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Active site mutations change the cleavage specificity of neprilysin.

Sexton T, Hitchcook LJ, Rodgers DW, Bradley LH, Hersh LB - PLoS ONE (2012)

Bottom Line: We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563) and Ser(546).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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America.

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.

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Related in: MedlinePlus

Time course of NEP mediated hydrolysis of Aß1–40.Time course measurements were carried out by incubation of NEP with 24 µM Aß1–40 in 20 mM MES, pH 6.5, at 37°. 100 µL aliquots were removed at each time point and 10 µL of 5% TFA was added to stop the reaction. Samples were analyzed as described in Figure 1. Numbers under each peak indicate the identification of the peptide by sequence.
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pone-0032343-g003: Time course of NEP mediated hydrolysis of Aß1–40.Time course measurements were carried out by incubation of NEP with 24 µM Aß1–40 in 20 mM MES, pH 6.5, at 37°. 100 µL aliquots were removed at each time point and 10 µL of 5% TFA was added to stop the reaction. Samples were analyzed as described in Figure 1. Numbers under each peak indicate the identification of the peptide by sequence.

Mentions: To further study the effect of mutations on cleavage site specificity, time course assays were also performed for the hydrolysis of the physiological substrate Aß1–40. Like the analysis of insulin B chain, a time course assay was first done with NEP to identify cleavage products, Figure 3andTable 6. After 15% hydrolysis of Aß1–40 (150 min.) by NEP there were six discernable product peaks corresponding to Aß1–16, Aß1–17, Aß10–17, Aß20–28, Aß20–29, and Aß20–30. At 25% hydrolysis (240 min.), peaks corresponding to Aß1–9, Aß4–16, and Aß4–17 were observed, while at 40% hydrolysis peaks Aß4–9 and Aß10–16 appeared. Peak Aß12–17 was the last peak to be observed at 45% hydrolysis of Aß1–40 (360 min.). Missing from the HPLC analysis were the C-terminal products resulting from the cleavages at K28-G29, G29-A30, and A30-I31. These products are derived from the trans-membrane region of the amyloid precursor protein (APP) from which Aß is formed and are rather hydrophobic. It is likely that these peptides were not eluted in the gradient we employed.


Active site mutations change the cleavage specificity of neprilysin.

Sexton T, Hitchcook LJ, Rodgers DW, Bradley LH, Hersh LB - PLoS ONE (2012)

Time course of NEP mediated hydrolysis of Aß1–40.Time course measurements were carried out by incubation of NEP with 24 µM Aß1–40 in 20 mM MES, pH 6.5, at 37°. 100 µL aliquots were removed at each time point and 10 µL of 5% TFA was added to stop the reaction. Samples were analyzed as described in Figure 1. Numbers under each peak indicate the identification of the peptide by sequence.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285688&req=5

pone-0032343-g003: Time course of NEP mediated hydrolysis of Aß1–40.Time course measurements were carried out by incubation of NEP with 24 µM Aß1–40 in 20 mM MES, pH 6.5, at 37°. 100 µL aliquots were removed at each time point and 10 µL of 5% TFA was added to stop the reaction. Samples were analyzed as described in Figure 1. Numbers under each peak indicate the identification of the peptide by sequence.
Mentions: To further study the effect of mutations on cleavage site specificity, time course assays were also performed for the hydrolysis of the physiological substrate Aß1–40. Like the analysis of insulin B chain, a time course assay was first done with NEP to identify cleavage products, Figure 3andTable 6. After 15% hydrolysis of Aß1–40 (150 min.) by NEP there were six discernable product peaks corresponding to Aß1–16, Aß1–17, Aß10–17, Aß20–28, Aß20–29, and Aß20–30. At 25% hydrolysis (240 min.), peaks corresponding to Aß1–9, Aß4–16, and Aß4–17 were observed, while at 40% hydrolysis peaks Aß4–9 and Aß10–16 appeared. Peak Aß12–17 was the last peak to be observed at 45% hydrolysis of Aß1–40 (360 min.). Missing from the HPLC analysis were the C-terminal products resulting from the cleavages at K28-G29, G29-A30, and A30-I31. These products are derived from the trans-membrane region of the amyloid precursor protein (APP) from which Aß is formed and are rather hydrophobic. It is likely that these peptides were not eluted in the gradient we employed.

Bottom Line: We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563) and Ser(546).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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biochemistry, The Center for Structural Biology, University of Kentucky, Lexington, Kentucky, United States of America.

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.

Show MeSH
Related in: MedlinePlus