Bile salt-dependent lipase biosynthesis in rat pancreatic AR 4-2 J cells. Essential requirement of N-linked oligosaccharide for secretion and expression of a fully active enzyme.
This study used the rat pancreatic AR 4-2 J cell line as a model system to investigate the role of glycosylation in bile salt-dependent lipase (BSDL) secretion and esterolytic activity. Results indicated that AR 4-2 J cells synthesized BSDL, the esterolytic activity of which was the greater part of the cell activity toward 4-nitrophenylcaproate. The protein thus expressed was glycosylated and had a molecular mass approximately 74 kDa. Exposure of these cells to tunicamycin significantly decreased [3H]mannose incorporation, while [35S]methionine incorporation in trichloroacetic acid-precipitable material was not modified. Tunicamycin treatment of AR 4-2 J cells lead to a lower molecular mass form (approximately 70 kDa) of BSDL which did not incorporate [3H]mannose. The nonglycosylated low M(r) form of the enzyme was not secreted as shown by the decreasing activity in cell-free medium which paralleled the time-dependent secretion of the enzyme. Tunicamycin had no effect on BSDL synthesis. Nevertheless, the nonglycosylated BSDL was apparently not degraded in any cell compartment as shown in part by the enzyme activity accumulation within cells upon brefeldin A treatment. The BSDL expressed by AR 4-2 J cells was characterized by a Km of 68 +/- 30 microM and a kcat of 106 +/- 19 min-1. The sequestrated BSDL due to tunicamycin treatment of cells presents a significant increase of Km of over 10 times to 757 +/- 303 microM. kcat was affected by a factor of approximately 4-445 +/- 22 min-1. These data correlated with an approximately 2.5-fold decrease of the esterolytic activity following inhibition of the N-glycosylation of the protein. The nonglycosylated BSDL was less stable to temperature than the native form. Processing inhibitors (castanospermine, 1-deoxymannojirimycin, and swainsonine) had no effect either on the enzyme activity or on its secretion. Results suggested first that the transfer of the oligosaccharide precursor to the nascent BSDL is essential for the folding of the fully active BSDL. Second that the glycan structure is required for the enzyme secretion.
Abouakil
N
N
Institut National de la Santé et de la Recherche Médicale U-260, Faculté de Médecine, Unité de Recherche de Physiopathologie des Régulations Hormono-Nutritionnelles, Marseille, France.
Mas
E
E
Bruneau
N
N
Benajiba
A
A
Lombardo
D
D
D017485:1-Deoxynojirimycin / Q000494:pharmacology; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D050260:Carbohydrate Metabolism; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D005959:Glucosidases / Q000037:antagonists & inhibitors; D006031:Glycosylation; D007212:Indolizines / Q000494:pharmacology; D007700:Kinetics; D008049:Lipase / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010190:Pancreatic Neoplasms; D051381:Rats; D002787:Sterol Esterase; D013462:Sulfur Radioisotopes; D017026:Swainsonine / Q000494:pharmacology; D013816:Thermodynamics; D014316:Tritium; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology; D054836:Indolizidines
D017485:1-Deoxynojirimycin; D000596:Amino Acids; D005959:Glucosidases; D007212:Indolizines; D008049:Lipase; D008358:Mannose; D008715:Methionine; D002787:Sterol Esterase; D013462:Sulfur Radioisotopes; D017026:Swainsonine; D014316:Tritium; D014415:Tunicamycin; C054784:bile salt-stimulated lipase; C037806:castanospermine; D054836:Indolizidines