Source of heterogeneity in secreted interferon-gamma. A study on products of translation in vitro.
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.
Bulleid
N J
NJ
Biological Laboratory, University of Kent, Canterbury, U.K.
Curling
E
E
Freedman
R B
RB
Jenkins
N
N
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D004285:Dogs; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D011994:Recombinant Proteins; D014158:Transcription, Genetic
D011994:Recombinant Proteins; D007371:Interferon-gamma; D004247:DNA