Molecular Identification and Characterization of Novel Membrane-bound Metalloprotease, the Soluble Secreted Form of Which Hydrolyzes a Variety of Vasoactive Peptides

Reed

Journal of Biological Chemistry

2001

320

245

Deposition of ␤-amyloid (A␤) peptides in the brain is an early and invariant feature of all forms of Alzheimer's disease. As with any secreted protein, the extracellular concentration of A␤ is determined not only by its production but also by its catabolism. A major focus of Alzheimer's research has been the elucidation of the mechanisms responsible for the generation of A␤. Much less, however, is known about the mechanisms responsible for A␤ removal in the brain. In this report, we describe the identification of endothelin-converting enzyme-1 (ECE-1) as a novel A␤-degrading enzyme. We show that treatment of endogenous ECE-expressing cell lines with the metalloprotease inhibitor phosphoramidon causes a 2-3-fold elevation in extracellular A␤ concentration that appears to be due to inhibition of intracellular A␤ degradation. Furthermore, we show that overexpression of ECE-1 in Chinese hamster ovary cells, which lack endogenous ECE activity, reduces extracellular A␤ concentration by up to 90% and that this effect is completely reversed by treatment of the cells with phosphoramidon. Finally, we show that recombinant soluble ECE-1 is capable of hydrolyzing synthetic A␤40 and A␤42 in vitro at multiple sites.

Section: Resultsmentioning

confidence: 99%

Degradation of the Alzheimer's Amyloid β Peptide by Endothelin-converting Enzyme

Reed

Journal of Biological Chemistry

2001

320

245

Journal of Biological Chemistry

“…Therefore, it constitutes a poor template, seeing it is a distant bacterial enzyme (from Bacillus stearothermophilus) that does not contain all of the catalytically important residues of NEP, and is significantly shorter in length than NEP and its homologues (316 amino acids for thermolysin versus 743 for hNEP). The recent disclosure of the structure of the ectodomain of NEP obtained by x-ray crystallography (33) provides a new and relevant template to model closely related members of the M13 subfamily and, in particular, the recently characterized NEP2 neuropeptidase (1)(2)(3). The obtained active site model of NEP2 (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Neprilysin 2 (SEP and NL-1) is a recently identified type II membrane-bound zinc-dependent metalloprotease (1)(2)(3). It is part of the M13 family of metalloproteases, which also comprises neprilysin (NEP, 1 EC 3.4.24.11) (4 -6), the endothelinconverting enzymes, ECE-1 (EC 3.4.24.71) (7) and ECE-2 (8), the Kell blood group antigen (9), the phosphate-regulating neutral endopeptidase on the X chromosome (10) and X-converting enzyme (11).…”

mentioning

confidence: 99%

A Three-dimensional Model of the Neprilysin 2 Active Site Based on the X-ray Structure of Neprilysin

Voisin

Rognan

Gros

et al. 2004

Neprilysin 2 (NEP2), a recently identified member of the M13 subfamily of metalloproteases, shares the highest degree of homology with the prototypical member of the family neprilysin. Whereas the study of the in vitro enzymatic activity of NEP2 shows that it resembles that of NEP as it cleaves the same substrates often at the same amide bonds and binds the same inhibitory compounds albeit with different potencies, its physiological role remains elusive because of the lack of selective inhibitors. To aid in the design of these novel compounds and better understand the different inhibitory patterns of NEP and NEP2, the x-ray structure of NEP was used as a template to build a model of the NEP2 active site. The results of our modeling suggest that the overall structure of NEP2 closely resembles that of NEP. The model of the active site reveals a 97% sequence identity with that of NEP with differences located within the S 2 subsite of NEP2 where Ser 133 and Leu 739 replace two glycine residues in NEP. To validate the proposed model, site-directed mutagenesis was performed on a series of residues of NEP2, mutants expressed in AtT20 cells, and their ability to bind various substrates and inhibitory compounds was tested. The results confirm the involvement of the conserved Arg 131 and Asn 567 in substrate binding and catalytic activity of NEP2 and further show that the modifications in its S 2 pocket, particularly the presence therein of Leu 739 , account for a number of differences in inhibitor binding between NEP and NEP2.Neprilysin 2 (SEP and NL-1) is a recently identified type II membrane-bound zinc-dependent metalloprotease (1-3). It is part of the M13 family of metalloproteases, which also comprises neprilysin (NEP, 1 EC 3.4.24.11) (4 -6), the endothelinconverting enzymes, ECE-1 (EC 3.4.24.71) (7) and ECE-2 (8), the Kell blood group antigen (9), the phosphate-regulating neutral endopeptidase on the X chromosome (10) and X-converting enzyme (11). Not only do all of the members of this family of metalloproteases share considerable sequence identities, suggesting a common ancestor as well as a common fold, NEP2, by far the closest homologue of NEP displays over 50% of overall protein sequence identity with NEP, a degree of homology that also suggests a common function (12).The best characterized member of the family, NEP (13, 14), was first identified as a kidney brush-border neutral endopeptidase slowly hydrolyzing [ 125 I]iodoinsulin B chain (15) and rediscovered as an "enkephalinase" of cerebral membranes hydrolyzing the Gly 3 -Phe 4 bond of enkephalins (16). Thereafter, it was shown to hydrolyze a number of biologically active peptides into inactive fragments in vitro, such as tachykinins (17), bradykinin (18), and atrial natriuretic peptides (19,20). The physiological implication of NEP in the inactivation of these messenger peptides was confirmed in vivo by the design and use of selective and potent inhibitors, e.g. thiorphan (21-24). These functions of NEP and the design of specific inhibitors thereof are of ...

