May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Effect of Crystallins on Human Caspase 6
Author Affiliations & Notes
  • K. Sharma
    University of Missouri, Columbia, Missouri
    Ophthalmology and Biochemistry,
  • S. Yellamaraju
    University of Missouri, Columbia, Missouri
    Ophthalmology,
  • G. Rao
    University of Missouri, Columbia, Missouri
    Ophthalmology,
  • Footnotes
    Commercial Relationships  K. Sharma, None; S. Yellamaraju, None; G. Rao, None.
  • Footnotes
    Support  NIH grants EY09855, EY 14795 and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4100. doi:
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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)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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.

Conclusions: : γ-Crystallin has human caspase-6 inhibitory activity and may be involved in regulating caspase-6 activity in lens fiber cells.

Keywords: proteolysis • crystallins • protein purification and characterization 
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