Visualization of in situ intracellular aggregation of two cataract-associated human gamma-crystallin mutants: lose a tail, lose transparency.
OBJECTIVE
To understand the molecular features underlying autosomal dominant congenital cataracts caused by the deletion mutations W156X in human gammaD-crystallin and W157X in human gammaC-crystallin.


METHODS
Normal and mutant cDNAs (with the enhanced green fluorescent protein [EGFP] tag in the front) were cloned into the pEGFP-C1 vector, transfected into various cell lines, and observed under a confocal microscope for EGFP fluorescence. Normal and W156X gammaD cDNAs were also cloned into the pET21a(+) vector, and the recombinant proteins were overexpressed in the BL-21(DE3)pLysS strain of Escherichia coli, purified, and isolated. The conformational features, structural stability, and solubility in aqueous solution of the mutant protein were compared with those of the wild type using spectroscopic methods. Comparative molecular modeling was performed to provide additional structural information.


RESULTS
Transfection of the EGFP-tagged mutant cDNAs into several cell lines led to the visualization of aggregates, whereas that of wild-type cDNAs did not. Turning to the properties of the expressed proteins, the mutant molecules show remarkable reduction in solubility. They also seem to have a greater degree of surface hydrophobicity than the wild-type molecules, most likely accounting for self-aggregation. Molecular modeling studies support these features.


CONCLUSIONS
The deletion of C-terminal 18 residues of human gammaC- and gammaD-crystallins exposes the side chains of several hydrophobic residues in the sequence to the solvent, causing the molecule to self-aggregate. This feature appears to be reflected in situ on the introduction of the mutants in human lens epithelial cells.
Talla
Venu
V
Brien Holden Research Center, Hyderabad Eye Research Foundation, LV Prasad Eye Institute, Hyderabad, India.
Srinivasan
Narayanaswamy
N
Balasubramanian
Dorairajan
D
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells / Q000378:metabolism; D002386:Cataract / Q000151:congenital / Q000235:genetics*; D018929:Cell Culture Techniques; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003459:Crystallins / Q000737:chemistry / Q000235:genetics*; D004847:Epithelial Cells / Q000378:metabolism; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology; D018613:Microscopy, Confocal; D008875:Middle Aged; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins; D012165:Retinal Ganglion Cells / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D038222:gamma-Crystallins / Q000737:chemistry / Q000235:genetics*
C504903:CRYGD protein, human; C522806:Crygc protein, human; D003459:Crystallins; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D038222:gamma-Crystallins; D049452:Green Fluorescent Proteins