A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase

Abstract

Angiotensin I-converting enzyme (ACE) is one of a number of integral membrane proteins that is proteolytically shed from the cell surface by a zinc metallosecretase. Mutagenesis of Asn 631 to Gln in the juxtamembrane stalk region of ACE resulted in more efficient secretion of the mutant protein (ACE NQ ) as determined by pulse-chase analysis. In contrast to the wild-type ACE, the cleavage of ACE NQ was not blocked by the metallosecretase inhibitor batimastat but by the serine protease inhibitor, 1,3-dichloroisocoumarin. Incubation of the cells at 15°C revealed that ACE NQ was cleaved in the endoplasmic reticulum, and mass spectrometric analysis of the secreted form of the protein indicated that it had been cleaved at the Asn 635 -Ser 636 bond, three residues N-terminal to the normal secretase cleavage site at Arg 638 -Ser 639 . These data clearly show that a point mutation in the juxtamembrane region of an integral membrane protein can invoke the action of a mechanistically and spatially distinct secretase. In light of this observation, previous data on the effect of mutations in the juxtamembrane stalk of shed proteins being accommodated by a single secretase having a relaxed specificity need to be re-evaluated.

Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding

“…Thus our data are strongly suggestive that it is the same, or a very closely related, zinc metalloproteinase that cleaves both ACE and MDP-STM ACE . This is in contrast with the results with a mutant of ACE containing a single amino acid change in the juxtamembrane stalk, which is cleaved by a mechanistically and spatially distinct secretase [34].…”

“…The transmembrane region is in italics, although there is uncertainty about its precise start, and VGQ is an alternative [20,24]. MDP (wtMDP), were stably expressed in the IMR32 cell line that we have used previously for the characterization of the ACE secretase [17,34]. The ACE N-terminal domain mutant was designed to test whether there may be a secretase recognition motif in the C-terminal domain of ACE that is absent in the Nterminal domain.…”

Angiotensin I-Converting Enzyme Mutation (Trp1197Stop) Causes a Dramatic Increase in Blood ACE

“…We think that some of the changes in pattern of ACE immunoprecipitation by 16 mAbs seen in mutants lacking transmembrane anchor are likely due to the changes in glycosylation pattern of mutated ACE. The time of traffic of mutated ACEs through endoplasmic reticulum is much shorter than a wild-type ACE [41]–[42], [29] and as a result the glycosylation pattern of mutated ACEs is different from wild-type ACE; this could result in differential binding of some mAbs (i1A8, i2H5, and 5F1), because glycosylation sites on Asn residues are included in their epitopes [40], [43]–[45].…”

Autolytic Processing at Glu586-Ser587within the Cysteine-rich Domain of Human Adamalysin 19/Disintegrin-Metalloproteinase 19 Is Necessary for Its Proteolytic Activity

“…3B), which is consistent with some reports showing that the shedding of MT5-MMP, MT6-MMP, and IL-6 receptor is not affected by metalloproteinase inhibitors (47,52,62). One possibility, we speculate, is that shedding, in most cases, occurs at membrane-proximal regions on the cell surface, which are easily accessible to hydroxamate-based inhibitors and TIMP-3 (5,25,60,63). Human ADAM19 processing takes place at a region distal from the transmembrane domain in the secretory pathway, which is less accessible to GM6001 and TIMP-3 (Fig.…”