Abstract
Purpose :
Immunohistochemical localization of CNGB1 (cGMP-gated cation channel β-subunit) with a C-terminal antibody revealed a strong signal in the inner plexiform layer of the retina. The purpose of this study was to identify and characterize the inner retina protein(s) that are recognized by this antibody.
Methods :
Peptide affinity-purified polyclonal antibodies were generated against amino acids 1-15 (N-term Ab), 1253-1266 (C-term Ab) of mouse CNGB1. The N-terminal sequence is highly conserved across species and the C-terminal sequence is unique to mouse CNGB1. Antibodies against GABA, Islet-1, and glycine were used in double labeling studies. Immunohistochemistry (IHC) was performed on frozen sections using standard protocols for fixation, cryoprotection and labeling. Western blotting was performed using standard protocols for NIR secondary antibody detection. Immunoprecipitation was done in RIPA buffer using tosyl-activated sepharose magnetic beads or protein A sepharose. For protein identification, SDS gel bands were reduced/alkylated and digested overnight with trypsin. Extracted samples were were analyzed by reverse phase chromatography. Protein Pilot vs 4.5 (SCIEX, Foster City, CA) was used for protein identifications using the Mouse UniProt database.
Results :
IHC staining with the N-term antibody of human, monkey, and mouse retinas revealed strong labeling that was limited to the rod outer segments (ROS). Staining with the C-terminal Ab showed strong labeling in mouse ROS, but also in the inner plexiform layer and in a subset of somatic membranes in the inner nuclear layer (INL). Colocalization analysis indicated that the labeled cells in the INL included a subset of GABA immunoreactive amacrine cells. Western analysis identified three distinct protein bands in the size range 34-54 kDa. Immunoprecipitation using magnetic beads or protein A sepharose revealed 6 bands in the identified size range. Sequence analysis showed that multiple proteins were contained within each band and none of the proteins identified are closely related to the Cngb1 locus.
Conclusions :
A proline, glutamate rich peptide of mouse CNGB1 shares antigenic properties with protein(s) in the inner retina. The relationship of these proteins to CNGB1 in the ROS could not be determined. Further characterization with additional antibodies will be needed to assess the importance of this finding.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.