title: Demonstration of a calcium requirement for secretory protein processing and export. Differential effects of calcium and dithiothreitol.
abstract: HepG2 cells were employed as model system to investigate potential relationships between early protein processing and Ca2+ storage by the endoplasmic reticulum. Ca2+ was required for glycoprotein processing and export by intact cells. The processing and export of alpha 1-antitrypsin and the secretion of complement factor 3, which are glycosylated proteins, were inhibited by the Ca2+ ionophore ionomycin whereas the export of albumin, a non-glycoprotein, was little affected. Ionomycin blocked processing of alpha 1-antitrypsin at the conversion from the high mannose to the complex glycosylated form without affecting ATP or GTP contents. Pre-existing inhibition of intracellular processing of alpha 1-antitrypsin by ionomycin was fully reversible upon removal of the ionophore with fatty acid-free bovine serum albumin. This reversal required Ca2+. After reversal the arrested form of alpha 1-antitrypsin was fully converted to the mature form and exported to the medium. Inhibitions of alpha 1-antitrypsin processing and complement factor 3 secretion by the metalloendoprotease antagonist Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) were strongest at low extracellular Ca2+ but were reduced or prevented by high extracellular Ca2+. Processing and secretion of alpha 1-antitrypsin were reduced upon incubation in low Ca2+ medium. Exposure to dithiothreitol reduced albumin export while affecting alpha 1-antitrypsin export minimally. Suppression of amino acid incorporation into total cellular proteins of HepG2 cells accompanied inhibitions of protein processing by agents depleting sequestered Ca2+ stores or by dithiothreitol. Putative control of rates of translational initiation by the endoplasmic reticulum through linkage to rates of early protein processing is discussed.
authors.affiliation: Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
authors.forename: G
authors.initials: G
authors.lastname: Kuznetsov
authors.forename: M A
authors.initials: MA
authors.lastname: Brostrom
authors.forename: C O
authors.initials: CO
authors.lastname: Brostrom
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D003176:Complement C3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D004151:Dipeptides / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D015759:Ionomycin / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000378:metabolism; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D012709:Serum Albumin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000378:metabolism
chemical_list: D000596:Amino Acids; D003176:Complement C3; D004151:Dipeptides; D006023:Glycoproteins; D009363:Neoplasm Proteins; D012709:Serum Albumin; D000515:alpha 1-Antitrypsin; C019156:carbobenzoxyglycylphenylalanine; D004533:Egtazic Acid; D015759:Ionomycin; D002118:Calcium; D004229:Dithiothreitol