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

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Department of Medical Biochemistry, University of Cape Town Medical School, Observatory 7925, South Africa, Department of Biochemistry, University of Cape Town, Rondebosch 7700, South Africa, and Center for Biochemical and Biophysical Sciences and Medicine, Harvard Medical School, Boston, Massachusetts 02115
Cite this: Biochemistry 1996, 35, 29, 9549–9559
Publication Date (Web):July 23, 1996
Copyright © 1996 American Chemical Society

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    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-JMΔ17, -JMΔ24, and -JMΔ47, 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-JMΔ17 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- JMΔ17) 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-JMΔ17, at distances of 24 and 10 residues from the membrane, respectively. Data for ACE-JMΔ24 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 are in close agreement with expected masses of 4371 and 4542 for peptides ending at Ala-628 and Gly-630, respectively, indicating cleavages at 17 or 15 residues from the membrane. These data indicate that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, cleavage at a distance of 10 residues from the membrane is more favorable, as ACE-JMΔ17 is cleaved 12-fold faster than WT-ACE. In contrast, ACE-JMΔ24 is released 17-fold slower, suggesting that a minimum distance from the membrane must be preserved. This is supported by results with the ACE-JMΔ47 mutant, which is membrane-bound but not cleaved, likely because the entire stalk has been deleted. Finally, soluble full-length (anchor-plus) WT-ACE is not cleaved when incubated with various CHO cell fractions or intact CHO cells. On the basis of these and other data, we propose that the CHO cell MPSP that solubilizes ACE (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain; and (4) may have a weak preference for cleavage at Arg/Lys-X bonds.

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     Supported by a grant from the South African Medical Research Council (M.R.W.E.), NIH Grant HL34704 (J.F.R.), a grant from the Foundation for Research Development (W.F.B.), the Honors Program, University of MichiganFlint (G.A.M.), and grants from the Nellie Atkinson, Latham and Caporn, and Cancer Research Trusts.


     To whom correspondence should be addressed. Tel:  +27 21 406-6335. FAX:  +27 21 47-7669. E-mail:  [email protected].

     Department of Medical Biochemistry.

     Present address:  Department of Biochemistry and Institute for Molecular Virology, University of WisconsinMadison, Madison, WI 53706.


     Department of Biochemistry.

     Harvard Medical School.

     Abstract published in Advance ACS Abstracts, June 15, 1996.

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