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 pro teolysis. We have constructed stalk glycosylation mutants of angiotensin-co nverting enzyme (ACE), a type I ectoprotein that is vigorously shed when ex pressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation d id not significantly inhibit release. Introduction of an N-linked glycan di rectly 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 th e Phe-640/Leu-641 bond. Substitution of the wildtype 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 gly cosylation may have been due to membrane proximity. Relative to levels of c ell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL wer e enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr b ond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristic s of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3,4-d ichloroisocoumarin. These data indicate that (1) stalk glycosylation modula tes but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-gl ycosylated stalk directs cleavage, at least in part, by an alternative shed ding protease, which may resemble an activity recently described in TNF-alp ha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].