Conformational Fingerprinting Using Monoclonal Antibodies (on the Example of Angiotensin I-Converting Enzyme-ACE)

Abstract

⎯During the past 30 years my laboratory has generated 40+ monoclonal antibodies (mAbs) directed to structural and conformational epitopes on human ACE as well as ACE from rats, mice and other species. These mAbs were successfully used for detection and quantification of ACE by ELISA, Western blotting, flow cytometry and immunohistochemistry. In all these applications mainly single mAbs were used. We hypothesized that we can obtain a completely new kind of information about ACE structure and function if we use the whole set of mAbs directed to different epitopes on the ACE molecule. When we finished epitope mapping of all mAbs to ACE (and especially, those recognizing conformational epitopes), we realized that we had obtained a new tool to study ACE. First, we demonstrated that binding of some mAbs is very sensitive to local conformational changes on the ACE surface-due to local denaturation, inactivation, ACE inhibitor or mAbs binding or due to diseases. Second, we were able to detect, localize and characterize several human ACE mutations. And, finally, we established a new concept-conformational fingerprinting of ACE using mAbs that in turn allowed us to obtain evidence for tissue specificity of ACE, which has promising scientific and diagnostic perspectives. The initial goal for the generation of mAbs to ACE 30 years ago was obtaining mAbs to organ-specific endothelial cells, which could be used for organ-specific drug delivery. Our systematic work on characterization of mAbs to numerous epitopes on ACE during these years has lead not only to the generation of the most effective mAbs for specific drug/gene delivery into the lung capillaries, but also to the establishment of the concept of conformational fingerprinting of ACE, which in turn gives a theoretical base for the generation of mAbs, specific for ACE from different organs. We believe that this concept could be applicable for any glycoprotein against which there is a set of mAbs to different epitopes.

Epitope mapping of novel monoclonal antibodies to human angiotensin I‐converting enzyme

“…The ACE fingerprint could thus potentially identify the cells from which ACE originates. 13,16 We also previously proved that the conformation is tissue-specific by comparing the conformational fingerprint of lung and seminal fluid ACEs using a set of 17 mAbs to epitopes of human ACE. Patterns of binding to ACE isolated from the lungs and seminal fluid were dramatically different, 15 reflecting differences in the local conformations of these ACE isoforms, likely due to different patterns of ACE glycosylation in the lung endothelial cells and in the epithelial cells of the epididymis/prostate.…”

“…and C domains, all expressed in CHO cells (Figure 1d) and showed that the pattern of ACE activity precipitation for WTΔ ACE (soluble ACE without transmembrane anchor 27 ), also differed (for four mAbs) from the pattern observed for lung ACE (Figure S1A), confirming that glycosylation of ACE (as well as the pattern of mAb binding) is both cell-and tissue-specific. 13,15,16 Paradoxically, it was found 35 that ACE activity was increased in the cerebrospinal fluid of patients with Alzheimer's diseases (AD), which was confirmed by several studies of ACE activity in the AD brain, while ACE immunoreactive protein in the CSF of patients with AD was significantly decreased. In the Miners et al study, ACE immunoreactive protein was quantified by R&D ACE assay, where a monoclonal antibody specific for human ACE had been pre-coated onto a microplate and to precipitate ACE from CSF or brain homogenates.…”

“…Binding of mAb 1B3 to ACE from a patient with the P1199L mutation decreased several folds, confirming previous results. 16 Decrease of mAb 1A2 binding to ACE from both patients could be considered as an indication that the epitope for 1A2 is sensitive to conformational changes in the C terminal end of soluble ACE due to different mutations in the juxtamembrane stalk region.…”

“…We previously generated and characterized a panel of 16 mAbs against conformational epitopes on the both the N and C domains of ACE. This set of mAbs have been extensively mapped, which allowed us to establish the novel approach for ACE characterization, denoted conformational fingerprinting, [13][14][15][16] to facilitate the study of various aspects of ACE structure and biology as well as ACE-related pathologies, such as sarcoidosis, 13 uremia, 14 Gaucher's disease, 17 prostate and lung cancers. 18,19 Moreover, epitope mapping of these mAbs helped to identify several important functional mutations of ACE-reviewed in Reference 20.…”

“…21 For example, mAb 3A5 induces shedding and has an anti-catalytic effect, while mAb 1B3 allows for the detection of an ACE mutation in the juxtamembrane stalk region, and mAbs 6A12/1G12 can be used to detect ACE inhibitors in patient blood samples. 16 The study on the effect of mAbs on ACE ectodomain shedding or dimerization in reverse micelles have revealed a region in the N domain that suppresses ACE shedding, and may be involved in dimerization. 22 The characterization of mAb epitopes to ACE at the molecular level is clearly an invaluable tool for investigating ACE structure and function.…”