Detection of protein-protein interactions among lens crystallins in a mammalian two-hybrid system assay

Abstract

α-Crystallin consists of two subunits, αA and αB, and each can form an oligomer by itself or with the other. The aggregation arises from interdomain interactions. However, it is not known whether such interactions also exist among α-, β-, and γ-crystallins. This heterogeneous crystallin interaction is far weaker than the homogeneous crystallin interaction and is difficult to detect by conventional spectroscopic measurements. We used a mammalian two-hybrid system in this study. The major crystallin components, αA-, αB-, βB2-, and γC-crystallin genes, were subcloned into the DNA binding domain and transcription activation domain vectors of the two-hybrid system, and they were cotransfected along with a chloramphenicol acetyltransferase (CAT) reporter vector into HeLa cells. Chloramphenicol acetyltransferase activity indicated that there were interactions between αA- (or αB-) and βB2- or γC-crystallins but with an intensity of one-third that of αA-βB interactions. Hsp27, a member of the 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. These results show that the twohybrid system can detect interactions among various crystallins and may be used in mapping interaction domains.