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Hanane Koteiche, Sanjay Mishra, Hassane Mchaourab; Danio rerio αB1-crystallin encodes for an activated chaperone. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5740.
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© ARVO (1962-2015); The Authors (2016-present)
The chaperone activity of α-crystallin, defined by substrate affinity and binding capacity, has been evolutionary optimized to the stress challenges of the species or tissue wherein it is expressed. The zebrafish lens expresses two αB-crystallin isoforms, αB1 and αB2. The latter is expressed both within and outside the lens mimicking the expression pattern of the human ortholog. Using T4 lysozyme destabilized mutants, we compared the binding affinity of zebrafish α-crystallins to their human counterpart.
Zebrafish and human α-crystallins were expressed in E. coli. T4 lysozyme was site specifically labeled with the fluorescent probe bimane. Binding isotherms were obtained by monitoring the anisotropy of bimane-labeled T4L incubated with increasing concentration of α-crystallins.
Binding isotherms were fit to a single mode binding model yielding an apparent KD and the number of bound T4L per α- subunit. The data demonstrates that human and zebrafish αA-crystallin affinities to T4L are within a factor of four. In contrast, there is a 2 order of magnitude increase in the apparent affinity of zebrafish αB1 relative to the human αB-crystallin. In fact, the affinity of the zebrafish ortholog to T4 lysozyme destabilized mutants approaches that of phosphorylated human αB-crystallin.
We propose that zebrafish αB1 sequence encodes for a permanently activated chaperone. Consistent with this hypothesis, αB1 does not have two of the serine phosphorylation sites found in the human ortholog. We are currently using homolog scanning mutagenesis to identify sequence and structural elements associated with activation of the zebrafish ortholog.
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