Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme.
Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.
Naperova
Irina A
IA
Chemistry Faculty, Moscow State University, Russia, Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL 60612, USA.
Balyasnikova
Irina V
IV
Schwartz
David E
DE
Watermeyer
Jean
J
Sturrock
Edward D
ED
Kost
Olga A
OA
Danilov
Sergei M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000032:analysis*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D000939:Epitopes; D006031:Glycosylation; D006801:Humans; D008251:Macaca; D008958:Models, Molecular; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000276:immunology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007703:Peptidyl-Dipeptidase A