Active site mutations change the cleavage specificity of neprilysin

Travis Sexton; Lisa J. Hitchcook; David W. Rodgers; Luke H. Bradley; Louis B. Hersh

doi:10.1371/journal.pone.0032343

Active site mutations change the cleavage specificity of neprilysin

Travis Sexton, Lisa J. Hitchcook, David W. Rodgers, Luke H. Bradley, Louis B. Hersh

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17 Scopus citations

Abstract

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe ⁵⁶³ and Ser ⁵⁴⁶. Among the mutants studied in detail we observed changes in their activity towards leucine ⁵-enkephalin, insulin B chain, and amyloid β _1-40. For example, NEP ^F563I displayed an increase in preference towards cleaving leucine ⁵-enkephalin relative to insulin B chain, while mutant NEP ^S546E was less discriminating than neprilysin. Mutants NEP ^F563L and NEP ^S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß _1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

Original language	English
Article number	e32343
Journal	PLoS ONE
Volume	7
Issue number	2
DOIs	https://doi.org/10.1371/journal.pone.0032343
State	Published - Feb 23 2012

Bibliographical note

Funding Information:
Cleavage of physiological peptides was measured via reverse phase high performance liquid chromatography (HPLC) following incubation of the purified NEP or its mutants with 15 µM insulin B chain (Sigma Aldrich), 24 µM Aß (Anaspec), or 64 µM leu-ENK (Sigma) in 100 µL of 20 mM MES, pH 6.5, at 37°C. Reactions were run in triplicate. HPLC was carried out in a Vydac C4 column using a linear gradient from 0.1% trifluoroacetic acid (TFA) in 95% water, 5% acetonitrile to 0.1% TFA in 50% acetonitrile/water at a flow rate of 1 mL/min. Peptides and hydrolysis products were detected at 214 nm and quantified by measuring peak areas. Peptides obtained from HPLC were analyzed on an Applied Biosystems 4800 MALDI TOF/TOF Proteomics Analyzer at the University of Kentucky Proteomics core. This facility is supported in part by grant P20RR020171 from the NIH/NCRR. 1–40

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title = "Active site mutations change the cleavage specificity of neprilysin",

abstract = "Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe 563 and Ser 546. Among the mutants studied in detail we observed changes in their activity towards leucine 5-enkephalin, insulin B chain, and amyloid β 1-40. For example, NEP F563I displayed an increase in preference towards cleaving leucine 5-enkephalin relative to insulin B chain, while mutant NEP S546E was less discriminating than neprilysin. Mutants NEP F563L and NEP S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid {\ss} 1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.",

author = "Travis Sexton and Hitchcook, {Lisa J.} and Rodgers, {David W.} and Bradley, {Luke H.} and Hersh, {Louis B.}",

note = "Funding Information: Cleavage of physiological peptides was measured via reverse phase high performance liquid chromatography (HPLC) following incubation of the purified NEP or its mutants with 15 µM insulin B chain (Sigma Aldrich), 24 µM A{\ss} (Anaspec), or 64 µM leu-ENK (Sigma) in 100 µL of 20 mM MES, pH 6.5, at 37°C. Reactions were run in triplicate. HPLC was carried out in a Vydac C4 column using a linear gradient from 0.1% trifluoroacetic acid (TFA) in 95% water, 5% acetonitrile to 0.1% TFA in 50% acetonitrile/water at a flow rate of 1 mL/min. Peptides and hydrolysis products were detected at 214 nm and quantified by measuring peak areas. Peptides obtained from HPLC were analyzed on an Applied Biosystems 4800 MALDI TOF/TOF Proteomics Analyzer at the University of Kentucky Proteomics core. This facility is supported in part by grant P20RR020171 from the NIH/NCRR. 1–40 ",

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AU - Sexton, Travis

AU - Hitchcook, Lisa J.

AU - Rodgers, David W.

AU - Bradley, Luke H.

AU - Hersh, Louis B.

N1 - Funding Information: Cleavage of physiological peptides was measured via reverse phase high performance liquid chromatography (HPLC) following incubation of the purified NEP or its mutants with 15 µM insulin B chain (Sigma Aldrich), 24 µM Aß (Anaspec), or 64 µM leu-ENK (Sigma) in 100 µL of 20 mM MES, pH 6.5, at 37°C. Reactions were run in triplicate. HPLC was carried out in a Vydac C4 column using a linear gradient from 0.1% trifluoroacetic acid (TFA) in 95% water, 5% acetonitrile to 0.1% TFA in 50% acetonitrile/water at a flow rate of 1 mL/min. Peptides and hydrolysis products were detected at 214 nm and quantified by measuring peak areas. Peptides obtained from HPLC were analyzed on an Applied Biosystems 4800 MALDI TOF/TOF Proteomics Analyzer at the University of Kentucky Proteomics core. This facility is supported in part by grant P20RR020171 from the NIH/NCRR. 1–40

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N2 - Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe 563 and Ser 546. Among the mutants studied in detail we observed changes in their activity towards leucine 5-enkephalin, insulin B chain, and amyloid β 1-40. For example, NEP F563I displayed an increase in preference towards cleaving leucine 5-enkephalin relative to insulin B chain, while mutant NEP S546E was less discriminating than neprilysin. Mutants NEP F563L and NEP S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß 1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

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Active site mutations change the cleavage specificity of neprilysin

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