2005
DOI: 10.1021/pr049859w
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Localization of an N-Domain Region of Angiotensin-Converting Enzyme Involved in the Regulation of Ectodomain Shedding Using Monoclonal Antibodies

Abstract: ACE chimeric proteins and N domain monoclonal antibodies (mAbs) were used to determine the influence of the N domain, and particular regions thereof, on the rate of ACE ectodomain shedding. Somatic ACE (having both N and C domains) was shed at a rate of 20%/24 h. Deletion of the C domain of somatic ACE generated an N domain construct (ACEDeltaC) which demonstrated the lowest rate of shedding (12%). However, deletion of the N domain of somatic ACE (ACEDeltaN) dramatically increased shedding (212%). Testicular A… Show more

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Cited by 27 publications
(75 citation statements)
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“…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.…”
Section: Resultsmentioning
confidence: 99%
See 1 more Smart Citation
“…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.…”
Section: Resultsmentioning
confidence: 99%
“…After washing with PBS containing 0.05% Tween 20, the wells were incubated with different anti-ACE mAbs (2 µg/ml) directed to 16 different epitopes on the N and C domain of ACE [15][16], [30], [32][34] in PBS + 0.1 mg/ml BSA for 2 hrs at RT and washed. Wells were then incubated with 50 µl of soluble ACE secreted from CHO cells transfected with wild type or mutant ACE or with lysates from these cells, resembling a membrane-bound form of ACE (wt and mutant).…”
Section: Methodsmentioning
confidence: 99%
“…Both have buried surface areas, encompassing helices 13, 14 and 35 on the one hand and 26 and 27 on the other with areas of 2310.2 Å 2 and 2811.5 Å 2 , respectively. A contact region that might be involved in dimerisation has been proposed on the basis of epitope mapping, 18 but the arrangement of the monomers in the asymmetric unit is somewhat different. There is only partial overlap between the interface around helices 13,14 and 35 and the area proposed to be involved by Balyasnikova et al However, the dimer interaction could be mediated by a carbohydrate recognition domain 17 and the N domain carbohydrates in the crystal structure were enzymatically truncated to facilitate crystallisation.…”
Section: Resultsmentioning
confidence: 99%
“…18 Substrates such as the hemoregulatory peptide AcSDKP, 19 angiotensin 1-7, 20 and the enkephalin precursor Met 5 -EnkArg 6 -Phe 721 are specific for the N domain, whereas the physiological substrates bradykinin and angiotensin I are hydrolysed with similar catalytic efficiency as the C domain. Interestingly, the N domain preferentially hydrolyses the Ab peptide of the amyloid precursor protein resulting in inhibition of Ab aggregation and cytotoxicity in cellbased assays, 22 although this is not necessarily the case in vivo.…”
Section: Introductionmentioning
confidence: 99%
“…We think that some of the changes in pattern of ACE immunoprecipitation by 16 mAbs seen in mutants lacking transmembrane anchor are likely due to the changes in glycosylation pattern of mutated ACE. The time of traffic of mutated ACEs through endoplasmic reticulum is much shorter than a wild-type ACE [41][42], [29] and as a result the glycosylation pattern of mutated ACEs is different from wild-type ACE; this could result in differential binding of some mAbs (i1A8, i2H5, and 5F1), because glycosylation sites on Asn residues are included in their epitopes [40], [43][45].…”
Section: Resultsmentioning
confidence: 99%