Deschuyteneer M, Eckhardt A E, Roth J, Hill R L
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
J Biol Chem. 1988 Feb 15;263(5):2452-9.
Antibodies prepared against enzymatically deglycosylated porcine submaxillary gland mucin (apomucin), which were unreactive with native mucin and its partially deglycosylated derivatives, were used to immunolocalize apomucin in situ. Electron microscopy of sections of Lowicryl K4M-embedded tissue reacted successively with antibodies and protein A-gold complexes showed apomucin exclusively in mucous cells within the rough endoplasmic reticulum, transitional elements of the endoplasmic reticulum, and vesicles at the cis side of the Golgi apparatus. The Golgi apparatus, forming mucous droplets, and mucous droplets contained no apomucin. Although the rough endoplasmic reticulum contained most of the apomucin in mucous cells, some cisternae of the endoplasmic reticulum and the nuclear envelope were devoid of apomucin. Examination of tissue sections treated with the glycosidases used to prepare apomucin revealed immunolabel for apomucin throughout the secretory pathway. Colloidal gold coated with Helix pomatia lectin was used to detect nonreducing N-acetylgalactosamine residues. In mucin-producing cells lectin-gold was found in the mucous droplets, the forming mucous droplets, and throughout the Golgi apparatus but mostly in the cis portion of this organelle. In tissue sections reacted successively with lectin-gold and anti-apomucin/protein A-gold, both types of gold complex could be found in the cis side of the Golgi apparatus. These data indicate that the O-glycosylation of mucin is a posttranslational event that occurs in the Golgi apparatus and begins in the cis side of the Golgi apparatus.
针对经酶促去糖基化的猪下颌下腺粘蛋白（脱辅基粘蛋白）制备的抗体，与天然粘蛋白及其部分去糖基化衍生物无反应，用于原位免疫定位脱辅基粘蛋白。对用Lowicryl K4M包埋的组织切片进行电子显微镜观察，切片先后与抗体和蛋白A-金复合物反应，结果显示脱辅基粘蛋白仅存在于粗面内质网内的粘液细胞、内质网的过渡元件以及高尔基体顺面的囊泡中。形成粘液滴的高尔基体和粘液滴中不含脱辅基粘蛋白。尽管粗面内质网含有粘液细胞中大部分的脱辅基粘蛋白，但内质网的一些扁平囊和核膜不含脱辅基粘蛋白。对用制备脱辅基粘蛋白所用糖苷酶处理的组织切片进行检查，发现整个分泌途径中均有脱辅基粘蛋白的免疫标记。用苹果蜗牛凝集素包被的胶体金用于检测非还原性N-乙酰半乳糖胺残基。在产生粘蛋白的细胞中，凝集素-金存在于粘液滴、正在形成的粘液滴以及整个高尔基体中，但主要存在于该细胞器的顺面部分。在先后与凝集素-金和抗脱辅基粘蛋白/蛋白A-金反应的组织切片中，两种类型的金复合物均可在高尔基体的顺面发现。这些数据表明，粘蛋白的O-糖基化是一种发生在高尔基体中的翻译后事件，且始于高尔基体的顺面。