The American Journal of Pathology

“…endoplasmic reticulum. 7,11 However, Oh-Hashi and colleagues 12 did find murine NEP2 activity at the cell surface. Studies of NEP2 in mice and rats have demonstrated that it is expressed primarily neurons and shows mild expression in the cerebral cortex, mild to moderate expression in the hippocampus, and strong expression in numerous thalamic, hypothalamic, and brainstem nuclei.…”

mentioning

confidence: 99%

“…NEP2 (also known as MMEL1/2, SEP, NL1, NE-PLP) possesses a 55% sequence identity to NEP and has been shown to degrade vasoactive peptides. 7,8 In addition, the membrane-bound ␣-splice form of murine NEP2 has demonstrated A␤-degrading properties in membrane fractions. 9 In transduced HEK293T cells, our research group previously showed that cell surface human NEP2 (␤-splice form) was able to degrade both A␤ 42 and A␤ 40 peptides.…”

mentioning

confidence: 99%

Neprilysin-2 Is an Important β-Amyloid Degrading Enzyme

Hafez

Huang

Huynh

et al. 2011

Proteases that degrade the amyloid-␤ peptide (A␤) are important in protecting against Alzheimer's disease (AD), and understanding these proteases is critical to understanding AD pathology. Endopeptidases sensitive to inhibition by thiorphan and phosphoramidon are especially important, because these inhibitors induce dramatic A␤ accumulation (ϳ30-to 50-fold) and pathological deposition in rodents. The A␤-degrading enzyme neprilysin (NEP) is the best known target of these inhibitors. However, genetic ablation of NEP results in only modest increases (ϳ1.5-to 2-fold) in A␤, indicating that other thiorphan/phosphoramidon-sensitive endopeptidases are at work. Of particular interest is the NEP homolog neprilysin 2 (NEP2), which is thiorphan/ phosphoramidon-sensitive and degrades A␤. We investigated the role of NEP2 in A␤ degradation in vivo through the use of gene knockout and transgenic mice. Mice deficient for the NEP2 gene showed significant elevations in total A␤ species in the hippocampus and brainstem/diencephalon (ϳ1.5-fold). Increases in A␤ accumulation were more dramatic in NEP2 knockout mice crossbred with APP transgenic mice. In NEP/NEP2 double-knockout mice, A␤ levels were marginally increased (ϳ1.5-to 2-fold), compared with NEP Alzheimer's disease (AD) is a neurodegenerative disorder currently affecting more than 26 million people worldwide and, as advances in modern medicine prolong lifespan, this number is expected to quadruple by 2050. 1 A major factor believed to be involved in the progression of AD pathology is the accumulation of amyloid-␤ peptide (A␤). Studying the mechanisms of A␤ clearance is, therefore, very important to understanding AD.Currently, enzymatic degradation is thought to play an integral role in the removal of A␤. Of the A␤-degrading enzymes, neprilysin (NEP) has been shown to be highly critical for cerebral A␤ control.2 NEP expression has also been inversely correlated with amyloid pathology in humans and mice, and NEP gene transfer has been reported to reduce amyloid pathology in transgenic mice (reviewed by Marr and Spencer 3 ). Despite the importance of NEP-mediated A␤ degradation, NEP knockout (KO) mice show only moderately elevated A␤ levels (1.5-to 2-fold), insufficient to cause plaque deposition. 4 However, when treated with thiorphan, an NEP endopeptidase inhibitor, mice and rats demonstrate pathological accumulations of A␤ after only 1 month. 2,5 This was also found in mice treated with phosphoramidon, another NEP inhibitor. 6 These results indicate that there may exist additional NEPlike endopeptidases in the metalloprotease 13 (M13) family that are central to the A␤ clearance pathway.The NEP homolog neprilysin 2 (NEP2) is one such endopeptidase. NEP2 (also known as MMEL1/2, SEP, NL1, NE-PLP) possesses a 55% sequence identity to NEP and has been shown to degrade vasoactive peptides. 7,8 In addition, the membrane-bound ␣-splice form of murine NEP2 has demonstrated A␤-degrading properties in membrane fractions. 9 In transduced HEK293T cells, our research group previously sho...