Howell D N, Ross J R, Miller S E, Sanfilippo F P
Department of Pathology, Durham VA Medical Center, NC 27705.
Invest Ophthalmol Vis Sci. 1994 Jul;35(8):3308-18.
To isolate and characterize ciliary body epithelial antigens reactive with a monoclonal antibody, 2B4.14.1.
A mouse monoclonal antibody generated against human corneal endothelium, 2B4.14.1 reacts with nonpigmented epithelium of human and guinea pig ciliary bodies. The ciliary body proteins reactive with 2B4.14.1 were identified by Western blotting and were partially purified by affinity chromatography with 2B4.14.1 coupled to a solid support matrix. Carbohydrate components of the antigenic molecules were analyzed by lectin chromatography and by digestion with the enzymes N-glycosidase F and endoglycosidase F. The cellular and subcellular distribution of the antigens was examined by immunoperoxidase staining and by immunoelectron microscopy of ultracryotome sections of ciliary body.
2B4.14.1 reacted with families of guinea pig and human ciliary body glycoproteins with estimated 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 ligands from both guinea pig and human ciliary body were bound by immobilized lectins, including concanavalin A, Datura stramonium lectin, and Lens culinaris hemagglutinin, which recognize components of N-linked oligosaccharides. Guinea pig ciliary body antigens digested with N-glycosidase F and endoglycosidase F failed to react with 2B4.14.1 in Western blots, confirming the presence of N-linked oligosaccharide chains and indicating that they form an integral part of the 2B4.14.1-reactive antigenic site. Molecular weight shifts of glycosidase-digested antigens were consistent with the presence of two to four N-linked oligosaccharide units. In immunoperoxidase-stained sections of guinea pig and human ciliary body, 2B4.14.1 reacted primarily with nonpigmented epithelial cells. Staining of guinea pig epithelial cells was fairly uniform; staining of human epithelial cells was concentrated on the basal surface. By immunoelectron microscopy, a majority of the 2B4.14.1 antigenic reactivity was localized immediately external to the nonpigmented epithelial cell plasma membrane.
2B4.14.1 reacts with a novel family of high molecular weight glycoproteins associated with the nonpigmented epithelial cell surface in guinea pig and human ciliary body.
分离并鉴定与单克隆抗体2B4.14.1反应的睫状体上皮抗原。
一种针对人角膜内皮产生的小鼠单克隆抗体2B4.14.1，可与人及豚鼠睫状体的无色素上皮反应。通过蛋白质印迹法鉴定与2B4.14.1反应的睫状体蛋白，并用与固相支持基质偶联的2B4.14.1进行亲和层析对其进行部分纯化。通过凝集素层析以及用N -糖苷酶F和内切糖苷酶F消化来分析抗原分子的碳水化合物成分。通过免疫过氧化物酶染色以及对睫状体超薄冷冻切片进行免疫电子显微镜检查来检测抗原的细胞和亚细胞分布。
2B4.14.1与豚鼠和人睫状体糖蛋白家族反应，估计分子量范围为250至325kD。在电泳前还原的样品的蛋白质印迹中，高分子量条带被115至130kD和210kD的弱反应条带取代，表明2B4.14.1配体具有二硫键。来自豚鼠和人睫状体的2B4.14.1配体被固定化凝集素结合，包括伴刀豆球蛋白A、曼陀罗凝集素和菜豆凝集素，这些凝集素识别N -连接寡糖的成分。用N -糖苷酶F和内切糖苷酶F消化的豚鼠睫状体抗原在蛋白质印迹中未能与2B4.14.1反应，证实了N -连接寡糖链的存在，并表明它们构成了2B4.14.1反应性抗原位点的一个组成部分。糖苷酶消化抗原的分子量变化与存在两到四个N -连接寡糖单元一致。在豚鼠和人睫状体的免疫过氧化物酶染色切片中，2B4.14.1主要与无色素上皮细胞反应。豚鼠上皮细胞的染色相当均匀；人上皮细胞的染色集中在基底表面。通过免疫电子显微镜观察，大部分2B4.14.1抗原反应性定位于无色素上皮细胞质膜紧邻的外部。
2B4.14.1与豚鼠和人睫状体中与无色素上皮细胞表面相关的一个新的高分子量糖蛋白家族反应。
