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Synthesis and Intracellular Transport of Common Antigens of Secretory Granule Membrane.

分泌颗粒膜常见抗原的合成和细胞内转运。

基本信息

批准号：
01570018
负责人：
YAMASHITA Shuji
金额：
$ 1.34万
依托单位：
Keio University, School of Medicine, Department of Anatomy
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570018/
关键词：
Common antigens of secretory granule membrane Monoclonal antibody Immunohistochemistry Recycling of secretory granule membrane Immunoelectron microscopy Rat Rabbit 分泌果粒共通抗原

项目摘要

Monoclonal antibodies (mAbs) were generated to the secretory granule membrane of rat and rabbit parotid gland, respectively. The mAbs recognized integral membrane glycoproteins localized on the luminal side of the secretory granules with N-linked carbohydrates : molecular weight ; 98-65 kD in the rat, 55-65 kD in the rabbit.Immunohistochemical studies indicated that both the secretory granule membrane antigens were found in the secretory granules of both exocrine and endocrine cells, and in the lysosomes of various cells in the rat and rabbit. These results indicated that common antigens are present in the secretory granule membranes. Immunoelectron microscopy with immunogold revealed that antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles and condensing vacuoles in pancreatic and parotid acinar cells and AR-42J rat pancreatic tumor cells ; the Golgi stacks exhibited no immunoreaction. In the rabbit, the antigen was localized exclusively along the membranes of secretory granules in the parotid acinar cells, exocrine pancreatic cells and adenohypophyseal cells.Employing HRP labeled mAb, we followed the retrieval of the antigen after exocytosis in AR42J cells. The mAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody. These results indicated that the common antigen of secretory granule memrane is reused after exocytosis, and suggested that the pool of the antigen might present in the trans-Golgi network.

分别制备了抗大鼠和兔腮腺分泌颗粒膜的单克隆抗体。免疫组化结果表明，分泌颗粒膜抗原在大鼠和家兔的外分泌细胞和内分泌细胞的分泌颗粒中均有分布，并存在于各种细胞的溶酶体中。这些结果表明，共同抗原存在于分泌颗粒膜。免疫金免疫电镜显示，抗原存在于胰腺和腮腺腺泡细胞和AR-42 J大鼠胰腺肿瘤细胞的分泌颗粒、溶酶体、高尔基体囊泡和凝聚空泡的膜上;高尔基体堆叠未显示出免疫反应。在兔腮腺腺泡细胞、胰腺外分泌细胞和腺垂体细胞中，该抗原仅沿着于分泌颗粒的膜上。mAb通过抗原介导的内吞作用特异性内化。它被运输到内体，随后到trans-Golgi网络，然后包装成分泌颗粒。然而，高尔基体堆叠显示标记的抗体没有摄取。这些结果表明，分泌颗粒膜的共同抗原在胞吐后被重新利用，并提示抗原池可能存在于高尔基体网络中。