CHARACTERIZATION OF A NOVEL FAMILY OF CILIARY BODY GLYCOPROTEINS

Purpose. To isolate and characterize ciliary body epithelial antigens reactive with a monoclonal antibody, 2B4.14.1. Methods. A mouse monocl onal antibody generated against human corneal endothelium, 2B4.14.1 re acts with nonpigmented epithelium of human and guinea pig ciliary bodi es. The ciliary body proteins reactive with 2B4.14.1 were identified b y Western blotting and were partially purified by affinity chromatogra phy with 2B4.14.1 coupled to a solid support matrix. Carbohydrate comp onents of the antigenic molecules were analyzed by lectin chromatograp hy and by digestion with the enzymes N-glycosidase F and endoglycosida se F. The cellular and subcellular distribution of the antigens was ex amined by immunoperoxidase staining and by immunoelectron microscopy o f ultracryotome sections of ciliary body. Results. 2B4.14.1 reacted wi th families of guinea pig and human ciliary body glycoproteins with es timated molecular weights ranging from 250 to 325 kD. In Western blots of samples reduced before electrophoresis, the high molecular weight bands were replaced by weakly reactive bands at 115 to 130 and 210 kD, indicating that the 2B4.14.1 ligands have disulfide bonds. 2B4.14.1 l igands from both guinea pig and human ciliary body were bound by immob ilized lectins, including concanavalin A, Datura stramonium lectin, an d Lens culinaris hemagglutinin, which recognize components of N-linked oligosaccharides. Guinea pig ciliary body antigens digested with N-gl ycosidase F and endoglycosidase F failed to react with 2B4.14.1 in Wes tern blots, confirming the presence of N-linked oligosaccharide chains and indicating that they form an integral part of the 2B4.14.1-reacti ve antigenic site. Molecular weight shifts of glycosidase-digested ant igens were consistent with the presence of two to four N-linked oligos accharide units. In immunoperoxidase-stained sections of guinea pig an d human ciliary body, 2B4.14.1 reacted primarily with nonpigmented epi thelial cells. Staining of guinea pig epithelial cells was fairly unif orm; staining of human epithelial cells was concentrated on the basal surface. By immunoelectron microscopy, a majority of the 2B4.14.1 anti genic reactivity was localized immediately external to the nonpigmente d epithelial cell plasma membrane. Conclusions. 2B4.14.1 reacts with a novel family of high molecular weight glycoproteins associated with t he nonpigmented epithelial cell surface in guinea pig and human ciliar y body.