Biochemical and histochemical studies of the photoreceptor cells and the interphotoreceptor matrix of the bovine retina

Much interest has been shown recently in the components of the surfaces of the rod and cone photoreceptor cells in the mammalian retina and in the constituents of the interphotoreceptor matrix (IPM) which interact with the surfaces of the photoreceptor cells. Such molecules are likely to assist in- maintaining the structures of the photoreceptor cells and /or to have functional roles in the continual interactions between the pigmented epithelium cell layer and the neural retina. Abnormalities in the composition of such molecules may lead to disease states in which such structures and functions have broken down.One approach to identifying constituents of the surfaces of cells is to use cell -specific probes in the form of monoclonal antibodies which can be raised against cell- specific surface molecules, and may be used to identify these molecules and to allow comparisons to be made between normal and diseased tissue.Mice immunised with partially purified surface molecules of photoreceptor outer segments or with a synthetic peptide corresponding to the N- terminus of a cone -specific rhodopsin -like protein, gave rise to the production of monoclonal antibodies with only general immunoreactivity against the retina.Immunisation with interphotor-eceptor matrix or crude photoreceptor outer segment preparations resulted in the production of hybridomas which secreted monoclonal antibodies 1001.A1 and .1001.A3.100l.A1 binds to the interstitial retinol-binding protein (IRBP), associated with the rod photoreceptor cells and the IPM, as demonstrated by immunohistochemistry and by Western blotting and dot blotting of IPM and purified IRBP.1001.A3 binds a soluble high molecular weight chondroitin sulphate proteoglycan. Immunohistochemistry indicated the antigen to be present in the form of distinct sheath -like structures surrounding the photoreceptor cells. Gel filtration chromatography both in native conditions and in the presence of guanidinium chloride showed the antigen to have an apparent molecular mass of greater than 2000kDaltons and indicated the antigen was not a loosely associated aggregate of smaller components. Binding of .1007.A3 to fixed tissue sections of the bovine retina was completely abolished by their prior treatment with either chondroitinase ABC, chondroitinase AC, hyaluronidase (testicular) or trypsin. Prior treatment of tissue sections with either heparinase or neuraminidase had no effect on binding. Treatment of tissue sections with hyaluronidase (Streptomyces) had no effect on the ability of 1001.A3 to bind, but the structure of the antigen was altered. The sheath -like structure surrounding the photoreceptors was broken down and immunoreactivity was seen in the same area of the 1PM, adjacent to the photoreceptors but with no defined structure. In conclusion, the antigen is a chondroitin sulphate molecule which is associated with hyaluronic acid molecules and which together form a defined sheath -like structure surrounding the photoreceptor cells