Analysis of the endoplasmic reticular Ca2+ requirement for α1-antitrypsin processing and transport competence

Abstract

Depletion of Ca²⁺ sequestered within the endoplasmic reticulum (ER) of HepG2 hepatoma cells results in the luminal accumulation of immature α₁-antitrypsin possessing Man_8–9 GlcNAc₂ oligosaccharide side chains. This study explores the basis for this arrest and describes consequent alterations in the size and rate of secretion of the complex endoglycosidase H-resistant form of the protein. Inhibition of glucosidase I and II with castanospermine or α-1,2-mannosidase with 1-deoxymannojirimycin produced altered ER processing intermediates that were rapidly secreted. Subsequent mobilization of ER Ca²⁺ stores resulted in the appearance and retention of slightly larger related forms of these intermediates. Retention of glycosylated intermediates was not ascribable to an association with α1,2-mannosidase or lectin-like chaperones, the intermediates were not degraded and all evidence of ER retention or size alterations produced by Ca²⁺ depletion was quickly reversed by Ca²⁺ restoration. Cells that were Ca²⁺ depleted for 2 h slowly secreted an abnormal slightly smaller complex oligosaccharide form of α₁-antitrypsin at approximately the same rate as the non-glycosylated protein generated by treatment with tunicamycin. The hypothesis that Ca²⁺ affects the folding and ER transport competence of glycosylated forms of α₁-antitrypsin is discussed.