title: Two C-terminal cysteines are necessary for proper folding of the peptidase neprilysin/CD10.
abstract: Neprilysin (NEP) consists of 749 amino acids with two conserved cysteines (734, 746) and a putative CAAX motif (residues 746-749, CRVW) at the C-terminus. To investigate the role of the C-terminal conserved cysteine residues, three NEP mutants (C734S, C746S, and double mutant C734S/C746S) were constructed by use of site-directed mutagenesis. Western blot analysis of lysates of transfected cells revealed the presence of three NEP forms in wild type and mutants with a different glycosylation pattern. Point mutations of C734 as well as C746 by serine dramatically diminished the plasma membrane association of NEP as detected by flow cytometry and laser scanning microscopy. Endoprotease enzyme activity was slightly diminished in the C746S-NEP variant and was not detectable in the C734S-form of NEP suggesting a pivotal role of the C734 in the proper folding of the enzyme. Prenylation of NEP was not detected in an in vivo assay.
authors.affiliation: ACGT ProGenomics AG, Halle, Germany.
authors.forename: Alexander
authors.initials: A
authors.lastname: Navarrete Santos
authors.forename: Jens
authors.initials: J
authors.lastname: Wulfänger
authors.forename: Grit
authors.initials: G
authors.lastname: Helbing
authors.forename: Tanja
authors.initials: T
authors.lastname: Blosz
authors.forename: Jürgen
authors.initials: J
authors.lastname: Langner
authors.forename: Dagmar
authors.initials: D
authors.lastname: Riemann
mesh_terms: D001483:Base Sequence; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism*; D017931:DNA Primers; D006801:Humans; D016297:Mutagenesis, Site-Directed; D015260:Neprilysin / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017368:Protein Prenylation
chemical_list: D017931:DNA Primers; D015260:Neprilysin; D003545:Cysteine