Localization of an N-domain region of angiotensin-converting enzyme involved in the regulation of ectodomain shedding using monoclonal antibodies.
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 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 ACEDeltaN. 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 ACEDeltaN 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.
Balyasnikova
Irina V
IV
Department of Anesthesiology, University of Illinois at Chicago, IL 60612, USA.
Woodman
Zenda L
ZL
Albrecht
Ronald F
RF
Natesh
Ramanathan
R
Acharya
K Ravi
KR
Sturrock
Edward D
ED
Danilov
Sergei M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011323:Primates; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D017931:DNA Primers