Cellular and molecular biological study of biosynthesis of human gastric mucin.

我们研究了粘蛋白的生物合成在人类胃中使用的抗羧基末端的脱粘蛋白抗体。用[^ S]蛋氨酸标记人胃粘膜<35>，追踪3 h。在胞内产物中检测到约60 kD的人胃粘蛋白前体蛋白亚基及其二聚体、三聚体和四聚体（120 kD、180 kD、240 kD）和高分子量的成熟粘蛋白。细胞外产物仅含有成熟的粘蛋白。用衣霉素抑制N-糖基化对60千道尔顿亚基及其寡聚体的合成和成熟粘蛋白的分泌没有影响。一些腺癌合成粘蛋白或粘蛋白相关产物并将其作为癌相关抗原分泌。但对所涉及的细胞内转运和分泌机制知之甚少。采用脉冲追踪法研究了胃癌细胞株（Hs 746 T）粘蛋白相关产物的合成和分泌。用脱粘蛋白单克隆抗体的抗羧基末端免疫沉淀细胞内和细胞外产物。在Hs 746 T细胞中，在胞内产物中检测到约55 kD的前体蛋白、110 kD的二聚体蛋白和高分子量的粘蛋白样产物。胞外产物包括二聚体和少量高分子量粘蛋白。用衣霉素或endo-b-N-乙酰氨基葡萄糖苷酶H处理对55 kD蛋白及其二聚体没有影响，但衣霉素抑制二聚体的分泌。这些发现表明，N-糖基化可能参与二聚体蛋白作为一种不成熟产物的分泌机制。

We studied the biosynthesis of mucin in the human stomach using an anti-carboxyl terminal of the apomucin antibody. Human stomach mucosa was labeled with [^<35>S]methionine,and chased for 3 h. Approximately a 60 kilo-daltons subunit of human gastric mucin precursor protein,and its dimer,trimer,and tetramer(120,180,240kilo-daltons), and high molecular weight mature mucin were detected in the intracellular product. Extracellular products contained only the mature mucin. Inhibition of N-glycosylation with tunicamycin had no effects on the synthesis of 60 kilo-daltons subunit and its oligomers,and the secretion of the mature mucin. Some adenocarcinomas synthesize mucin or mucin-related products and secrete them as carcinoma-associated antigens. But very little is known about the intracellular transport and secretion mechanisms involved. We studied the biosynthesis and secretion of mucin-related products in a gastric cancer cell line(Hs746T) by pulse-chase experiments. Intracellular and extracellular products were immunoprecipitated with an anti-carboxyl terminal of the apomucin monoclonal antibody. In Hs746T cells,an approximately 55kilo-daltons precursor protein, 110kilo-daltons protein as the dimer,and a high molecular weight mucin-like product were detected among the intracellular products. The extracellular products included the dimer and a small amount of high molecular weight mucin. Treatment with tunicamycin or endo-b-N-acetylglucosaminidase H had no effect on the 55kilo-daltons protein and its dimer,but tunicamycin inhibited secretion of the dimer. These findings suggest that N-glycosylation may be involved in the secretory mechanism of the dimer protein as an immature product.