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 termi nus. The enzymatically active extracellular domain of ACE is slowly re leased from the cell by cleavage-removal of its membrane-anchoring car boxyl-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 u sed an in vitro translation system, a permanently transfected mouse ce ll line, and human and Chinese hamster cells transiently transfected w ith vaccinia virus-T7 RNA polymerase-driven expression vectors. Sugar modifications of ACE were analyzed by testing its sensitivity to speci fic glycosidases. Cellular protein glycosylation was inhibited by usin g chemical inhibitors and a mutant cell line defective in protein glyc osylation. Our experiments demonstrated that newly synthesized ACE acq uires both N- and O-linked sugars before its cleavage-secretion and co mplete blockage of glycosylation results in rapid intracellular turnov er of underglycosylated ACE. However, ACE synthesized without N-linked complex sugars and O-linked sugars can undergo normal transport and c leavage-secretion, and the underglycosylated protein is enzymatically active.