Herrera H, Rodríguez E M
Instituto de Histología y Patología, Facultad de Medicina, Universidad Austral de Chile, Valdivia.
Histochemistry. 1990;93(6):607-15. doi: 10.1007/BF00272203.
Two experimental protocols were used to investigate the secretory glycoproteins of the subcommissural organ (SCO). Protocol I: Lectins, specific exoglycosidases and immunocytochemistry were sequentially applied to the same section or to adjacent semithin sections of the rat SCO fixed in Bouin's fluid and embedded in methacrylate. Lectins used: concanavalin A (con A), wheat germ agglutinin, Limulus polyphemus agglutinin, Ricinus communis agglutinin and Arachis hypogeae agglutinin. Glycosidases used: neuroaminidase, beta-galactosidase, alpha-mannosidase, alpha-glucosidase and beta-N-acetyl-glucosaminidase. For immunocytochemistry an antiserum against bovine Reissner's fiber (AFRU) was used. Lectins and glycosidases were used in sequences that allowed the cleaved sugar residue to be identified as well as that appearing exposed as a terminal residue. This approach led to the following conclusions: (1) the terminal sugar chain of the secreted glycoproteins has the sequence sialic acid-galactose-glucosamine-; (2) the con A-binding material present in the rough endoplasmic reticulum corresponds to mannose; (3) the apical secretory granules and Reissner's fibers displayed a strong con A affinity after removing sialic acid, thus indicating the presence of internal mannosyl residues in the secreted material; (4) after removing most of the sugar moieties the secretory material continued to be strongly immunoreactive with AFRU. Protocol II: Rats were injected into the lateral ventricle with Tunica-mycin and killed 12, 24, 50 and 60 h after the injection. The SCO of rats from the last two groups showed a complete absence of con A binding sites. The results from the two experiments confirm that the secretory glycoproteins of the rat SCO are N-linked complex-type glycoproteins with the conformation previously suggested (Rodríguez et al. 1986).
采用两种实验方案研究连合下器官（SCO）的分泌性糖蛋白。方案一：将凝集素、特异性外切糖苷酶和免疫细胞化学方法依次应用于固定在布因氏液中并包埋于甲基丙烯酸酯的大鼠SCO的同一切片或相邻半薄切片上。所用凝集素：伴刀豆球蛋白A（Con A）、麦胚凝集素、鲎试剂凝集素、蓖麻凝集素和花生凝集素。所用糖苷酶：神经氨酸酶、β-半乳糖苷酶、α-甘露糖苷酶、α-葡萄糖苷酶和β-N-乙酰氨基葡萄糖苷酶。免疫细胞化学使用抗牛赖氏纤维抗血清（AFRU）。凝集素和糖苷酶按一定顺序使用，以便识别被切割的糖残基以及作为末端残基暴露的糖残基。该方法得出以下结论：（1）分泌性糖蛋白的末端糖链序列为唾液酸-半乳糖-葡萄糖胺-；（2）粗面内质网中存在的Con A结合物质对应于甘露糖；（3）去除唾液酸后，顶端分泌颗粒和赖氏纤维显示出强烈的Con A亲和力，表明分泌物质中存在内部甘露糖基残基；（4）去除大部分糖基后，分泌物质仍与AFRU强烈免疫反应。方案二：向大鼠侧脑室内注射衣霉素，并在注射后12、24、50和60小时处死。最后两组大鼠的SCO显示完全没有Con A结合位点。这两个实验的结果证实，大鼠SCO的分泌性糖蛋白是N-连接的复合型糖蛋白，其构象如先前所示（Rodríguez等人，1986年）。
