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

Marwan Alfalah,Edward T Parkin,Ralf Jacob,Edward D Sturrock,Reinhard Mentele,Anthony J Turner,Nigel M Hooper,Hassan Y Naim

doi:10.1074/jbc.m100339200

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 degrees 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.

Highlights

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
Both constructs were expressed in the cells as determined by metabolic labeling with [35S]Met followed by immunoprecipitation from the cell lysate with the anti-ACE antibody (Fig. 2). wtACE was present in the medium, consistent with its release from the cell surface by the batimastat-sensitive secretase (Fig. 2)
ACENQ was detected in the medium from the cells, and the relative ratio of ACE protein in the medium compared with the cell lysate appeared greater for the mutant as compared with the wild-type protein

Summary

Secretase Cleavage of ACE

Indicating that the mutation has no general effects on the folding of ACE. Pulse-chase analysis revealed that ACENQ was more readily cleaved and secreted into the medium than the wild-type protein. Mass spectrometric analysis of the secreted protein and temperature block and inhibitor studies indicated that ACENQ was being cleaved between Asn635 and Ser636 in the endoplasmic reticulum (ER) by a serine protease and not between Arg638 and Ser639 at the cell surface by the batimastat-sensitive metallosecretase. In light of this observation, the conclusions of earlier studies investigating the sequence requirements of various secretases through analysis of the effect of mutations on the secretion of the substrate protein require re-evaluation and should not be interpreted solely in the context of a single protease activity having a relaxed specificity

EXPERIMENTAL PROCEDURES

RESULTS

Medium wtACE ACENQ

DISCUSSION