B-crystallins from H 2 O 2 -treated lenses had an adduct at Cys-109, partial oxidation at all 7 Met residues, and evidence for two disulfide bonds.Age-related cataract is one of the major causes of blindness in humans. The pathology of such a condition involves opacification and decreased transparency of the lens, which can lead to loss of vision. Although the mechanisms for age-related cataractogenesis are not understood, oxidation of the lens proteins, known as crystallins, is associated with cataract formation in humans (1, 2). Protein thiolation, which involves the formation of disulfide bonds between the cysteinyl residues of lens proteins and other low molecular weight thiols in the lens, is one of the modifications caused by oxidative stress of the lens (3). Thiols in the lens that participate in this reaction are GSH and cysteine, which form protein-S-S-glutathione (PSSG) 1 and protein-S-S-cysteine (PSSC), respectively (4). These proteinthiol products are referred to as mixed disulfides. The conformational changes caused by protein thiolation (5, 6) may allow some of the buried functional groups to be exposed and modified. This may cause proteins to aggregate and disrupt the close packing of the crystallins, decreasing their solubility (7) and leading to cataract formation. In the H 2 O 2 -induced cataract model, the progression of cataract is associated with sequential events involving first the formation of PSSG, followed by protein-protein disulfide cross-links, decreased protein solubility, and finally an increase in the formation of high molecular weight aggregates (8). Protein-thiol mixed disulfides accumulate in older human lenses (9) and in all types of human cataractous lenses ...

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“…The masses of these peptides were compared with masses of expected peptides calculated from the known sequence (35) (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

“…Peptides were identified by matching their molecular masses with the masses of peptides expected from tryptic digestion of ␥B-crystallin, which had been calculated from the known sequence of bovine ␥B-crystallin (35).…”

Section: Analysis Of Tryptic Peptides Of ␥B-crystallins By Esims-mentioning

confidence: 99%

Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

Hanson¹,

Chen

Smith³

et al. 1999

Journal of Biological Chemistry

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“…Using motif 1 of bovine ␥B-crystallin (8,23,25) as an archetype, the most important residues are G13, F or Y at positions 6 and 11, and S34. Other positions are also well conserved, particularly in B-type motifs, with their characteristic Tyr and Trp corners (26).…”

Section: Aim1mentioning

confidence: 99%

AIM1 , a novel non-lens member of the βγ-crystallin superfamily, is associated with the control of tumorigenicity in human malignant melanoma

Ray

Wistow

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

140

119

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AIM1 is a novel gene whose expression is associated with the experimental reversal of tumorigenicity of human malignant melanoma. The predicted protein product of the major 4.1-kb transcript shows striking similarity to the ␤␥-crystallin superfamily. All known members of this superfamily contain two or four characteristic motifs arranged as one or two symmetrical domains. AIM1, in contrast, contains 12 ␤␥ motifs, suggesting a 6-domain structure resembling a trimer of ␤-or ␥-crystallin subunits. The structure of the AIM1 gene shows remarkable similarity to ␤-crystallin genes, with homologous introns delineating equivalent protein structural units. AIM1 is the first mammalian member of the ␤␥ superfamily with a primarily non-lens role. Other parts of the predicted AIM1 protein sequence have weak similarity with filament or actin-binding proteins. AIM1 is a good candidate for the putative suppressor of malignant melanoma on chromosome 6, possibly exerting its effects through interactions with the cytoskeleton. AIM1 (absent in melanoma) is a novel gene whose expression is altered in association with tumor suppression in a model of human melanoma (1). The AIM1 gene is localized to 6q21, within the putative chromosome 6 tumor suppressor region for human melanoma (2). Characterization of the AIM1 gene and its major transcripts reveals, unexpectedly, that AIM1 belongs to the ␤␥-crystallin (␤␥) superfamily.The optical properties of the eye lens largely depend on abundant soluble structural proteins, the crystallins (3-5). Although some crystallins are enzymes, the result of relatively recent gene recruitment events in distinct evolutionary lineages, other crystallins, related to stress-inducible proteins, have more ancient origins and are represented ubiquitously in all vertebrates (6, 7). This ubiquitous class includes the ␤-and ␥-crystallins, which are closely related in sequence and in gene and protein structure (8).Three non-lens members of the ␤␥ superfamily have been identified through a conserved sequence signature: Protein S of Myxococcus xanthus, a sporulating bacterium (9, 10); spherulin 3a of the slime mold Physarum polycephalum (11,12); and an epidermis differentiation-specific protein (EDSP or ep37) of the amphibian Cynops pyrrhogaster (13-15).The structure of Protein S has been confirmed by NMR analysis (16). The structure of a yeast killer toxin (WmKT) has also revealed unexpected similarity to the folding pattern of the ␤␥ superfamily (17). Although an ancestral relationship has been discussed, WmKT may represent an interesting example of evolutionary convergence.Except for spherulin 3a, and possibly WmKT, with two motifs, all other identified members of the ␤␥ superfamily have a 4-fold repeat of the characteristic ␤␥ motif. In contrast, AIM1 contains 12 ␤␥ motifs. Furthermore, the exon͞intron structure of the AIM1 gene shows remarkable similarity with ␤-crystallin genes, with each motif coded by a separate exon.Protein S, a calcium-binding protein of the bacterial spore coat, and spherulin 3a, a ...

