Production and properties of a recombinant soluble form of aminopeptidase A.
Aminopeptidase A is a homodimeric membrane-bound zinc metallopeptidase anchored at the plasma membrane by a 22-amino-acid hydrophobic segment. The anchor segment separates a small N-terminal cytoplasmic domain from a large ectodomain that contains the active site. Site-directed mutagenesis was performed to investigate the role of the cytoplasmic domain of aminopeptidase A in membrane anchoring and routing of the enzyme. Expression in COS-7 cells of a mutant lacking the N-terminal cytoplasmic domain resulted in the efficient secretion of a catalytically active enzyme in the medium. The soluble mutated aminopeptidase A, purified from the medium of a stable cell line, exhibited similar biochemical features to those of the wild-type enzyme. Pulse/chase metabolic labeling experiments revealed that the soluble form is generated intracellularly at an early stage of biosynthesis, suggesting that the signal peptide/membrane anchor domain of aminopeptidase A is removed in the endoplasmic reticulum through the action of the signal peptidase.
INSERM Unit 36, Collège de France, Paris.
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Vazeux
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Wilk
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Wilk
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Corvol
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Llorens-Cortès
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D018076:DNA, Complementary; D043384:Glutamyl Aminopeptidase; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase