Modulation of Juxtamembrane Cleavage (“Shedding”) of Angiotensin-Converting Enzyme by Stalk Glycosylation:  Evidence for an Alternative Shedding Protease

• Published in: Biochemistry (Easton) 1999-08, Vol.38 (32), p.10388-10397
• Main Authors: Schwager, Sylva L. U, Chubb, Anthony J, Scholle, Renate R, Brandt, Wolf F, Mentele, Reinhard, Riordan, James F, Sturrock, Edward D, Ehlers, Mario R. W
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Carbohydrates - analysis; Cell-Free System - chemistry; Cell-Free System - metabolism; CHO Cells; Cricetinae; Glycosylation; Humans; Kinetics; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Sequence Data; Molecular Weight; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Peptidyl-Dipeptidase A - biosynthesis; Peptidyl-Dipeptidase A - chemistry; Peptidyl-Dipeptidase A - genetics; Peptidyl-Dipeptidase A - metabolism; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Sequence Deletion; Solubility
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi990357j

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-α convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765−27767].