EFFECT OF MUTATIONS OF MURINE LENS ALPHA-B-CRYSTALLIN ON TRANSFECTED NEURAL CELL VIABILITY AND CELLULAR TRANSLOCATION IN RESPONSE TO STRESS

We examined the influence of over-expressed native and mutant murine l ens alpha B crystallin on the response of a murine neural cell line to heat and ionic strength shock. Native and mutant (F27R and KK174/175L L) murine alpha B crystallin amplicons were subcloned into a Lac-Switc h IPTG-inducible RSV promoter eukaryotic vector, and transfected into N1E-115 cells using lipofectin, Expression was induced maximally 8 h a fter addition of IPTG (optimal final concentration 1 mM) to the medium , Cells grew normally after transfection with native and mutant murine alpha B crystallin. We demonstrated expression of the protein using s pecific anti-alpha crystallin antibodies. Viability under severe heat and ionic strength stress increased when cells had been transfected wi th native alpha B crystallin, but not with mutants F27R or KK174/175LL , Heat shock caused translocation of both native and mutant alpha B cr ystallins into the central region of the cells, These results show tha t mutations in alpha B crystallin that effect its chaperone-like activ ity may also influence viability of N1E-115 neural cells under stress, while not influencing the distribution of the protein within the cell , (C) 1998 Federation of European Biochemical Societies.