Somatic angiotensin converting enzyme (sACE) is comprised of two homologous domains (N and C domains), whereas the smaller germinal isoform (tACE) is identical to the C domain. Both isozymes share an identical stalk, transmembrane and cytoplasmic domain, and undergo ectodomain shedding by an as yet unknown protease. Here we present evidence for the role of regions distal and proximal to the cleavage site in human ACE shedding. First, because of intrinsic differences between the N and C domains, discrete secondary structures (alpha-helix 7 and 8) on the surface of tACE were replaced with their N domain counterparts. Surprisingly, neither alpha-helix 7 nor alpha-helix 8 proved to be an absolute requirement for shedding. In the proximal ectodomain of tACE residues H-610-L-614 were mutated to alanines and this resulted in a decrease in ACE shedding. An N-terminal extension of this mutation caused a reduction in cellular ACE activity. More importantly, it affected the processing of the protein to the membrane, resulting in expression of an underglycosylated form of ACE. When E-608-H-614 was mutated to the homologous region of the N domain, processing was normal and shedding only moderately decreased suggesting that this region is more crucial for the processing of ACE than it is for regulating shedding. Finally, to determine whether glycosylation of the asparagine proximal to the Pro1199-Leu polymorphism in sACE affected shedding, the equivalent (PL)-L-623 mutation in tACE was investigated. The (PL)-L-623 tACE mutant showed an increase in shedding and MALDI MS analysis of a tryptic digest indicated that (NWT)-W-620 was glycosylated. The absence of an N-linked glycan at N-620, resulted in an even greater increase in shedding. Thus, the conformational flexibility that the leucine confers to the stalk, is increased by the lack of glycosylation reducing access of the sheddase to the cleavage site. (C) 2016 Elsevier Inc. All rights reserved.