Insertion of hydrophilic amino acid residues in the signal peptide/membrane anchor domain of neprilysin (neutral endopeptidase-24.11) results in its cleavage: role of the position of insertion.
We have expressed in COS-1 cells mutants of neprilysin (neutral endopeptidase-24.11; NEP) in which the hydrophilic sequence S-Q-N-S was either substituted for V42-T-M-I or inserted after T38 in the signal peptide/membrane anchor (SA) domain. These mutations were introduced in full-length NEP (mutants NEP(H1) and NEP(H2), respectively) and a form of NEP lacking its cytosolic tail (mutants NEP delta cyto(H1) and NEP delta cyto(H2), respectively). Immunoblotting showed that NEP(H1) was membrane-bound while NEP delta cyto(H1), NEP(H2), and NEP delta cyto(H2) were secreted. Furthermore, carbonate treatment of isolated intracellular membranes suggested that cleavage of the SA domain was performed in the endoplasmic reticulum, presumably by signal peptidase. Sequencing of the secreted proteins indicated that cleavage of the SA domain mostly occurred at the carboxy side of Ala46 but also at the carboxy side of Ala41 in NEP(H2) and NEP delta cyto(H2). We conclude that the position of the S-Q-N-S sequence influences the accessibility of the cleavage site and, in the case of NEP(H1) and NEP(H2), the efficiency of cleavage of the SA domain.
Yang
X F
XF
Département de biochimie, Faculté de médecine, Université de Montréal, Canada.
Chatellard
C
C
Lazure
C
C
Crine
P
P
Boileau
G
G
D000595:Amino Acid Sequence; D000818:Animals; D002254:Carbonates / Q000494:pharmacology; D002522:Chlorocebus aethiops; D010450:Endopeptidases / Q000378:metabolism; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D012995:Solubility; D013329:Structure-Activity Relationship
D002254:Carbonates; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D008666:Metalloendopeptidases; D015260:Neprilysin