Role of glycosylation in the biosynthesis and activity of rabbit testicular angiotensin-converting enzyme.
Angiotensin-converting enzyme (ACE) is a type I glycoprotein anchored in the plasma membrane by a hydrophobic domain near its carboxyl terminus. The enzymatically active extracellular domain of ACE is slowly released from the cell by cleavage-removal of its membrane-anchoring carboxyl-terminal region. In the present study, we investigated the role of N- and O-glycosylation in intracellular transport and extracellular cleavage-secretion of rabbit testicular ACE. For ACE expression, we used an in vitro translation system, a permanently transfected mouse cell line, and human and Chinese hamster cells transiently transfected with vaccinia virus-T7 RNA polymerase-driven expression vectors. Sugar modifications of ACE were analyzed by testing its sensitivity to specific glycosidases. Cellular protein glycosylation was inhibited by using chemical inhibitors and a mutant cell line defective in protein glycosylation. Our experiments demonstrated that newly synthesized ACE acquires both N- and O-linked sugars before its cleavage-secretion and complete blockage of glycosylation results in rapid intracellular turnover of underglycosylated ACE. However, ACE synthesized without N-linked complex sugars and O-linked sugars can undergo normal transport and cleavage-secretion, and the underglycosylated protein is enzymatically active.
Kasturi
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Department of Cardiovascular Biology, Cleveland Clinic Foundation Research Institute, Ohio 44195.
Jabbar
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Sen
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Sen
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D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015870:Gene Expression; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology; D006367:HeLa Cells; D006596:Hexosaminidases / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013737:Testis / Q000201:enzymology*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins
D014764:Viral Proteins; D014415:Tunicamycin; D008985:Monensin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D006596:Hexosaminidases; D007703:Peptidyl-Dipeptidase A