Detection of protein-protein interactions among lens crystallins in a mammalian two-hybrid system assay.
alpha-Crystallin consists of two subunits, alphaA and alphaB, 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 alpha-, beta-, and gamma-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, alphaA-, alphaB-, betaB2-, and gammaC-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 alphaA- (or alphaB-) and betaB2- or gammaC-crystallins but with an intensity of one-third that of alphaA-alphaB interactions. Hsp27, a member of the family of the small heat-shock proteins, showed a similar interaction property with alphaB-crystallin. Using the N- and C-terminal domain-truncated mutants, we demonstrated that both domains were important in the alphaA-crystallin self-interaction, but that only the C-terminal domain was important in the alphaB-crystallin self-interaction. These results show that the two-hybrid system can detect interactions among various crystallins and may be used in mapping interaction domains.
Fu
Ling
L
Center for Ophthalmic Research, Brigham and Women's Hospital, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Liang
Jack J-N
JJ
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003459:Crystallins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
D003459:Crystallins; D017931:DNA Primers; D006360:Heat-Shock Proteins; D015500:Chloramphenicol O-Acetyltransferase