Interaction sites in human alphaB crystallin mediate multiple functions

Abstract

Abstract: : Introduction: αB crystallin, the archetype of small heat shock proteins (sHSPs) has three known functions that include subunit assembly, filament organization and maintaining the solubility of proteins through its chaperone–like function. The sites for filament organization and molecular chaperone activity remain to be identified. Methods: Sequential peptides corresponding to the primary sequence of human αB crystallin were immobilized on a protein pin array in a 96–well microtiter plate format. The protein pin array was used to identify sequences that interacted with filamentous proteins including actin, desmin, vimentin, glial–fibrillary acidic protein. Similarly, sequences were identified that interacted with endogenous ligand proteins that included ß crystallin, γ crystallin and exogenous chaperone target proteins that included alcohol dehydrogenase, α lactalbumin, citrate synthase and insulin. The binding of both filament and chaperone ligand proteins to the peptides of the human αB crystallin protein pin array was measured at 22°C and at 45°C to demonstrate the effect of temperature on the interactions occurring at ambient temperature (22°C) as well as interactions induced by stress conditions (45°C) that result in the unfolding of proteins. Results: The sequences ₄₁STSLSPFYLRPPSFLRAPSWFDTGL₆₅ belonging to the N–terminus, ₇₃DRFSVNLDVK₈₂, ₁₃₁LTITSSLSSDGVLTVN₁₄₆ and ₁₁₃FISRE₁₁₇ belonging to the "α–crystallin core domain" and ₁₅₅PERTIPITREE₁₆₅, belonging to the C–terminus extension were identified as sites involved in the interaction of human αB crystallin with its various ligands including crystallins, filaments and chaperone targets. The interaction between filament proteins and the αB crystallin protein pin array peptides was stronger at 22°C than at 45°C. In contrast, the interaction between chaperone target proteins the αB crystallin protein pin array peptides was stronger at 45°C than at 22°C. Conclusion: Filament proteins and chaperone ligand proteins bound the same four sequences that were earlier identified as subunit–subunit interaction sites (Ghosh & Clark, ARVO 2003). These results suggest that the five interactive sites of αB crystallin may have common functions in complex assembly, filament organization and molecular chaperone activity. Supported by NEI grant EY04542.

Keywords: crystallins • chaperones • cataract