Dekker J, Van Beurden-Lamers W M, Strous G J
Department of Cell Biology, State University of Utrecht, School of Medicine, The Netherlands.
J Biol Chem. 1989 Jun 25;264(18):10431-7.
We have studied the biosynthesis of rat gastric mucin in stomach segments using an antiserum against rat gastric mucin specific for peptide epitopes. Pulse-chase experiments were performed with [35S]methionine, [3H]galactose, and [35S]sulfate to label mucin precursors in different stages of biosynthesis, which were analyzed after immunoprecipitation. The earliest mucin precursor that could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was a 300-kDa protein. The occurrence of N-linked "high-mannose" oligosaccharides on this protein was shown by susceptibility to degradation by endo-beta-N-acetylglucosaminidase H. This precursor could be labeled with [35S]methionine and not with [3H]galactose or [35S]sulfate. The 300-kDa precursor was converted into mature mucin after extensive glycosylation and sulfation. The mature mucin but not the 300-kDa precursor was in part secreted into the medium. Specific inhibition of sulfation with sodium chlorate had no effect on rate and amount of mucin secretion. In addition, we show that two core proteins are expressed in rats, slightly varying in Mr among individual animals.
我们使用针对大鼠胃粘蛋白肽表位的抗血清，研究了胃段中大鼠胃粘蛋白的生物合成。用[35S]甲硫氨酸、[3H]半乳糖和[35S]硫酸盐进行脉冲追踪实验，以标记生物合成不同阶段的粘蛋白前体，免疫沉淀后对其进行分析。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳能够检测到的最早的粘蛋白前体是一种300 kDa的蛋白质。内切β-N-乙酰葡糖胺糖苷酶H对该蛋白质的降解敏感性表明其上存在N-连接的“高甘露糖”寡糖。这种前体可用[35S]甲硫氨酸标记，而不能用[3H]半乳糖或[35S]硫酸盐标记。300 kDa的前体在广泛糖基化和硫酸化后转化为成熟粘蛋白。成熟粘蛋白部分分泌到培养基中，而300 kDa的前体则不会。用氯酸钠特异性抑制硫酸化对粘蛋白分泌的速率和量没有影响。此外，我们发现大鼠中表达了两种核心蛋白，个体动物之间的相对分子质量略有差异。
