SOURCE OF HETEROGENEITY IN SECRETED INTERFERON-GAMMA - A STUDY ON PRODUCTS OF TRANSLATION INVITRO

A cDNA clone coding for human interferon-γ (IFN-γ) 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 M(r) to pre-IFN-γ (18 000) and one with a lower M(r) (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-γ occurred, as judged by protection from exogenous proteinases. The resultant changes in the M(r) of the translation products were indicative of signal-peptide cleavage and heterogeneous core glycosylation. When translation products were treated with N-glycanase, the higher-M(r) produts 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-γ. 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-γ glycosylation variants are determined at an early stage in protein synthesis and that product variants may occur when IFN-γ is expressed in cells derived from different tissues.