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. 1990 Jun 15;268(3):777–781. doi: 10.1042/bj2680777

Source of heterogeneity in secreted interferon-gamma. A study on products of translation in vitro.

N J Bulleid 1, E Curling 1, R B Freedman 1, N Jenkins 1
PMCID: PMC1131508  PMID: 2114101

Abstract

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.

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