A point mutation in the juxtamembrane stalk of human angiotensin I-converting enzyme invokes the action of a distinct secretase.
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.
Alfalah
M
M
Department of Physiological Chemistry, School of Veterinary Medicine, D-30559 Hannover, Germany.
Parkin
E T
ET
Jacob
R
R
Sturrock
E D
ED
Mentele
R
R
Turner
A J
AJ
Hooper
N M
NM
Naim
H Y
HY
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma; D009474:Neurons; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000494:pharmacology; D017354:Point Mutation; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013876:Thiophenes / Q000494:pharmacology; D014162:Transfection
D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D013876:Thiophenes; D010649:Phenylalanine; C080985:batimastat; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D007703:Peptidyl-Dipeptidase A; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human