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Updated: Sep 30, 2026

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis
Published on: March 17, 2010
Two C-terminal cysteines are necessary for proper folding of the peptidase neprilysin/CD10
Alexander Navarrete Santos1, Jens Wulfänger, Grit Helbing
1ACGT ProGenomics AG, Halle, Germany.
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.
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