Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme

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dc.contributor.authorDanilov, S M
dc.contributor.authorWatermeyer, J M
dc.contributor.authorBalyasnikova, I V
dc.contributor.authorGordon, K
dc.contributor.authorKugaevskaya, E V
dc.contributor.authorElisseeva, Y E
dc.contributor.authorAlbrecht, R F
dc.contributor.authorSturrock, E D
dc.date.accessioned2016-09-05T19:00:30Z
dc.date.available2016-09-05T19:00:30Z
dc.date.issued2007
dc.date.updated2016-09-05T12:48:02Z
dc.description.abstractAngiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with α-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE.en_ZA
dc.identifierhttp://dx.doi.org/10.1021/bi700489v
dc.identifier.apacitationDanilov, S. M., Watermeyer, J. M., Balyasnikova, I. V., Gordon, K., Kugaevskaya, E. V., Elisseeva, Y. E., ... Sturrock, E. D. (2007). Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme. <i>Biochemistry</i>, http://hdl.handle.net/11427/21675en_ZA
dc.identifier.chicagocitationDanilov, S M, J M Watermeyer, I V Balyasnikova, K Gordon, E V Kugaevskaya, Y E Elisseeva, R F Albrecht, and E D Sturrock "Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme." <i>Biochemistry</i> (2007) http://hdl.handle.net/11427/21675en_ZA
dc.identifier.citationDanilov, S. M., Watermeyer, J. M., Balyasnikova, I. V., Gordon, K., Kugaevskaya, E. V., Elisseeva, Y. E., ... & Sturrock, E. D. (2007). Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme. Biochemistry, 46(31), 9019-9031.en_ZA
dc.identifier.issn0006-2960en_ZA
dc.identifier.ris TY - Journal Article AU - Danilov, S M AU - Watermeyer, J M AU - Balyasnikova, I V AU - Gordon, K AU - Kugaevskaya, E V AU - Elisseeva, Y E AU - Albrecht, R F AU - Sturrock, E D AB - Angiotensin I-converting enzyme (ACE, peptidyl dipeptidase, EC 3.4.15.2) is a key enzyme in cardiovascular pathophysiology. A wide spectrum of monoclonal antibodies to different epitopes on the N and C domains of human ACE has been used to study different aspects of ACE biology. In this study we characterized the monoclonal antibody (mAb) 5F1, developed against the N domain of human ACE, which recognizes both the catalytically active and the denatured forms of ACE. The epitope for mAb 5F1 was defined using species cross-reactivity, synthetic peptide (PepScan technology) and phage display library screening, Western blotting, site-directed mutagenesis, and protein modeling. The epitope for mAb 5F1 shows no overlap with the epitopes of seven other mAbs to the N domain described previously and is localized on the other side of the N domain globule. The binding of mAb 5F1 to ACE is carbohydrate-dependent and increased significantly as a result of altered glycosylation after treatment with α-glucosidase-1 inhibitor, N-butyldeoxynojirimycin (NB-DNJ), or neuraminidase. Out of 17 species tested, mAb 5F1 showed strict primate ACE specificity. In addition, mAb 5F1 recognized human ACE in Western blots and on paraffin-embedded sections. The sequential part of the epitope for mAb 5F1 is created by the N-terminal part of the N domain, between residues 1 and 141. A conformational region of the epitope was also identified, including the residues around the glycan attached to Asn117, which explains the sensitivity to changes in glycosylation state, and another stretch localized around the motif 454TPPSRYN460. Site-directed mutagensis and inhibition assays revealed that mAb 5F1 inhibits ACE activity at high concentrations due to binding of residues on both sides of the active site cleft, thus supporting a hinge-bending mechanism for substrate binding of ACE. DA - 2007 DB - OpenUCT DP - University of Cape Town J1 - Biochemistry LK - https://open.uct.ac.za PB - University of Cape Town PY - 2007 SM - 0006-2960 T1 - Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme TI - Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme UR - http://hdl.handle.net/11427/21675 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/21675
dc.identifier.vancouvercitationDanilov SM, Watermeyer JM, Balyasnikova IV, Gordon K, Kugaevskaya EV, Elisseeva YE, et al. Fine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzyme. Biochemistry. 2007; http://hdl.handle.net/11427/21675.en_ZA
dc.language.isoeng
dc.publisherAmerican Chemical Societyen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.sourceBiochemistryen_ZA
dc.source.urihttp://pubs.acs.org/journal/bichaw
dc.titleFine epitope mapping of monoclonal antibody 5F1 reveals anticatalytic activity toward the N domain of human angiotensin-converting enzymeen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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