Protein–protein interactions involving congenital cataract T5P γC-crystallin mutant: A confocal fluorescence microscopy study

The human lens crystallin gene CRYGC T5P is associated with Coppock-like cataract and has a phenotype of a dust-like opacity of the fetal lens nucleus and deep cortical region. Previous in vitro mutation studies indicate that the protein has changed conformation, solubility, and stability, which may make it susceptible to aggregation, as seen in cataractous lens and cell culture expression. To investigate the mechanisms leading to these events, we studied protein–protein interactions using confocal fluorescence resonance energy transfer (FRET) microscopy. The method detects protein–protein interactions in the natural environment of living cells. Crystallin genes (CRYGC T5P, CRYGC, and CRYAA) were fused to either the green fluorescence protein (GFP) or red fluorescence protein (DsRED or RFP) vector. Each of the following GFP–RFP (donor–acceptor) plasmid pairs was cotransfected into HeLa cells: γC–γC, γC–γCT5P, γCT5P–γCT5P, αA–γC, and αA–γCT5P. After culture, confocal fluorescence cell images were taken. Protein–protein interactions in the form of net FRET were evaluated. The confocal fluorescence images show that cells expressing T5P γC-crystallin contain many protein aggregates, but cells co-expressing with either γC- or αA-crystallin reduce the aggregation considerably. FRET determination indicates that γCT5P–γCT5P shows less protein–protein interaction than either γC–γC or γC–γCT5P. Cotransfection with αA-crystallin (αA–γC or αA–T5PγC) increases nFRET compared with γC–γC or γC–T5PγC. Our results demonstrate that T5P γC-crystallin shows more protein aggregates and less protein–protein interaction than WT γC-crystallin. Chaperone αA-crystallin can rescue T5P γC-crystallin from aggregation through increased protein interaction. The formation of congenital cataract may be due to reduced protein–protein interactions and increased aggregation from an insufficient amount of α-crystallin for protection.