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Y Kim, TR Shearer, LL David, HP Bachinger, DM Kapfer, KJ Lampi; Modified betaB1-crystallins Have Decreased Stability and Altered Interaction With alphaA-crystallin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4652.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: ßB1-crystallin is deamidated and truncated during aging of the human lens. The purpose of this study was to determine how these post-translational modifications affect stability of ßB1 crystallin as measured by urea or heat denaturation. Methods: Proteins tested were recombinant wild-type ßB1 (WT), deamidated ßB1 (Q204E), truncated WT (trWT), and truncated ßB1Q204E (trQ204E). WT and Q204E were proteolyzed by recombinant calpain II to generate proteins missing 47 residues from the N- and 5 from the C-termini of ßB1. Denaturation/renaturation of proteins in urea was measured by spectrofluorimetry and circular dichroism (CD). To test heat stability, proteins were incubated at 55C, and turbidity was followed at 405 nm. To measure chaperone activity, the heat stability assay was also performed in the presence of recombinant αA-crystallin. Results: Modified ßB1 crystallins were less stable than WT during either urea or heat denaturation. Both fluorescence and CD results showed that Q204E underwent 50% unfolding at 4.75 M compared to WT 50% unfolding at 5.60 M. Q204E showed a 2-step unfolding transition during urea-induced denaturation, while WT showed a 1-step transition. CD measurements revealed that Q204E did not completely refold to native form during renaturation, while WT refolded to its native form. TrWT and trQ204E showed similar urea-induced denaturation patterns as WT and Q204E, respectively. During heating, the rate of precipitation was greatest for trQ204E and then Q204E when compared to either WT or trWT. Although trWT did not show a significant difference in heat-induced precipitation compared to WT, twice as much αA was required to prevent turbidity of trWT compared to WT. Both Q204E and trQ204E required 3-4 molar excess of αA to prevent precipitation. Conclusion: Deamidation of glutamine in a hydrophobic region of ßB1 and/or truncations of the C- & N-terminal extensions caused instability and changed the ability of αA-crystallin to chaperone. These changes may increase susceptibility of lenses to cataract.
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