Abstract
Purpose: :
The goal of this study was to identify, clone, characterize and localize an unknown membrane glycoprotein in bovine rod outer segment preparations as a first step in elucidating its function in photoreceptor cell biology.
Methods: :
Two monoclonal antibodies, Mab 3C4 and BD8, were used to label western blots of bovine outer segment proteins separated on SDS gels and bovine retina cryosections for immunofluorescence microscopy. A 70 kDa glycoprotein was purified from bovine rod outer segments by immunoaffinity chromatography and subjected to trypsin digestion. The tryptic peptides were analyzed by mass spectrometry for protein identification. Bovine embigin was cloned from bovine retinal cDNA by RT-PCR and 3’RACE.
Results: :
Monoclonal antibodies 3C4 and BD8 labelled a 70 kDa glycoprotein in bovine rod outer segment preparations by western blotting. The protein was identified by mass spectrometry as embigin, a member of the immunoglobulin superfamily that has not been previously reported to be expressed in the retina. Bovine embigin was cloned and expressed in HEK293T cells to confirm the specificity of the antibodies. Both antibodies detected embigin in different states of glycosylation after digestion with deglycosylation enzymes. Immunofluorescence microscopy localized embigin to rod photoreceptor outer and inner segments as well as the outer nuclear layer. Biochemical studies further localized embigin to the rod outer segment plasma membrane.
Keywords: photoreceptors • glycoconjugates/glycoproteins • protein purification and characterization