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Increase in surface hydrophobicity of the cataract-associated P23T mutant of human gammaD-crystallin is responsible for its dramatically lower, retrograde solubility.

ABSTRACT: The cataract-associated Pro23 to Thr (P23T) mutation in human gammaD-crystallin (HGD) has a variety of phenotypes and is geographically widespread. Therefore, there is considerable interest in understanding the molecular basis of cataract formation due to this mutation. We showed earlier [Pande, A., et al. (2005) Biochemistry 44, 2491-2500] that the probable basis of opacity in this case is the severely compromised, retrograde solubility and aggregation of P23T relative to HGD. The dramatic solubility change occurs even as the structure of the mutant protein remains essentially unchanged in vitro. We proposed that the retrograde solubility and aggregation of P23T were mediated by net hydrophobic, protein-protein interactions. On the basis of these initial findings for P23T and related mutants, and the subsequent finding that they show atypical phase behavior [McManus, J. J., et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 16856-16861], we concluded that the protein clusters formed in solutions of the mutant proteins were held together by net hydrophobic, anisotropic interactions. Here we show, using chemical probes, that the surface hydrophobicities of these mutants are inversely related to their solubility. Furthermore, by probing the isolated N-terminal domains of HGD and P23T directly, we find that the increase in the surface hydrophobicity of P23T is localized in the N-terminal domain. Modeling studies suggest the presence of sticky patches on the surface of the N-terminal domain that could be engaged in the formation of protein clusters via hydrophobic protein-protein interactions. This work thus provides direct evidence of the dominant role played by net hydrophobic and anisotropic protein-protein interactions in the aggregation of P23T.

SUBMITTER: Pande A

PROVIDER: S-EPMC2913551 | biostudies-other | 2010 Jul

REPOSITORIES: biostudies-other

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Increase in surface hydrophobicity of the cataract-associated P23T mutant of human gammaD-crystallin is responsible for its dramatically lower, retrograde solubility.

Pande Ajay A Ghosh Kalyan S KS Banerjee Priya R PR Pande Jayanti J

Biochemistry 20100701 29

The cataract-associated Pro23 to Thr (P23T) mutation in human gammaD-crystallin (HGD) has a variety of phenotypes and is geographically widespread. Therefore, there is considerable interest in understanding the molecular basis of cataract formation due to this mutation. We showed earlier [Pande, A., et al. (2005) Biochemistry 44, 2491-2500] that the probable basis of opacity in this case is the severely compromised, retrograde solubility and aggregation of P23T relative to HGD. The dramatic solu ...[more]

PMID: 20553008

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Project description:Cataract is the most common cause of visual loss in humans. A spontaneously occurred, autosomal dominant mouse mutant Secc, which displayed combined features of small eye, cataract and closed eyelid was discovered in our laboratory. In this study, we identified the mutation and characterized the cataract phenotype of this novel Secc mutant. The Secc mutant mice have eyelids that remain half-closed throughout their life. The mutant lens has a significant reduction in size and with opaque spots clustered in the centre. Histological analysis showed that in the core region of the mutant lens, the fiber cells were disorganized and clefts and vacuoles were observed. The cataract phenotype was evident from new born stage. We identified the Secc mutation by linkage analysis using whole genome microsatellite markers and SNP markers. The Secc locus was mapped at chromosome 1 flanked by SNPs rs3158129 and rs13475900. Based on the chromosomal position, the candidate cataract locus γ-crystallin gene cluster (Cryg) was investigated by sequencing. A single base deletion (299delG) in exon 3 of Cryga which led to a frame-shift of amino acid sequence from position 91 was identified. As a result of this mutation, the sequences of the 3rd and 4th Greek-key motifs of the γA-crystallin are replaced with an unrelated C-terminal peptide of 75 residues long. Coincidentally, the point mutation generated a HindIII restriction site, allowing the identification of the CrygaSecc mutant allele by RFLP. Western blot analysis of 3-week old lenses showed that the expression of γ-crystallins was reduced in the CrygaSecc mutant. Furthermore, in cell transfection assays using CrygaSecc mutant cDNA expression constructs in 293T, COS-7 and human lens epithelial B3 cell lines, the mutant γA-crystallins were enriched in the insoluble fractions and appeared as insoluble aggregates in the transfected cells. In conclusion, we have demonstrated that the Secc mutation leads to the generation of CrygaSecc proteins with reduced solubility and prone to form aggregates within lens cells. Accumulation of mutant proteins in the lens fibers would lead to cataract formation in the Secc mutant.

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Increase in surface hydrophobicity of the cataract-associated P23T mutant of human gammaD-crystallin is responsible for its dramatically lower, retrograde solubility.

Publications

Increase in surface hydrophobicity of the cataract-associated P23T mutant of human gammaD-crystallin is responsible for its dramatically lower, retrograde solubility.

Similar Datasets

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