Bulleid N J, Curling E, Freedman R B, Jenkins N
Biological Laboratory, University of Kent, Canterbury, U.K.
Biochem J. 1990 Jun 15;268(3):777-81. doi: 10.1042/bj2680777.
A cDNA clone coding for human interferon-gamma (IFN-gamma) was subcloned into a transcription-translation vector. When the mRNA transcribed in vitro was added to a rabbit reticulocyte-lysate system, two polypeptides were synthesized: one corresponding in Mr to pre-IFN-gamma (18,000) and one with a lower Mr (12,000) which corresponds to a polypeptide arising from incorrect initiation of translation. When microsomal vesicles isolated from dog pancreas or Chinese-hamster ovary (CHO) cells were added to the translation system, translocation of the pre-IFN-gamma occurred, as judged by protection from exogenous proteinases. The resultant changes in the Mr of the translation products were indicative of signal-peptide cleavage and heterogeneous core glycosylation. When translation products were treated with N-glycanase, the higher-Mr products were no longer observed, consistent with removal of all oligosaccharide side chains, leaving a single core polypeptide. Glycosylation of the synthesized protein yielded both singly and doubly glycosylated products compatible with the glycosylation variants seen in secreted IFN-gamma. Quantitative differences were seen in the relative amounts of singly and doubly glycosylated products synthesized by dog pancreatic compared with CHO-derived microsomes. These data indicate that the relative amounts of IFN-gamma glycosylation variants are determined at an early stage in protein synthesis and that product variants may occur when IFN-gamma is expressed in cells derived from different tissues.
将编码人γ干扰素（IFN-γ）的cDNA克隆亚克隆到一个转录-翻译载体中。当将体外转录的mRNA加入兔网织红细胞裂解物系统时，合成了两种多肽：一种的相对分子质量（Mr）与前体IFN-γ（18,000）相对应，另一种Mr较低（12,000），它对应于因翻译起始错误而产生的一种多肽。当将从犬胰腺或中国仓鼠卵巢（CHO）细胞分离的微粒体囊泡加入翻译系统时，根据对外源蛋白酶的抗性判断，前体IFN-γ发生了转位。翻译产物Mr的最终变化表明信号肽被切割且发生了不均一的核心糖基化。当用N-糖苷酶处理翻译产物时，不再观察到较高Mr的产物，这与所有寡糖侧链被去除、仅留下单一核心多肽一致。合成蛋白的糖基化产生了单糖基化和双糖基化产物，这与分泌型IFN-γ中所见的糖基化变体相符。与CHO来源的微粒体相比，犬胰腺来源的微粒体合成的单糖基化和双糖基化产物的相对量存在定量差异。这些数据表明，IFN-γ糖基化变体的相对量在蛋白质合成的早期阶段就已确定，并且当IFN-γ在源自不同组织的细胞中表达时可能会出现产物变体。
