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 alpha 1-antitrypsin (alpha 1-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 alpha 1-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 alpha 1-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 alpha 1-AT but not their secretion nor the degradation of PiZ alpha 1-AT. Those results suggest that PiZ alpha 1-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 alpha 1-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 alpha 1-AT in a similar way as it affects retention of naturally endoplasmic-reticulum-resident proteins, suggesting that the small proportion of PiZ alpha 1-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.
Ciccarelli
E
E
Department of Applied Genetics, Université Libre de Bruxelles, Nivelles, Belgium.
Alonso
M A
MA
Cresteil
D
D
Bollen
A
A
Jacobs
P
P
Alvarez
F
F
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport / Q000187:drug effects; D020126:Brefeldin A; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D008985:Monensin / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D000515:alpha 1-Antitrypsin / Q000235:genetics* / Q000378:metabolism
D003517:Cyclopentanes; D009838:Oligodeoxyribonucleotides; D000515:alpha 1-Antitrypsin; D014415:Tunicamycin; D020126:Brefeldin A; D008985:Monensin