Epitope-specific antibody-induced cleavage of angiotensin-converting enzyme from the cell surface.
Angiotensin I-converting enzyme (ACE; CD143, EC 3.4.15.1) 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 degrees 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.
Balyasnikova
Irina V
IV
Department of Anesthesiology, University of Illinois at Chicago, 1819 W. Polk St. (M/C 519), Chicago, IL 60612, U.S.A.
Karran
Eric H
EH
Albrecht
Ronald F
RF
Danilov
Sergei M
SM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D003374:Coumarins / Q000494:pharmacology; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D049934:Isocoumarins; D007700:Kinetics; D007703:Peptidyl-Dipeptidase A / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D014162:Transfection; D014471:Umbilical Veins
D000911:Antibodies, Monoclonal; D003374:Coumarins; D000939:Epitopes; D049934:Isocoumarins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C046235:3,4-dichloroisocoumarin; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; C077675:angiotensin converting enzyme secretase