Epitope-Dependent Blocking of the Angiotensin-Converting Enzyme Dimerization by Monoclonal Antibodies to the N-Terminal Domain of ACE:  Possible Link of ACE Dimerization and Shedding from the Cell Surface

Abstract

In a biomembrane modeling system, reverse micelles, somatic ACE forms dimers via carbohydrate-mediated interaction, providing evidence for the existence of a carbohydrate-recognizing domain on the ACE molecule. We localized this putative region on the N-domain of ACE using monoclonal antibodies (mAbs) to seven different epitopes of ACE. Two mAbs, 9B9 and 3G8, directed to distinct, but overlapping, epitopes of the N-domain of ACE shielded the CRD. Only "simple" ACE-antibody complexes were found in the system. Five mAbs allowed the formation of "double" antibody-ACE-ACE-antibody complexes via carbohydrate-mediated interactions. The results were confirmed using the ACE N- and C-domains. Testicular ACE was unable to form carbohydrate-mediated ACE dimers in the reverse micelles, while the N-domain of ACE, obtained by limited proteolysis of the parent full-length ACE, retained the ability to form dimers. Furthermore, mAb 3G8, which blocked ACE dimerization in micelles, significantly inhibited ACE shedding from the surface of ACE-expressing cells. Galactose prevented ACE dimerization in reverse micelles and also affected antibody-induced ACE shedding in an epitope-dependent manner. Restricted glycosylation of somatic ACE, obtained by the treatment of CHO-ACE cells with the glucosidase inhibitor N-butyldeoxynojirimycin, significantly increased the rate of basal ACE shedding and altered antibody-induced ACE shedding. A chemical cross-linking approach was used to show that ACE is present (at least in part) as noncovalently linked dimers on the surface of CHO-ACE cells. These results suggest a possible link between putative ACE dimerization on the cell surface and the proteolytic cleavage (shedding) of ACE.

Angiotensin-Converting Enzyme (ACE) Dimerization Is the Initial Step in the ACE Inhibitor-Induced ACE Signaling Cascade in Endothelial Cells

“…None of the antibodies tested were able to prevent the ramiprilat-induced dimerization of ACE in ACE-overexpressing porcine aortic endothelial cells (Fig. 5c), excluding the previously speculated correlation between ACE dimerization and shedding (Kost et al, 2003).…”

“…We next assessed the role of the putative carbohydrate recognition domain (Kost et al, 2003), a lectin-like structure residing within the N-terminal portion of somatic ACE, in enzyme dimerization. Because ACE dimer formation in vitro in inverse micelles has been reported to be inhibited by galactose and other carbohydrates (Kost et al, 2000), we incubated ACE-overexpressing porcine aortic endothelial cells with 10 M galactose, 10 M glucose, or 10 M mannitol (as an osmotic control) for 30 min, before the addition of 100 nM ramiprilat for 2 min.…”

“…Porcine aortic endothelial cells stably overexpressing human ACE were used as a positive control (ϩve CTL). and thus shielded by the monoclonal antibody 9B9 (Kost et al, 2003). We therefore determined the effect of different monoclonal ACE antibodies (9B9, 3A5, 5F1, and C78) directed to different epitopes within the N domain (Danilov et al, 1994) on ACE dimer formation.…”

“…Other forms of human somatic ACE exist, such as an immature form found in intracellular compartments (ϳ170 kDa) and a soluble form (ϳ175 kDa) found in the plasma representing a C-terminally truncated form of the enzyme that is generated by the proteolytic cleavage of its stalk region and the subsequent release from the plasma membrane . Aggregates of ACE that might reflect enzyme dimers and/or higher oligomers have been found during purification of ACE from human lung (Nishimura et al, 1977) as well as in in vitro experiments by using inverse micelles and ACE-expressing COS cells (Kost et al, 2000(Kost et al, , 2003.…”

“…It is however not clear how the binding of an ACE inhibitor is able to initiate the processes described. Because ACE can exist in a dimeric form in vitro (Kost et al, 2003), the aim of the present investigation was to determine whether or not ACE dimerizes in endothelial cells and whether dimerization is involved in ACE inhibitor-induced ACE signaling.…”