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

Abstract

Background: The 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.

Results: The 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.

Conclusion: The 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.

Figure 1

A section of a multiple sequence alignment of M13 peptidases. A multiple sequence alignment of 111 protein sequences was generated using MUSCLE [32,33] and was as the basis for the analysis presented. A highly conserved section of the alignment, representing residues 541 to 652 of human neprilysin, contains important catalytic residues. These residues include the HExxH zinc binding motif and the catalytically important GENIAD and VNAFY motifs which are coloured blue. For full alignment see additional file 2.

Figure 2

Phylogenetic analysis of M13 peptidases. Majority consensus tree of all three methods of phylogenetic reconstruction. The tree was generated using CTree [65]. See additional file 3 for bootstrap values. Key to species; Dme Drosophila melanogaster, Aga Anopheles gambiae, Dps Drosophila pseudoobscura, Lmi Locusta migratoria, Bmo Bombyx mori, Ame Apis mellifera, Hsa Homo sapiens, Rno Rattus norvegicus, Mmu Mus musculus, Fru Fugu rubripes, Pfl Perca flavescens, Xla Xenopus laevus, Ocu Oryctolagus cuniculus, Bta Bos taurus, Cpo Cavia porcelllus, Cin Ciona intestinalis, Cel Caenorhabditis elegans and Cbr Caenorhabditis briggsae.

Figure 3

Utilisation of residues by ligand binding subsites of M13 peptidases. The utilisation of the twenty amino acids in both the S1' and S2' subsites was examined. The percentage contribution of each amino acid to either binding site was calculated and residues were placed in the order of descending frequency for the S1' subsite.