Monoclonal antibodies to denatured human ACE (CD 143), broad species specificity, reactivity on paraffin sections, and detection of subtle conformational changes in the C‐terminal domain of ACE

Abstract

Two new mouse monoclonal antibodies (mAbs) were generated to denatured human angiotensin-converting enzyme (ACE, CD143). The clones 2E2 and 3C5, each of the IgG1 kappa chain isotype, detect ACE with high sensitivity, respectively, at 20 ng and 2 ng of protein per lane in Western blotting. They both recognize different epitopes on the C-domain of ACE located between amino acid residues 740 and 992. In formalin-fixed and paraffin-embedded human tissues, immunohistochemistry revealed all known expression sites of ACE, e.g. the epithelial brush borders of proximal kidney tubules, epithelial cells of epididymis, endothelial cells, activated macrophages as well as germ cells during spermatogenesis. In contrast to other mAbs to denatured human ACE, mAbs 2E2 and 3C5 demonstrate cross-reactivity with a broad spectrum of animal species such as monkey, rat, rabbit, cattle, dog, cat, and guinea pig. In addition, mAb 2E2 recognized mouse ACE in Western blotting and on paraffin sections. Our findings suggest that mAbs 2E2 and 3C5 are useful for identifying even subtle changes in ACE conformation resulting from denaturation. These mAbs are also sensitive tools for the detection of minimal amounts of ACE in biological fluids and tissues using proteomics approaches. Their reactivity in routinely processed tissues of various species may prove useful for correlation of ACE expression in animal models to human diseases.

Intrarenal Distributions and Changes of Angiotensin-Converting Enzyme and Angiotensin-Converting Enzyme 2 in Feline and Canine Chronic Kidney Disease

“…Intrarenal localization of ACE has been investigated previously, and the brush border of proximal tubules is a principal site of ACE expression in human kidney [2]. A similar distribution of ACE was demonstrated in canine kidney [1], and the findings from the present study match those of the previous report. Although the localization of ACE in the feline kidney has been unclear, the present study demonstrated strong ACE immunoreactivity in the brush border of the proximal tubules.…”

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

“…For the initial characterization of mutant ACE produced by CHO cells, we performed Western blotting of cell lysates from CHO-WT-ACE and CHO-Q1069R-ACE using several mAbs to denatured human ACE: mouse mAb 1D8, 3C5, and 2E2, which recognize sequential epitopes in the C domain [35]–[37], as well as rat mAb 4G6, that recognizes an epitope on the mouse and human N domain [38]. Fig.…”

An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

“…After electrophoretic transfer of proteins to microporous PVDF-Plus membranes, each membrane was incubated in 10 mM Tris-HCl (pH 8.0) buffer containing 150 mM NaCl, 0.05% Tween 20, and 5% dry milk prior to incubation overnight at 4°C with mouse mAbs to sequential epitopes on human ACE, suitable for detection of the denatured ACE - 3C5, 1D8, 5C8 [45]–[47]. Subsequent steps were carried out with the biotin/streptavidin system (Amresco, Solon, OH) and peroxidase activity was developed using WestPico Super Signal Chemiluminescense substrate (Pierce, Rockford, IL).…”