Purchase this article with an account.
H Zhang, N Cuenca, J Church-Kopish, T Ivanova, JM Frederick, PR MacLeish, W Baehr; Monoclonal Antibody 7G6 Recognizes Primate and Bovine Cone Arrestin . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1414.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Purpose:Monoclonal antibody (mAb) 7G6 was produced previously (by PRM) against crude monkey retinal homogenate. Our aim was to identify the antigen recognized by 7G6 that is expressed specifically in primate and bovine cones. Methods:Cryosections of human and bovine retina were analyzed by confocal immunocytochemistry. SDS-PAGE and immunoblots were performed using extracts from whole retina versus macula. Peptide sequences were determined by Edman degradation and LC-MS/MS. Results:In human and bovine retinas, mAb 7G6 labeled cone photoreceptors strongly and specifically, and particularly cone outer segments (COS). Preliminary experiments using dark- vs. light-adapted bovine retinas suggest that COS immunolabeling is independent of light conditions. Western blots of a crude human macular extract indicated that 7G6 labeled a protein of mobility ∼ 47 kD. Further studies showed that the antigen recognized by 7G6 could be extracted by hypotonic buffer (20 mM Tris, 1 mM EDTA, pH 8.0) suggesting that it is not an integral membrane protein. To identify the antigen, hypotonic human macular extract was incubated with mAb 7G6 and the antigen was precipitated by protein G-coupled agarose. The polypeptide corresponding to the antigen was excised from SDS gels, subjected to tryptic digestion and tryptic peptides were separated by HPLC. Edman degradation of one peptide revealed a sequence that matched human cone arrestin. An additional 20 peptide sequences acquired by LC-MS/MS matched human cone arrestin. Immunoblots with recombinant human cone arrestin (gift of Dr. Cheryl Craft, USC) and probed using mAb 7G6 were positive, verifying the antigen's identity. Human, mouse, and zebrafish cone arrestin gene contigs were retrieved (Genbank) and the gene structures derived in silico. Consisting of 17 exons, the arrestin genes reveal high conservation of exon/intron arrangement. The C-terminal regions of human and bovine cone arrestins are conserved, but deviate in other species. Conclusion:Identification of the 7G6 antigen as cone arrestin (X-arrestin) is consistent with hypotonic extraction of the antigen and with specific labeling of human and bovine cones. The antigenic determinant of 7G6 is likely the C-terminal region of cone arrestin that is conserved in bovine and primate. The human X-arrestin gene, located on chromosome Xq13.2-21.1, is a candidate gene for X-linked cone dystrophies.
This PDF is available to Subscribers Only