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K. Sharma, S. Yellamaraju, G. Rao; Effect of Crystallins on Human Caspase 6. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4100.
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© ARVO (1962-2015); The Authors (2016-present)
Caspases play an important role in lens development. Previous studies have shown that both αA- and αB-crystallins have caspase inhibitory activity. This study was designed to investigate the role of γ-crystallins in controling caspase 6 activity.
Bovine lens crystallins were isolated by gel filtration chromatography and γ-crystallin was fractionated by ion-exchange method. Recombinant human caspase 6 (Calbiochem) was assayed with commercially available substrate in presence and absence γ-crystallins. The binding of caspase-6 to γ-crystallin was assessed by pull-down assay and western blot.
Recombinant human caspase 6 was inhibited by bovine γ-crystallin (50% with100µg) whereas bovine αA-, αB-, βL- or recombinant human αA- and αB-crystallins were found to show <10% inhibition of the activity. γ-Crystallin fractions showed inhibition of caspase 6 activity ranging between 20-40%. Incubation of recombinant caspase 6 with γ-crystallin did not show any cleavage of the protein by the enzyme confirming that the inhibition observed was not due to substrate competition. Kinetics studies revealed that inhibition of caspase 6 by γ-crystallin occurred by uncompetitive mechanism with a decrease in both Km and Vmax. Immunoprecipitation studies using γ-crystallin antibody immobilized on protein-G support showed co-precipitation of caspase 6 with γ-crystallin.
γ-Crystallin has human caspase-6 inhibitory activity and may be involved in regulating caspase-6 activity in lens fiber cells.
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