Role of Glycosylation in the Biosynthesis and Activity of Rabbit Testicular Angiotensin-Converting Enzyme

Abstract

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.

N-Domain Angiotensin I-Converting Enzyme With 80 kDa as a Possible Genetic Marker of Hypertension

“…The molecular mass found for the ACE forms S-1/SP-1/ Sen-1 (80 kDa), S-2/SP-2/Sen-2 (65 kDa) from urine of SHR, SHR-SP, SRHen rats; C-2 (65 kDa) from urine of 1K1C rats; DC-2/D2 (65 kDa) from urine of DOCA salt control and D-2 DOCA salt and BN-2 (65 kDa) from urine of BN rats was much lower than testicular ACE (90 to 100 kDa), which is heavily glycosylated, although they were similar to that of the nonglycosylated form of the single-domain tACE (76 to 84 kDa) 36,37 and the N-domain ACE (65 to 68 kDa) found recently in human urine 19 and ileal fluid. 38 When we compared the forms from WKY rats and ACE BN-1, with 190 kDa and BN-2, with 65 kDa, the data indicated that different strains presented the same enzyme forms.…”

Identification of N-Linked Glycosylation Sites in Human Testis Angiotensin-converting Enzyme and Expression of an Active Deglycosylated Form

“…DISCUSSION Glycosylation is an essential feature of the biosynthesis of ACE. Rabbit tACE, transiently expressed in human HeLa cells in the presence of tunicamycin, a chemical inhibitor of N-linked glycosylation, appeared only in the cytosol and was degraded rapidly (4). Studies of partial glycosylation of tACE by mutation of potential N-linked glycosylation sites indicated that the oligosaccharide chains at each site make different contributions to in vivo stability and localization (6).…”

“…Expression of ACE in human HeLa cells in the presence of tunicamycin resulted in complete inhibition of glycosylation, rapidly degraded intracellular ACE, and no enzyme released in the medium (4). An enzymatically active ACE was produced with partial glycosylation in a mutant Chinese hamster ovary (CHO) cell line (ldlD), although it was released to a lesser extent (4). Similarly, it was reported (5) that inhibitors of glucosidases I and II in the endoplasmic reticulum (ER) and mannosidase I in the cis-Golgi reduced the amount of oligosaccharide attached to human intestinal ACE and delayed protein release significantly.…”

“…Expression of ACE in human HeLa cells in the presence of tunicamycin resulted in complete inhibition of glycosylation, rapidly degraded intracellular ACE, and no enzyme released in the medium (4). An enzymatically active ACE was produced with partial glycosylation in a mutant Chinese hamster ovary (CHO) cell line (ldlD), although it was released to a lesser extent (4).…”

“…It is not known whether the two additional N-linked glycosylation sequons in human tACE are utilized, although studies have shown heterogeneity across different species, both in the sites of oligosaccharide attachment and the types of carbohydrate components (3). tACE from all three species contains a serine/threonine-rich NH 2 -terminal motif that is heavily glycosylated, although no apparent function for the O-glycosylation has been demonstrated (4,7).…”