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“…An aliquot of this fraction was digested with trypsin and analyzed by HPLC and FABMS. Computer-assisted analysis was used to compare the molecular weights of these tryptic peptides with those expected from bovine crystallins with known amino acid sequences (van der Ouderaa et al, 1973(van der Ouderaa et al, , 1974Hogg et al, 1986;Hay et al, 1987;Smith et al, 1991). The presence of many peptides with molecular weights expected for peptides of aA-and aB-crystallins indicated the presence of major portions of both of these proteins ( Table 2).…”

Section: Identification Of Crystallins By Esimsmentioning

confidence: 99%

Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

et al. 1992

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A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and posttranslational modifications of aB-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The aB-crystallin primary gene product, its mono-and diphosphorylated forms, its N-and C-terminal truncated forms, as well as other lens proteins unrelated to the aB-crystallins were identified by their molecular weights.Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that aB-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of a-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.Keywords: crystallins; lens proteins; mass spectrometry; protein phosphorylationThe lens crystallins are long-lived structural proteins of particular interest in understanding cataractogenesis (Harding, 1985) and chemical processes associated with aging (Harding & Dilley, 1976). There are three primary groups of lens crystallins, a-, P-, and y-crystallins, which can be separated by the size of aggregates they form. a-crystallins form aggregates with molecular weights over 500,000; &crystallin aggregates have molecular weights of 40,000-200,000; y-crystallins remain as monomers of 20,000. The molecular weights of the monomers of aand P-crystallins are also approximately 20,000. The nomenclature of the m-and P-crystalIins describes whether they are acidic (e.g., aA2, and PA3) or basic (aB2 and f3B2). Early investigators of the lens crystallins attributed the altered electrophoretic mobility of several a-crystallins to deamidation of asparagine (Bloemendal et al., 1972;Stauffer et al., 1974) and C-terminal degradation (Van Kleef et al., 1976). In 1985, Spector et al. (1985) showed that radiolabeled phosphate could be incorporated into both &A-and aB-crystallins, with evidence that serine was the only phosphorylated residue. Their as- sumption that electrophoretic behavior originally attributed to deamidation is due to phosphorylation (Chiesa et al., 1987(Chiesa et al., , 1988) was substantiated by quantification of phosphorus in a-crystallins (Voorter et al., 1989). The C-terminaI degradation and phosphorylation of aA-crystallins were confirmed recently by mass spectrometry . The fact that the a-crystallins could be phosphorylated suggested that they might have biological functions other than their contribution to lens structure. Using radioactively labeled phosphate Chiesa et al. (1987Chiesa et al. ( , 1988 determined that the phosphorylation of aBcrystal...

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cDNA Clones encoding bovine γ-crystallins

Cited by 39 publications

References 14 publications

Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

Thiolation of the γB-Crystallins in Intact Bovine Lens Exposed to Hydrogen Peroxide

AIM1 , a novel non-lens member of the βγ-crystallin superfamily, is associated with the control of tumorigenicity in human malignant melanoma

Identification of the posttranslational modifications of bovine lens αB‐crystallins by mass spectrometry

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