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L Fu, JJ Liang; Detection of Protein-Protein Interactions Among Lens Crystallins in a Mammalian Two-Hybrid System Assay . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4798.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study protein-protein interactions among crystallins using a mammalian two-hybrid system. Methods: The major crystallin components, αA-, αB-, ß2-, and γC-crystallin genes, were subcloned into the DNA binding domain and transcription activation domain vectors of the two-hybrid system, and were co-transfected along with a reporter gene chloramphenicol acetyltransferase (CAT). Results: CAT activity indicated that there were homo- and heterogeneous interactions among αA- (or αB-), ßB2- and γC-crystallin but intensities for heterogeneous interactions of αA-ßB2, αA-γC and ßB2-γC, and homogeneous interactions of ßB2-ßB2 and γC-γC were about one-third those of αA-αA, αB-αB and αA-αB interactions. Hsp27, a member of family of the small heat-shock proteins, showed a similar interaction property with αB-crystallin. Using the N- and C-terminal domain truncated mutants, we demonstrated that both domains were important in the αA-crystallin self-interaction but that only the C-terminal domain was important in the αB-crystallin self-interaction. Additionally, we included some cataract mutant genes in the study and found changes in the interaction properties. Conclusion: These results showed that the two-hybrid system could detect interactions, both homo- and heterogeneous systems, among various crystallins.
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