INTRACELLULAR RETENTION AND DEGRADATION OF HUMAN MUTANT VARIANT OF A ALPHA-1-ANTITRYPSIN IN STABLY TRANSFECTED CHINESE-HAMSTER OVARY CELL-LINES

Normal (PiM) and mutant (PiZ) variants of human alpha1-antitrypsin (alpha1-AT) cDNA, cloned into the pTnd eucaryotic expression vector, were used to derive recombinant Chinese hamster ovary cell lines permanently expressing the corresponding proteins. Secretion, accumulation and glycosylation of PiM and PiZ alpha1-AT proteins were studied in the presence of various transport-impairing drugs. Pulse-chase, followed by immunoprecipitation as well as immunofluorescence experiments showed that the PiZ alpha1-AT undergoes continuous degradation that was prevented by Brefeldin A but not by incubation of cells at 16-degrees-C. Moreover, monensin partially impaired the glycosylation of both PiM and PiZ alpha1-AT but not their secretion nor the degradation of PiZ alpha1-AT. Those results suggest that PiZ alpha1-AT degradation occurs in the cis-Golgi network, a compartment located between the endoplasmic reticulum and the Golgi stack. The process did not apparently involve lysosomes since it was insensitive to chloroquine. In addition, inhibition of PiM and PiZ alpha1-AT glycosylation and secretion by tunicamycin did not result in the accumulation of the protein, but instead in its rapid lag-free degradation. Treatment of cells with the A23187 ionophore, for a short (60 min) but not a long (24 h) period, improved the secretion of PiZ alpha1-AT in a similar way as it affects retention of naturally endoplasmic-reticulum-resident proteins, suggesting that the small proportion of PiZ alpha1-AT which is not degraded or secreted, but accumulates in the endoplasmic reticulum, is back transported as a partially glycosylated species from the post endoplasmic reticulum compartment in which degradation takes place.