In vitro and in vivo studies on transthyretin oligomerization.
The oligomerization of transthyretin has been studied in vitro and in vivo. The results showed that wild-type transthyretin synthesized in vitro in the absence of microsomes did not form dimers and tetramers, but in the presence of microsomes the mature transthyretin which had been translocated into the microsomal lumen formed dimers and a small amount of tetramers which could be detected only by using a cross-linking reagent. Efficiency of tetramer formation depends upon the source of microsomes; in fact the amount of tetramer formed in liver microsomes was much higher than that in pancreas microsomes. Transthyretin synthesized in HepG2 cells appeared after SDS-PAGE analysis in mostly tetrameric form, while that synthesized in transfected COS-1 cells appeared mainly as dimers. Brefeldin A treatment and pulse-chase experiments in HepG2 cells showed that transthyretin tetramer was formed in the endoplasmic reticulum. These results strongly indicate that transthyretin tetramer is formed most efficiently in the endoplasmic reticulum lumen of hepatocytes. Transthyretin without the signal peptide [S(-)] and transthyretin with a mutation that prevents processing of the signal peptide Msc failed to form dimers and tetramers in vitro. In the transfected COS-1 cells, however, S(-) transthyretin did form dimers while Msc transthyretin failed to oligomerize. These results show that the cleavage of the signal peptide and some cellular factors are required for transthyretin oligomerization.
Bellovino
D
D
Istituto Nazionale della Nutrizione, Via Ardeatina 546, Rome, 00178, Italy.
Morimoto
T
T
Pisaniello
A
A
Gaetani
S
S
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001704:Biopolymers; D020126:Brefeldin A; D019556:COS Cells; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D004285:Dogs; D018197:Hepatoblastoma / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D018942:Macrolides; D008862:Microsomes, Liver / Q000378:metabolism*; D009154:Mutation; D010179:Pancreas / Q000378:metabolism; D011228:Prealbumin / Q000235:genetics / Q000378:metabolism*; D051381:Rats
D000900:Anti-Bacterial Agents; D001704:Biopolymers; D003517:Cyclopentanes; D018942:Macrolides; D011228:Prealbumin; D020126:Brefeldin A