Angiotensin I-converting enzyme (ACE; CD143, EC 22.214.171.124) is a type-1 integral membrane protein that can also be released into extracellular fluids (such as plasma, and seminal and cerebrospinal fluids) as a soluble enzyme following cleavage mediated by an unidentified protease(s), referred to as ACE secretase, in a process known as ‘shedding'. The effects of monoclonal antibodies (mAbs) to eight different epitopes on the N-terminal domain of ACE on shedding was investigated using Chinese hamster ovary cells (CHO cells) expressing an ACE transgene and using human umbilical vein endothelial cells. Antibody-induced shedding of ACE was strongly epitope-specific: most of the antibodies increased the shedding by 20–40%, mAbs 9B9 and 3A5 increased the shedding by 270 and 410% respectively, whereas binding of mAb 3G8 decreased ACE shedding by 36%. The ACE released following mAb treatment lacked a hydrophobic transmembrane domain anchor. The antibody-induced shedding was completely inhibited at 4°C and by zinc chelation using 1,10-phenanthroline, suggesting involvement of a metalloprotease in this process. A hydroxamate-based metalloprotease inhibitor (batimastat, BB-94) was 15 times more efficacious in inhibiting mAb-induced ACE shedding than basal (constitutive) ACE release. Treatment of CHO-ACE cells with BB-94 more effectively prevented elevation in antibody-dependent (but not basal) ACE release induced by 3,4-dichloroisocoumarin and iodoacetamide. These data suggest that different secretases might be responsible for ACE release under basal compared with antibody-induced shedding. Further experiments with more than 40 protease inhibitors suggest that calpains, furin and the proteasome may participate in this process.
Abbreviations used: ACE, angiotensin I-converting enzyme; mAb, monoclonal antibody; CHO, Chinese hamster ovary; HUVEC, human umbilical vein endothelial cell; BB-94, batimastat; DCI, 3,4-dichloroisocoumarin; Tos-Phe-CH2Cl, N-tosyl-l-phenylalanylchloromethane; IA, iodoacetamide; DTT, dithiothreitol; LDH, lactate dehydrogenase; PKC, protein kinase C; ZIE, benzyloxycarbonyl-Ile-Glu(otBu)Ala-Leu-aldehyde; Dec-RVKR-cmk, decanoyl-Arg-Val-Lys-Arg-chloromethylketone; Hip-His-Leu, hippuryl-His-Leu.
Present address: Cardiovascular Therapeutic Area, Pfizer Central Research, Sandwich, Kent CT13 9NJ, U.K.