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.

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.