Identification and light-dependent translocation of a cone-specific antigen, cone arrestin, recognized by monoclonal antibody 7G6.
ABSTRACT To elucidate the antigen recognized by monoclonal antibody (mAb) 7G6, a widely used cone-specific marker.
7G6 immunocytochemistry was performed on sections of human, primate, and bovine retina. The antigen was immunoprecipitated from human retinal lysates and purified with protein G. Edman degradation and liquid chromatography of tryptic peptides combined with tandem mass spectrometry (LC-MS/MS) identified the antigen.
Sequencing of peptides derived from the immunoprecipitated 7G6 antigen identified it as cone arrestin. The identity was confirmed by Western blot analysis with recombinant human cone arrestin and competition with the antibody in immunocytochemistry. Subcellular localization of cone arrestin in dark-adapted and bleached bovine retinas showed that cone arrestin accumulated in cone outer segments of light-adapted retina but was more concentrated in the inner segments of dark-adapted retina. By expression of truncated human cone arrestin mutants systematically deleting areas divergent from bovine and primate cone arrestins, the epitope of 7G6 was identified as a divergent loop exposed at the surface within the N-domain of cone arrestin.
Several independent methods established that the 7G6 antigen is cone arrestin. The 7G6 epitope is contained in a divergent loop, the sequence of which is conserved in bovine and primates but not other vertebrate species consistent with the specificity of the antibody. The light-dependent translocation of cone arrestin suggests a role in light-dark adaptation of cones. Because of the location of its gene on the X-chromosome, cone arrestin is a candidate gene for X-linked cone dystrophies.