Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding.
A panel of monoclonal antibodies (mAbs) raised against both the N and C domains of angiotensin-I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) have been extensively mapped and have facilitated the study of various aspects of ACE structure and biology. In this study, we characterize two mAbs, 9B9 and 3G8, that recognize the N domain of ACE and that influence shedding and dimerization. Fine epitope mapping was performed, which mapped the epitopes for these mAbs to the N terminal region of the N domain where they overlap to a large extent, despite having different effects on ACE processing. The mAb 3G8 epitope appears to be shielded by the C domain and to be carbohydrate dependent as binding increased significantly as a result of underglycosylation, whereas these factors did not influence mAb 9B9 recognition. Three mutations within the overlapping region of these two epitopes, Q18H, L19E, and Q22A, which decreased mAb 3G8 binding to the soluble N domain, were introduced into full-length somatic ACE (sACE) to determine their influence on ACE expression and processing. Increased ACE expression, cell surface expression, and basal shedding were observed with all three mutations. Furthermore, cross-linking and western blotting of Chinese hamster ovary (CHO) cell lysates detected two distinct ACE dimers, a native and cross-linked dimer. Increasing amounts of the cross-linked dimer were observed for the mutant sACEQ22A, further implicating the overlapping region of the mAb 9B9 and 3G8 epitopes in ACE processing.
Gordon
K
K
Division of Medical Biochemistry, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
Balyasnikova
I V
IV
Nesterovitch
A B
AB
Schwartz
D E
DE
Sturrock
E D
ED
Danilov
S M
SM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D002241:Carbohydrates / Q000235:genetics / Q000276:immunology; D002462:Cell Membrane / Q000235:genetics / Q000276:immunology; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005260:Female; D009154:Mutation; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D055503:Protein Multimerization
D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D000939:Epitopes; D007703:Peptidyl-Dipeptidase A