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.
Kuznetsov
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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