Proteolytic Release of Membrane-Bound Angiotensin-Converting Enzyme: Role of the Juxtamembrane Stalk Sequence

作者：Mario R. Ehlers, Sylva L. U. Schwager, Renate R. Scholle, Gulam Abbas Manji, Wolf Friedrich BRANDT, James F Riordan · 发表于：Biochemistry · 年份：1996 · DOI：10.1021/bi9602425 · 被引用次数：69 · 研究领域：Protein Hydrolysis and Bioactive Peptides、Trace Elements in Health、Cholesterol and Lipid Metabolism

Many structurally and functionally diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors, and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE; human testis isozyme) or one of four juxtamembrane (stalk) mutants containing either deletions of 17, 24, and 47 residues (ACE-JM delta 17, -JM delta 24, and -JM delta 47, respectively) or a substitution of 26 stalk residues with a 20-residue sequence from the stalk of the low-density lipoprotein receptor (ACE-JMLDL). The C termini of released, soluble WT-ACE and ACE-JM delta 17 and -JMLDL were determined by MALDI-TOF mass spectrometry analyses of C-terminal peptides generated by CNBr cleavage. Observed masses of 4264 (WT-ACE) and 4269 (ACE-JM delta 17) are in good agreement with an expected mass of 4262 for the C-terminal CNBr peptide ending at Arg-627, indicating cleavage at the Arg-627/Ser-628 bond in both WT-ACE and ACE-JM delta 17, at distances of 24 and 10 residues from the membrane, respectively. Data for ACE-JM delta 24 are also consistent with cleavage at or near Arg-627. For ACE-JMLDL, in which the native cleavage site is absent, observed masses of 4372 and 4542 a...