Localization of an N-domain region of angiotensin-converting enzyme involved in the regulation of ectodomain shedding using monoclonal antibodies

Irina V. Balyasnikova, Zenda L. Woodman, Ronald F. Albrecht, Ramanathan Natesh, K. Ravi Acharya, Edward D. Sturrock, Sergei M. Danilov*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

28 Scopus citations

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 (ACEΔC) which demonstrated the lowest rate of shedding (12%). However, deletion of the N domain of somatic ACE (ACEAN) dramatically increased shedding (212%). Testicular ACE (tACE) having 36 amino acid residues (heavily O-glycosylated) at the N-terminus of the C domain shows a 4-fold decrease in the rate of shedding (49%) compared to that of ACEΔN. When the N-terminal region of the C domain was replaced with the corresponding homologous 141 amino acids of the N domain (N-delACE) the rate of shedding of the ACEΔN was only slightly decreased (174%), but shedding was still 3.5-fold more efficient than wild-type testicular ACE. Monoclonal antibodies specific for distinct, but overlapping, N-domain epitopes altered the rate of ACE shedding. The mAb 3G8 decreased the rate of shedding by 30%, whereas mAbs 9B9 and 3A5 stimulated ACE shedding 2- to 4-fold. Epitope mapping of these mAbs in conjunction with a homology model of ACE N domain structure, localized a region in the N-domain that may play a role in determining the relatively low rate of shedding of somatic ACE from the cell surface.

Original languageEnglish (US)
Pages (from-to)258-267
Number of pages10
JournalJournal of Proteome Research
Volume4
Issue number2
DOIs
StatePublished - Mar 1 2005

Keywords

  • Angiotensin l-converting enzyme
  • Epitope mapping
  • Monoclonal antibody
  • Shedding

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)

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