Modulation of juxtamembrane cleavage ("shedding") of angiotensin-converting enzyme by stalk glycosylation: evidence for an alternative shedding protease.
The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].
Schwager
S L
SL
Department of Medical Biochemistry and MRC Liver Research Centre, University of Cape Town Medical School, Observatory 7925, South Africa.
Chubb
A J
AJ
Scholle
R R
RR
Brandt
W F
WF
Mentele
R
R
Riordan
J F
JF
Sturrock
E D
ED
Ehlers
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D012995:Solubility
D002241:Carbohydrates; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007703:Peptidyl-Dipeptidase A