Active Site Mutations Change the Cleavage Specificity of Neprilysin

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, Phe563 and Ser546. Among the mutants studied in detail we observed changes in their activity towards leucine5-enkephalin, insulin B chain, and amyloid β1–40. For example, NEPF563I displayed an increase in preference towards cleaving leucine5-enkephalin relative to insulin B chain, while mutant NEPS546E was less discriminating than neprilysin. Mutants NEPF563L and NEPS546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß1–40 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

Time course of NEP mediated hydrolysis of Aß_1–40.

<p>Time course measurements were carried out by incubation of NEP with 24 µM Aß_1–40 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032343#pone-0032343-g001" target="_blank"><b>Figure 1</b></a>. Numbers under each peak indicate the identification of the peptide by sequence.</p

Comparison of insulin B chain cleavage between NEP, NEP^F563L, and NEP^S546E.

<p>A. HPLC profile of insulin B chain cleaved by NEP and NEP mutants at 30% hydrolysis. B. Rates of peak accumulation at each cleavage site normalized to that of NEP for mutant NEP^S546E. C. Rates of peak accumulation at each cleavage site normalized to that of NEP for mutant NEP^F563L. Dotted lines indicate the overall rate of hydrolysis of insulin B chain from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032343#pone-0032343-t002" target="_blank"><b>Table 2</b></a>. Reactions were carried out at 37°C with 15 µM insulin B chain in 20 mM MES, pH 6.5.</p

Relative rates of accumulation of peaks generated by the NEP and NEP mutant dependent hydrolysis of insulin B chain.

<p>Time course assays were carried out by incubation of NEP with insulin B chain using conditions as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032343#pone-0032343-t002" target="_blank"><b>Table 2</b></a>. At 0, 30, 60, 90, 120, and 180 min., aliquots of 100 µL were removed followed by the addition of 10 µL of 5% TFA to stop further hydrolysis. Each reaction mixture was subjected to HPLC analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032343#pone-0032343-t004" target="_blank"><b>Table 4</b></a> and peak areas measured. The rate of accumulation for each peak was calculated from the linear phase of the reaction.</p

The specific activity towards glutaryl-Ala-Ala-Phe-MNA cleavage is reduced in NEP mutants.

<p>All assays were conducted at 37°C at in 20 mM MES buffer, pH 6.5.</p><p>ND = not determined.</p><p>*The concentrations of NEP^S546A and NEP^S546T were too low to quantify, however, they were active enough to determine K_m.</p

Time course for NEP mediated hydrolysis of insulin B chain.

<p>Time course assays were conducted by incubation of NEP with 15 µM insulin B chain in 20 mM MES buffer, pH 6.5, at 37°C. A 100 µL aliquot was removed at each time point and 10 µL of 5% trifluoroacetic acid (TFA) was added to stop further hydrolysis. 95 µL were injected into a Vydac C4 column and developed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032343#s3" target="_blank">Materials and Methods</a>. Each product was isolated and subjected to mass spectral analysis. Numbers under each peak indicate the identification of the peptide by sequence. Peaks without numbers were not identified.</p

Rates of hydrolysis of physiological peptides by NEP mutants.

<p>Hydrolysis was carried out at 37°C at in 20 mM MES buffer, pH 6.5. Substrate concentrations were 15 µM insulin B chain, 24 µM Aß_1–40, and 64 µM leu-ENK. Activity was determined by following the disappearance of substrate by HPLC. Each reaction was run in at least triplicate. Statistical analysis was conducted using a two-tailed paired t-test with Prism4 software.</p

Leu-Enkephalin and Insulin B chain dual substrate assays.

<p>The numbers in parenthesis indicate activity relative to the uninhibited values.</p><p>Reactions were carried out at 37°C at in 20 mM MES buffer, pH 6.5 containing 15 µM insulin B chain and 64 µM leu-ENK. Activity was measured and statistical analysis carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032343#pone-0032343-t002" target="_blank"><b>Table 2</b></a>.</p