The effect of structural motifs on the ectodomain shedding of human angiotensin-converting enzyme.
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 (α-helix 7 and 8) on the surface of tACE were replaced with their N domain counterparts. Surprisingly, neither α-helix 7 nor α-helix 8 proved to be an absolute requirement for shedding. In the proximal ectodomain of tACE residues H610-L614were 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 E608-H614was 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 P623L mutation in tACE was investigated. The P623L tACE mutant showed an increase in shedding and MALDI MS analysis of a tryptic digest indicated that N620WT was glycosylated. The absence of an N-linked glycan at N620, 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.
Department of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory 7925, South Africa.
Nailah
N
Conrad
Department of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory 7925, South Africa.
Sylva L U
SL
Schwager
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, Brazil.
Adriana K
AK
Carmona
Department of Integrative Biomedical Sciences, Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Observatory 7925, South Africa. Electronic address: Edward.Sturrock@uct.ac.za.
Edward D
ED
Sturrock
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D055252:Cell-Derived Microparticles / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D003412:Cricetulus; D004789:Enzyme Activation; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D011485:Protein Binding; D011487:Protein Conformation; D000072417:Protein Domains; D013329:Structure-Activity Relationship
D008565:Membrane Proteins; D007703:Peptidyl-Dipeptidase A
Cellular processing; Cleavage; Ectodomain shedding; Glycosylation; Protein-protein interactions; Stalk