Bioinformatic analysis of the neprilysin (M13) family of peptidases reveals complex evolutionary and functional relationships

Abstract

BackgroundThe neprilysin (M13) family of endopeptidases are zinc-metalloenzymes, the majority of which are type II integral membrane proteins. The best characterised of this family is neprilysin, which has important roles in inactivating signalling peptides involved in modulating neuronal activity, blood pressure and the immune system. Other family members include the endothelin converting enzymes (ECE-1 and ECE-2), which are responsible for the final step in the synthesis of potent vasoconstrictor endothelins. The ECEs, as well as neprilysin, are considered valuable therapeutic targets for treating cardiovascular disease. Other members of the M13 family have not been functionally characterised, but are also likely to have biological roles regulating peptide signalling. The recent sequencing of animal genomes has greatly increased the number of M13 family members in protein databases, information which can be used to reveal evolutionary relationships and to gain insight into conserved biological roles.ResultsThe phylogenetic analysis successfully resolved vertebrate M13 peptidases into seven classes, one of which appears to be specific to mammals, and insect genes into five functional classes and a series of expansions, which may include inactive peptidases. Nematode genes primarily resolved into groups containing no other taxa, bar the two nematode genes associated with Drosophila DmeNEP1 and DmeNEP4. This analysis reconstructed only one relationship between chordate and invertebrate clusters, that of the ECE sub-group and the DmeNEP3 related genes. Analysis of amino acid utilisation in the active site of M13 peptidases reveals a basis for their biochemical properties. A relatively invariant S1' subsite gives the majority of M13 peptidases their strong preference for hydrophobic residues in P1' position. The greater variation in the S2' subsite may be instrumental in determining the specificity of M13 peptidases for their substrates and thus allows M13 peptidases to fulfil a broad range of physiological roles.ConclusionThe M13 family of peptidases have diversified extensively in all species examined, indicating wide ranging roles in numerous physiological processes. It is predicted that differences in the S2' subsite are fundamental to determining the substrate specificities that facilitate this functional diversity.

Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates

“…We have investigated the wt and the inactive mutants (H493A and E494A) of the metallopeptidase Zmp1, which is an important virulence factor of Mtb (Master et al , 2008 ). Zmp1 belongs to the M13 family (MEROPS database: http://merops.sanger.ac.uk/), such as NEP and ECE-1, whose enzymatic action is restricted to peptides with a strong preference for cleaving the amino terminal bond of hydrophobic residues (Bland et al , 2008 ). This preference for peptides is also confi rmed by the recently solved X-ray crystallographic structure of Zmp1, which reveals that the active site is located at the bottom of a hydrophobic channel, likely impairing the access of large macromolecular substrates in the proximity of the reaction centre (Ferraris et al , 2011 ).…”

Neprilysins: An Evolutionarily Conserved Family of Metalloproteases That Play Important Roles in Reproduction in Drosophila

“…V) at the P1' position (summarized from MEROPS; Rawlings et al 2012). In mammals, individual Neps tend to have very specific substrate affinities despite sharing conserved active site residues, suggesting that this specificity is based on other sequence features (Johnson et al 2002;Rose et al 2002;Bland et al 2008;Whyteside and Turner 2008). Although the cleavage specificities of Drosophila neprilysins are not as well known, there is evidence that Drosophila Nep2 is capable of cleaving locust, human, and fly tachykinins in vitro.…”

“…The D. melanogaster genome has 24 NEP-like genes, most of which are actively transcribed (Coates et al 2000;Chintapalli et al 2007;Bland et al 2008). However, little is known about their roles in vivo.…”

“…P ROTEASES play key roles in diverse physiological systems. One such family of metalloproteases, the M13 class of neutral endopeptidases, consists mainly of membranebound zinc proteases that are involved in the processing of neuropeptides and peptide hormones (reviewed in Turner et al 2000;Turner et al 2001;Bland et al 2008). In mammals, seven members of this family have been identified, of which neprilysin (NEP) and endothelin converting enzyme (ECE) are the best studied.…”

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…They are involved in a great number of biological processes, such as neurotransmission, reproduction, cancer progression, control of blood pressure; therefore, they are considered potential therapeutic targets in cardiovascular and inflammatory disorders (Bland et al, 2008;Turner et al, 2001) (Table 1). M13 enzymes display a short N-terminal cytoplasmic domain, a single transmembrane helix and a C-terminal extracellular domain containing the active site and they are generally selective inhibited by Streptomyces product phosphoramidon (Emoto and Yanagisawa, 1995;Turner and Nalivaeva, 2006).…”