Epitope mapping of mAbs to denatured human testicular ACE (CD143).
Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.
Balyasnikova
I V
IV
Department of Anesthesiology, University of Illinois at Chicago, Chicago, IL, USA.
Metzger
R
R
Franke
F E
FE
Conrad
N
N
Towbin
H
H
Schwartz
D E
DE
Sturrock
E D
ED
Danilov
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D003429:Cross Reactions / Q000276:immunology*; D018604:Epitope Mapping; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D019151:Peptide Library; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013737:Testis / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D019151:Peptide Library; D007703:Peptidyl-Dipeptidase A