Role of endoplasmic reticular calcium in oligosaccharide processing of alpha 1-antitrypsin.
Mobilization of Ca2+ from the endoplasmic reticulum (ER) suppresses translational initiation and inhibits post-translational processing and secretion of glycoproteins. This study explores the mechanism whereby ionomycin, a Ca2+ ionophore, and thapsigargin, an ER Ca(2+)-ATPase inhibitor, promote retention of alpha 1-antitrypsin (alpha 1-AT) bearing high mannose, endoglycosidase H (Endo H)-sensitive oligosaccharide side chains within the ER of HepG2 cells. Arrest occurred at the removal of mannose residues such that intermediates with Man7-9GlcNAc2 side chains accumulated with the Man8-9GlcNAc2 structures predominating. Maturation of alpha 1-AT bearing Man5-6GlcNAc2 side chains was unaffected. Inhibition of alpha 1-AT processing by ionomycin occurred independently of translational suppression. Forms of alpha 1-AT identical to those retained with ionomycin or thapsigargin were observed upon treatment with the alpha-1,2-mannosidase inhibitor 1-deoxymannojirimycin whereas castanospermine, an inhibitor of ER alpha-glucosidase I, produced different forms of the glycoprotein. Neither inhibitor impaired transport or secretion of alpha 1-AT. With brefeldin A, which causes redistribution of Golgi enzymes to the ER, alpha 1-AT was retained intracellularly but acquired resistance to Endo H. With ionomycin, thapsigargin, or 1-deoxymannojirimycin-treated cells, however, brefeldin A failed to promote further processing of the glycoprotein. Possible mechanisms for the suppression of alpha 1-AT processing at the alpha-1,2-mannosidase step by Ca(2+)-mobilizing agents are discussed. Excepting tunicamycin, traditional inhibitors of protein processing did not affect amino acid incorporation.
Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854.
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D017485:1-Deoxynojirimycin / Q000494:pharmacology; D020126:Brefeldin A; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002236:Carbohydrate Conformation; D006528:Carcinoma, Hepatocellular; D003517:Cyclopentanes / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D065089:Glycoside Hydrolase Inhibitors; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008113:Liver Neoplasms; D008358:Mannose / Q000032:analysis; D008361:Mannosidases / Q000037:antagonists & inhibitors; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009844:Oligosaccharides / Q000378:metabolism*; D013729:Terpenes / Q000494:pharmacology; D019284:Thapsigargin; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000378:metabolism*; D043323:alpha-Mannosidase; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D020126:Brefeldin A; D002118:Calcium; D000252:Calcium-Transporting ATPases; D003517:Cyclopentanes; D065089:Glycoside Hydrolase Inhibitors; D006596:Hexosaminidases; D007212:Indolizines; D015759:Ionomycin; D008358:Mannose; D008361:Mannosidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D009844:Oligosaccharides; D013729:Terpenes; D019284:Thapsigargin; D000515:alpha 1-Antitrypsin; D043323:alpha-Mannosidase; C037806:castanospermine; D054836:Indolizidines