Monoclonal antibodies 1B3 and 5C8 as probes for monitoring the integrity of the C-terminal end of soluble angiotensin-converting enzyme.
Angiotensin-converting enzyme (ACE) is a membrane-anchored ectoprotein that is proteolytically cleaved, yielding an enzymatically active soluble ACE. Two mouse monoclonal antibodies, MAbs 1B3 and 5C8, were generated to the C-terminal part of human soluble ACE. MAb 1B3 recognized the catalytically active ACE, as revealed by ELISA and precipitation assays, whereas Western blotting and immunohistochemisty on paraffin- embedded sections using MAb 5C8 detected denatured ACE. MAb 1B3 showed extensive cross-reactivity, recognizing 15 species out of the 16 tested. The binding of this MAb to ACE was greatly affected by conformational changes induced by adsorption on plastic, formalin fixation, and underglycosylation. Furthermore, MAb 1B3 binding to the mutated ACE (Pro1199Leu substitution in the juxtamembrane region, leading to a fivefold increase in serum ACE level) was markedly decreased. MAb 5C8 detected all the known expression sites of full-size ACE using formalin-fixed and paraffin-embedded human tissues. The sequential epitope for MAb 5C8 is formed by the last 11 amino acid residues of soluble ACE (Pro1193-Arg1203), whereas the conformational epitope for 1B3 is formed by a motif within these 11 amino acid residues and, in addition, by at least one stretch that includes Ala837-His839 located distal to the sequential epitope. Our findings demonstrated that MAbs 1B3 and 5C8 are very useful for the study of ACE shedding, for identification of mutations in stalk regions, and for studying alternatively spliced variants of ACE. In addition, binding of MAb 1B3 is a sensitive determinant of the integrity of soluble ACE.
Balyasnikova
Irina V
IV
Department of Anesthesiology, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
Sun
Zhu-Li
ZL
Franke
Folker E
FE
Berestetskaya
Yulia V
YV
Chubb
Antony J
AJ
Albrecht
Ronald F
RF
Sturrock
Edward D
ED
Danilov
Sergei M
SM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003429:Cross Reactions / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D005260:Female; D005470:Fluorometry; D006801:Humans; D006825:Hybridomas; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008297:Male; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D007703:Peptidyl-Dipeptidase A / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D016415:Sequence Alignment; D013045:Species Specificity; D013094:Spermatozoa / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D007703:Peptidyl-Dipeptidase A