Genetic heterogeneity of the Coppock-like cataract: a mutation in CRYBB2 on chromosome 22q11.2.
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PURPOSE: To identify the genetic defect for the Coppock-like cataract (CCL) affecting a Swiss family, which defect was unlinked to the chromosome 2q33-35 CCL locus. METHODS: A large family was characterized for linkage analysis by slit lamp examination or by the review of drawings made before cataract extraction. The affection status was attributed before genotyping, and the genotyping was masked to the affection status. Two-point and multipoint linkage analyses were performed using the MLINK and the LINKMAP components of the LINKAGE program package (ver. 5.1), respectively. Mutational analysis of candidate genes was performed by a combination of direct cycle sequencing and an amplification refractory mutation system assay. RESULTS: Ten individuals were affected with the CCL phenotype. The disease was autosomal dominant and appeared to be fully penetrant. A new CCL locus was identified on chromosome 22q11.2 within a 11.67-cM interval (maximum lod score [Zmax] = 4.14; theta = 0). Mutational analysis of the CRYBB2 candidate gene identified a disease-causing mutation in exon 6. This sequence change was identical with that previously described to be associated with the cerulean cataract, a clinically distinct entity. CONCLUSIONS: The CCL phenotype is genetically heterogeneous with a second gene on chromosome 22q11.2, CRYBB2. The CCL and the cerulean cataract are two distinct clinical entities associated with the same genetic defect. This work provides evidence for a modifier factor that influences cataract formation and that remains to be identified. (+info)
Expression of betaB(2)-crystallin mRNA and protein in retina, brain, and testis.
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PURPOSE: To evaluate the expression of betaB(2)-crystallin mRNA and protein in rat, bovine, and human nonlens and nonocular tissues. METHODS: betaB(2)-crystallin mRNA levels were detected by RT-PCR. betaB(2)-crystallin protein was purified from rat and bovine tissues by FPLC chromatography. FPLC fractions were analyzed by immunoblotting. The identity of betaB(2)-crystallin protein, isolated from the retina, was confirmed by protein microsequencing. RESULTS: betaB(2)-crystallin transcript was detected in rat brain, rat testis, and human retina by RT-PCR. betaB(2)-crystallin transcript was not found in rat lung, heart, ovary, spleen, thymus, kidney, and liver or in human brain and testis. betaB(2)-crystallin protein was partially purified from and its identity confirmed in rat brain, rat testis, and bovine retina. The bovine retinal protein was further confirmed to be authentic betaB(2)-crystallin by protein microsequencing. CONCLUSIONS: These results establish that betaB(2)-crystallin mRNA and protein are expressed in tissues outside of the lens and outside of the eye including retina, brain, and testis. Extralenticular and extraocular expression of betaB(2)-crystallin, coupled with its participation in phosphorylation pathways, suggests that it has nonrefractive functions in these tissues. (+info)
Aey2, a new mutation in the betaB2-crystallin-encoding gene of the mouse.
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PURPOSE: During an ethylnitrosourea (ENU) mutagenesis screen, mice were tested for the occurrence of dominant cataracts. One particular mutant was found that caused progressive opacity and was referred to as Aey2. The purpose of the study was to provide a morphologic description, to map the mutant gene, and to characterize the underlying molecular lesion. METHODS: Isolated lenses were photographed, and histologic sections of the eye were analyzed according to standard procedures. Linkage analysis was performed using a set of microsatellite markers covering all autosomal chromosomes. cDNA from candidate genes was amplified after reverse transcription of lens mRNA. RESULTS: The cortical opacification visible at eye opening progressed to an anterior suture cataract and reached its final phenotype as total opacity at 8 weeks of age. There was no obvious difference between heterozygous and homozygous mutants. The mutation was mapped to chromosome 5 proximal to the marker D5Mit138 (8.7 +/- 4.2 centimorgan [cM]) and distal to D5Mit15 (12.8 +/- 5.4 cM). No recombinations were observed to the markers D5Mit10 and D5Mit25. This position makes the genes within the betaA4/betaB-crystallin gene cluster excellent candidate genes. Sequence analysis revealed a mutation of T-->A at position 553 in the Crybb2 gene, leading to an exchange of Val for GLU: It affects the same region of the Crybb2 gene as in the Philly mouse. Correspondingly, the loss of the fourth Greek key motif is to be expected. CONCLUSIONS: The Aey2 mutant represents the second allele of Crybb2 in mice. Because an increasing number of beta- and gamma-crystallin mutations have been reported, a detailed phenotype-genotype correlation will allow a clearer functional understanding of beta- and gamma-crystallins. (+info)
Identification and properties of anti-chaperone-like peptides derived from oxidized bovine lens betaL-crystallins.
