N-terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme-1 and Neprilysin.

A Ian Smith,Helena C Parkington,Oded Kleifeld,Paul J Conroy,Bruno Lomonte,David H Small,Alexander J Spicer,Nkumbu L Sikanyika,Niwanthi W Rajapakse,Sanjaya Kuruppu,James C Whisstock,David M Kaye,Wayne C Hodgson

doi:10.1038/srep22413

Abstract

Neprilysin (NEP) and endothelin converting enzyme-1 (ECE-1) are two enzymes that degrade amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49-P1-20, from the venom of Bothrops asper which directly enhances the activity of both ECE-1 and NEP. This is evidenced by a 2- and 5-fold increase in the Vmax of ECE-1 and NEP respectively. The K49-P1-20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE-1 and NEP was 1.92 ± 0.07 and 1.33 ± 0.12 μM respectively. Using BLITZ biolayer interferometry we have shown that K49-P1-20 interacts directly with each enzyme. Intrinsic fluorescence of the enzymes change in the presence of K49-P1-20 suggesting a change in conformation. ECE-1 mediated reduction in the level of endogenous soluble amyloid beta 42 in cerebrospinal fluid is significantly higher in the presence of K49-P1-20 (31 ± 4% of initial) compared with enzyme alone (11 ± 5% of initial; N = 8, P = 0.005, unpaired t-test). K49-P1-20 could be an excellent research tool to study mechanism(s) of enzyme stimulation, and a potential novel drug lead in the fight against Alzheimer’s disease.

Highlights

It is of great interest to regulate the production of, and more importantly, the degradation of Aβ by stimulating the activity of these enzymes[2]
We report on the discovery of K49-P1-20, a 20 amino acid peptide from the venom of B. asper which stimulates the activity of both endothelin converting enzyme-1 (ECE-1) and NEP
Fractionation of venom confirmed that ECE-1 stimulation was mediated by the previously isolated B. asper myotoxin II (Fig. 1a), a lysine 49 (K49) type phospholipase A2 found in this venom which induces myonecrosis upon envenoming[8]

Summary

Introduction

It is of great interest to regulate the production of, and more importantly, the degradation of Aβ by stimulating the activity of these enzymes[2]. This in turn could reverse, prevent or at least halt the progression of Alzheimer’s disease. We report on the discovery of K49-P1-20, a 20 amino acid peptide from the venom of B. asper which stimulates the activity of both ECE-1 and NEP. The effect of this peptide on other closely related enzymes was examined

Objectives

Results

Conclusion