Abstract
Purpose: :
Alpha Crystallin is a major lens protein which consists of two subunits, αA and αB. Mutation of human αA-crystallin is known to cause congenital cataract. The present study was aimed to study in situ expressed seven different human αA-crystallin mutants and the effects of their interaction with native αA- and αB-crystallins in situ in living mammalian cells on protein aggregation using laser scanning confocal microscopy (LSM) and fluorescence resonance energy transfer (FRET).
Methods: :
QuickChange site-directed mutagenesis kit was used to generate mutants. HeLa cells were grown in 35 mm glass-bottom dishes and 80-90% confluent cells were transfected with lipofectamine and 2 microgram plasmid DNA of interest. YFP-tagged αA-wt or mutants transfected individually or co-transfected with CFP-tagged αAwt or αBwt constructs. After 48 hours transfection, a Zeiss Meta LSM 510 was used to visualize CFP and YFP fluorescence. Cells with significant level of protein aggregates were counted from aseveral fields of 50 cells. To examine whether protein-protein interaction is affected when each of the mutant interacts with αAwt and αBwt, FRET acceptor photobleaching method was employed. A series of pre-bleaching and post-bleaching donor and acceptor signal collection protocol were automated for the collection of FRET data
Results: :
Cells transfected with YFP-tagged αA-wt, R12C, R21L, R21W, R49C, R54C, R116C and R116H showed 2, 37,10, 26,32, 22, 52, 32 % of cells, respectively, had significant level of protein aggregates. After co-expression with αAwt, these mutants showed further increase in protein aggregation probably due to co-aggragatiomn with αAwt. Co-expression with αBwt, on other hand, had significantly decreased the number of cells carrying aggregates. However, for all the mutants the level of aggregation was still higher than in control αBwt- αAwt. According to in situ FRET data, interaction of all the mutants with αAwt and αBwt was significantly weaker than the control interactions of αAwt - αAwt and αBwt - αAwt
Conclusions: :
This is the first report on how cataract causing mutants of human αA-crystallin behaves in mammalian cells. It appears that the inability to interact adequately with native α-crystallin enhances protein aggregation. Although the structural and functional consequences of the mutants vastley varied (ARVO Abstract 2010) all the mutants showed higher level of protein aggregation.
Keywords: chaperones • cataract • protein structure/function