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Thermal aggregation of betaL-crystallin was higher in the presence of peptide fragments generated from oxidized and trypsin-digested betaL-crystallin compared with thermal aggregation of the control proteins without oxidized betaL-crystallin fragments. Increased aggregation of betaL-crystallin was also observed despite the presence of alpha-crystallin (which has anti-aggregating properties) in the system. Self-aggregation of the oxidized betaL-crystallin fragments per se was not observed under the experimental conditions. Reverse-phase HPLC analysis of the precipitate obtained after heating a mixture of betaL-crystallin and oxidized betaL-crystallin fragments revealed that more than one peptide co-precipitates with betaL-crystallin. Electrospray mass spectrometry analysis of the peptides revealed that the molecular weight(s) of the peptides ranged from 1400-1800. Tandem mass spectrometry and a data base search revealed that two of the peptides originated from betaA4-crystallin (LTIFEQENFLGR, residues 121-132) and betaB3-crystallin (AINGTWVGYEFPGYR, residues 153-167) respectively. Oxidized synthetic peptides representing the same sequence were also found to enhance the aggregation of betaL-crystallin in a manner similar to oxidized lens betaL-crystallin peptides. These data suggest that the polypeptides generated after oxidation and proteolysis of betaL-crystallins interact with denaturing proteins and facilitate their aggregation and light scattering, thus behaving like anti-chaperones. (+info)
Decreased heat stability and increased chaperone requirement of modified human betaB1-crystallins.
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PURPOSE: To determine how deamidation and partial loss of the N- and C-terminal extensions alter the heat stability of betaB1-crystallin. METHODS: Human lens betaB1, a deamidated betaB1, Q204E, and alphaA-crystallins were expressed. Truncated betaB1 was generated by proteolytic removal of part of its terminal extensions. The aggregation and precipitation of these proteins due to heating was monitored by circular dichroism and light scattering. The effect of heat on the stability of both monomers and oligomers was investigated. The flexibility of the extensions in wild type and deamidated betaB1 was assessed by 1H NMR spectroscopy. RESULTS: With heat, deamidated betaB1 precipitated more readily than wild type betaB1. Similar effects were obtained for either monomers or oligomers. Flexibility of the N-terminal extension in deamidated betaB1 was significantly reduced compared to the wild type protein. Truncation of the extensions further increased the rate of heat-induced precipitation of deamidated betaB1. The presence of the molecular chaperone, alphaA-crystallin, prevented precipitation of modified betaB1s. More alphaA was needed to chaperone the truncated and deamidated betaB1 than deamidated betaB1 or truncated betaB1. CONCLUSIONS: Deamidation and truncation of betaB1 led to destabilization of the protein and decreased stability to heat. Decreased stability of lens crystallins may contribute to their insolubilization and cataract formation. (+info)
A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q.
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Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the beta-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [theta] 0) and D22S1154 (Z=1.39 at theta=0), which closely flank the genes for betaB1-crystallin (CRYBB1) and betaA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G-->T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human betaB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans. (+info)
BetaB1-crystallin: identification of a candidate ciliary body uveitis antigen.
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PURPOSE: Perineuclear anti-neutrophil cytoplasmic antibody (pANCA), a marker antibody present in 12% of patients with anterior uveitis, recognizes cytoplasmic antigens in the nonpigmented ciliary body epithelium, a probable site of immunologic reactivity in this inflammatory disease. In this study, a recombinantly isolated pANCA monoclonal antibody was used to identify the corresponding antigenic target(s) in the ciliary body. METHODS: Proteins from microdissected eye bank ocular ciliary body tissue were used to identify the corresponding ANCA antigen. Parallel two-dimensional protein gels were used for simultaneous identification of candidate antigenic protein spots by Western blot analysis and as a source of material for proteomic analysis. Multiple independent methods including Western blot analysis, confocal microscopy, and RT-PCR were used to provide additional characterization of the candidate protein. RESULTS: Proteomic analysis suggested that beta B1 (betaB1)-crystallin is the primary ciliary body antigen. The presence of betaB1-crystallin in the human ciliary body was confirmed by Western blot with a betaB1 specific anti-peptide antibody. Confocal microscopy revealed colocalization of the antigenic reactivity of both anti-betaB1 antibody and monoclonal pANCA. RT-PCR confirmed the presence of betaB1-crystallin RNA in the ciliary body tissues. CONCLUSIONS: This study identified betaB1-crystallin as a new cytoplasmic ciliary body antigenic target of a marker autoantibody associated with uveitis. This characterization of betaB1-crystallin outside the lens raises questions about its extralenticular expression, intracellular role, and potential target of inflammation in uveitis. (+info)
Improving the performance of DomainParser for structural domain partition using neural network.
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Structural domains are considered as the basic units of protein folding, evolution, function and design. Automatic decomposition of protein structures into structural domains, though after many years of investigation, remains a challenging and unsolved problem. Manual inspection still plays a key role in domain decomposition of a protein structure. We have previously developed a computer program, DomainParser, using network flow algorithms. The algorithm partitions a protein structure into domains accurately when the number of domains to be partitioned is known. However the performance drops when this number is unclear (the overall performance is 74.5% over a set of 1317 protein chains). Through utilization of various types of structural information including hydrophobic moment profile, we have developed an effective method for assessing the most probable number of domains a structure may have. The core of this method is a neural network, which is trained to discriminate correctly partitioned domains from incorrectly partitioned domains. When compared with the manual decomposition results given in the SCOP database, our new algorithm achieves higher decomposition accuracy (81.9%) on the same data set. (+info)