Abstract
Purpose: :
To investigate the structural and functional consequences of the D140N mutation in alphaB-crystallin in comparison with other cataract inducing mutations.
Methods: :
The expression construct (pET23) for alphaB-crystallin from Zebrafish was constructed by a PCR, sequencing and subcloning strategy. This vector system allows the inducible expression of the cloned cDNAs in Escherishia coli by the addition of IPTG. Protein expression was monitored by SDS-PAGE. Like other alphaB-crystallin proteins expressed using this system the protein remained soluble and was purified to homogeneity by anion exchange chromatography. The functional integrity of the recombinantly expressed proteins was analysed by a series of structural assays, including CD, bisANS binding, size exclusion chromatography, in vitro chaperone assays, desmin filament binding assays and electron microscopy using negatively stained samples. The activity was compared to wild type alphaB-crystallin and compared to the effects of the R120G and 450delA mutations.
Results: :
The D140N mutation in alphaB-crystallin does not have as severe effects upon the oligomerisation and stability as the 450delA mutations, for instance. The effect upon in vitro client proteins (insulin and citrate synthase) indicated significant differences and these were compared to physiologically relevant clients (e.g. desmin).
Conclusions: :
The observed phenotypic differences for the various alphaB-crystallin mutations and particular tissue-specific effects observed for some (e.g. D140N and 450delA alphaB-crystallin) require further detail investigation to correlate with the available assay regimes.
Keywords: crystallins • chaperones • cytoskeleton