Shedding of somatic angiotensin-converting enzyme (ACE) is inefficient compared with testis ACE despite cleavage at identical stalk sites

Abstract

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg""$(\Leu""$)) clearly recognized soluble porcine somatic

Fine epitope mapping of monoclonal antibodies 9B9 and 3G8 to the N domain of angiotensin-converting enzyme (CD143) defines a region involved in regulating angiotensin-converting enzyme dimerization and shedding

“…Sheddases are known to require interaction with regions distal to the cleavage site for release of their substrates from the membrane (55); therefore, it is possible that a region within the ACE N domain is involved in contact between sACE and its cognate sheddase or this region participates in ACE dimerization, which in turn alters the rate of shedding. Orientation of the N domain with respect to the C domain could play a regulatory role in cleavage where, depending on its position, the N domain could either hamper or facilitate access of the sheddase, explaining why single domain tACE is more readily cleaved (56,57). Furthermore, as proposed previously (18,26), the same effect is produced in the presence of mAbs 9B9 and 3G8, where the induction of shedding observed with mAb 9B9 results from ACE:mAb interaction that positions the N domain in such a way to allow access of the sheddase, whereas mAb 3G8 has the opposite effect.…”

A disintegrin and metalloproteinase (ADAM)‐mediated ectodomain shedding of ADAM10

“…Therefore, it is more likely that ADAM10 is cleaved at a point N-terminal to Gly652 (i.e. the position at which the GPI-AD10-FLAG construct was truncated) which would explain why both WT-AD10-FLAG and GPI-AD10-FLAG were shed; a quite credible possibility as another known ADAM substrate, angiotensinconverting enzyme, is cleaved some 24 residues from the transmembrane domain (Woodman et al 2000). Future work to affinity purify shed WT-AD10-FLAG will enable Cterminal sequencing of the protein with a view to identifying the sheddase cleavage site.…”

The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity

“…Both sACE and tACE are type I ectoproteins that are shed by proteolytic cleavage at identical stalk cleavage sites by an unidentified sheddase. However, shedding of sACE is inefficient compared with that of tACE, but is increased 10-fold after deletion of the N domain [2,3].…”

“…An alternative explanation for the different rates of shedding observed for sACE and tACE [3] is that the N domain partially obstructs the stalk cleavage site. This concept finds some support from evidence for domain co-operativity in sACE.…”