Abstract
Neprilysin is a transmembrane zinc metallopeptidase that degrades a wide range of peptide substrates. It has received attention as a potential therapy for Alzheimer’s disease due to its ability to degrade the peptide amyloid beta. However, its broad range of peptide substrates has the potential to limit its therapeutic use due to degradation of additional peptides substrates that tightly regulate many physiological processes. We sought to generate a soluble version of the ectodomain of neprilysin with improved activity and specificity towards amyloid beta as a potential therapeutic for Alzheimer’s disease. Extensive amino acid substitutions were performed at positions surrounding the active site and inner surface of the enzyme and variants screened for activity on amyloid beta 1–40, 1–42 and a variety of other physiologically relevant peptides. We identified several mutations that modulated and improved both enzyme selectivity and intrinsic activity. Neprilysin variant G399V/G714K displayed an approximately 20-fold improved activity on amyloid beta 1–40 and up to a 3,200-fold reduction in activity on other peptides. Along with the altered peptide substrate specificity, the mutant enzyme produced a markedly altered series of amyloid beta cleavage products compared to the wild-type enzyme. Crystallisation of the mutant enzyme revealed that the amino acid substitutions result in alteration of the shape and size of the pocket containing the active site compared to the wild-type enzyme. The mutant enzyme offers the potential for the more efficient degradation of amyloid beta in vivo as a therapeutic for the treatment of Alzheimer’s disease.
Highlights
Neprilysin, or neutral endopeptidase (NEP), is an integral type II membrane-bound zinc-dependent peptidase of approximately 750 amino acid residues [1] that degrades a number of physiological peptides that are involved in processes such as blood pressure regulation and nociception
Because of the large substrate repertoires of many proteases, one of the key challenges to exploiting them therapeutically is the lack of specificity towards the target molecule, which may result in undesirable off-target activities
The substrate specificity of rat neurolysin has been altered by a single amino acid substitution, giving a 10-fold specificity switch [39]
Summary
Neprilysin, or neutral endopeptidase (NEP), is an integral type II membrane-bound zinc-dependent peptidase of approximately 750 amino acid residues [1] that degrades a number of physiological peptides that are involved in processes such as blood pressure regulation and nociception. NEP is composed of an ectodomain, which contains the catalytic site and belongs to the M13 family of peptidases/proteases, a transmembrane domain and a short intracellular domain. The larger N-terminal domain (domain 1), which is structurally related to the bacterial protease thermolysin, contains a single zinc atom that is critical for peptidase activity [2,3,4] and is coordinated by His and Glu residues [5]. Whilst the substrate specificity of the enzyme is quite broad, NEP has a strong preference for peptides over larger proteins. This specificity seems to result from the enclosed catalytic chamber and size-restricted access to that chamber. While structures of NEP in complex with different inhibitors display similar main-chain conformation, large ligand induced flexibility is observed for the side chains that form the substrate binding pockets (such as Arg102, Phe106, Arg110 and Trp693), which may explain the broad substrate specificity of the enzyme [5,7,8]
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