Monoclonal Antibodies 1B3 and 5C8 as Probes for Monitoring the Integrity of the C-Terminal End of Soluble Angiotensin-Converting Enzyme

Abstract

Angiotensin-converting enzyme (ACE) is a membrane-anchored ectoprotein that is proteolytically cleaved, yielding an enzymatically active soluble ACE. Two mouse monoclonal antibodies, MAbs 1B3 and 5C8, were generated to the C-terminal part of human soluble ACE. MAb 1B3 recognized the catalytically active ACE, as revealed by ELISA and precipitation assays, whereas Western blotting and immunohistochemisty on paraffin- embedded sections using MAb 5C8 detected denatured ACE. MAb 1B3 showed extensive cross-reactivity, recognizing 15 species out of the 16 tested. The binding of this MAb to ACE was greatly affected by conformational changes induced by adsorption on plastic, formalin fixation, and underglycosylation. Furthermore, MAb 1B3 binding to the mutated ACE (Pro1199Leu substitution in the juxtamembrane region, leading to a fivefold increase in serum ACE level) was markedly decreased. MAb 5C8 detected all the known expression sites of full-size ACE using formalin-fixed and paraffin-embedded human tissues. The sequential epitope for MAb 5C8 is formed by the last 11 amino acid residues of soluble ACE (Pro1193-Arg1203), whereas the conformational epitope for 1B3 is formed by a motif within these 11 amino acid residues and, in addition, by at least one stretch that includes Ala837-His839 located distal to the sequential epitope. Our findings demonstrated that MAbs 1B3 and 5C8 are very useful for the study of ACE shedding, for identification of mutations in stalk regions, and for studying alternatively spliced variants of ACE. In addition, binding of MAb 1B3 is a sensitive determinant of the integrity of soluble ACE.

Angiotensin I-Converting Enzyme Gln1069Arg Mutation Impairs Trafficking to the Cell Surface Resulting in Selective Denaturation of the C-Domain

“…Gln1069 (brown) is located right under the bump created by Lys1067 and Tyr1068 (purple) and shown by arrow. Dark blue indicates the first N-terminal amino acid residue (Asp616) seen in this structure; orange-indicates the C terminal end of the C-domain (and the epitope for mAb 1B3; [16], [36]. Light blue (P730) and red colored amino acid residues indicate highly immunogenic bumps on the surface of the C-domain.…”

“…Eight mAbs recognize epitopes on the catalytically active N-domain [15], [30], [32]–[34] and 8 mAbs recognize epitopes localized on the C-domain of ACE [16], [36]. We recently demonstrated that the pattern of precipitation of ACE activity (conformational fingerprinting of ACE) using this set of mAbs provides a sensitive means of identifying changes in local conformation of ACE due to inactivation, inhibition, mutations, etc.…”

Angiotensin I-Converting Enzyme Mutation (Trp1197Stop) Causes a Dramatic Increase in Blood ACE

“…We performed Western blotting of ACE purified from plasma of subject N1 using two mAbs to denatured human ACE: mAb 1D8, which recognizes a sequential epitope at the beginning of C domain [46], and mAb 5C8, which is directed to the C-terminal end of soluble ACE and of which binding was dramatically decreased to mutant P1199L ACE [47]. Binding of 5C8 was significantly abolished with both P1199L and W1197X mutants, whereas mAb 1D8 recognized similarly both wild-type and mutant ACE (Fig.…”

Detection of Mutated Angiotensin I-Converting Enzyme by Serum/Plasma Analysis Using a Pair of Monoclonal Antibodies

“…We recently observed reduced binding of soluble ACE in Dutch patients with a Pro1199Leu substitution detected by a new monoclonal antibody (mAb), 1B3, which recognizes a Pro 1199 -containing epitope in the C-terminal region of soluble ACE (25 ). We therefore set out to develop a method that would use mAb 1B3 in combination with mAb 9B9 to the central part of the N-domain of ACE (26 -28 ), which would enable us to distinguish persons with the Pro1199Leu mutation from patients with increased ACE attributable to other diseases, such as sarcoidosis and Gaucher disease.…”

“…For immunocapture studies, the following mAbs to human ACE were used: mAb 1B3, which recognizes a C-terminal part of soluble ACE (25 ), and mAbs 9B9 (26 -28 ) and 2B11, which recognize epitopes in the N-and C-domains of ACE, respectively. ACE activity in human serum or plasma was measured by a fluorometric assay (30,31 ).…”