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.

Because of its multiple targets, NEP has been the focus of numerous studies attempting to modulate its activity for therapeutic purposes. One such target is the use of NEP to reduce Aß peptide levels in Alzheimer's disease, since the oligomerization of Aß has been linked to the etiology of this disease [11]. Indeed, in studies with transgenic mice NEP expression decreases the level of Aß [12][13][14][15] and ameliorates cognitive deficits typically attributed to AD [16]. In yet another application inhibitors of NEP were developed to block its ''enkephalinase'' activity to increase the concentration of enkephalins in the brain and thus their analgesic effect [17]. Peripherally expressed NEP may have a role in appetite control and obesity. NEP deficient mice become obese [18], while a peripherally administered NEP inhibitor that does not cross the blood-brain barrier increased food intake and subsequently led to obesity. Recently, an NEP inhibitor was shown to increase female genitalia blood flow in rabbits by preventing vasoactive intestinal peptide (VIP) cleavage [19]. This could potentially lead to the use of NEP as a therapeutic agent in the treatment of female sexual arousal disorder.
While methods to modulate NEP activity have displayed the potential for therapeutic use, they also reveal a paradox to their usage. For example, using NEP to lower Aß may indeed decrease the amount of the target substrate; it may also have undesired consequences by removing other physiologically important products such as the enkephalins or vasopressin. Alternatively, inhibiting NEP to enhance opioid levels will likely cause an increase in Aß, which would result in an increased risk in the development of Alzheimer's disease.
A strategy to bypass the potential problems associated with the substrate promiscuity of NEP is to alter its specificity towards a target substrate thus reducing potential off-target effects. There is ample precedence to apply such a strategy. For example, substitutions within the active site of trypsin, although decreasing activity, shifted the relative preference for arginine versus lysine [20]. Similarly, a series of mutations in Rous sarcoma virus protease displayed altered amino acid preferences at particular substrate positions, allowing position-by-position control of substrate specificity [21]. Using thermolysin as a homology model, we were able to show that conversion of Val 573 to Leu produced a form of NEP which reacted with substrates with small P91 residues essentially the same as wild-type enzyme, yet substrates containing bulky P91 residues exhibited a decreased V max with little change in K m [22]. This study, although limited in scope, demonstrated the feasibility of altering NEP substrate specificity. The nomenclature of Schecter and Berger (Schechter I, Berger A. (1968) Biochem. Biophys. Res. Commun. 32: 898-902) is used where residues of the substrate C-terminal to the site of cleavage are designated P19, P29, P39, etc as they move away from the scissile bond and residues N-terminal to the scissile bond are designated P1, P2, P3, etc as they move away from the scissile bond. The corresponding binding sites on the enzyme are designated S19, S29, S39, and S1, S2, S3, etc. respectively.
By analyzing the crystal structure of NEP in complex with the inhibitor phosphoramidon [23], we have initiated a rational design approach to mutate NEP active site targeting residues likely to interact with substrates. In this study, we explore NEP substrate specificity by generating NEP mutant libraries of two active site residues, Phe 563 which is part of the S19 binding site and Ser 546 which appears to contribute to the S2/S3 binding site. A number of these mutants displayed differential changes in activity toward physiological substrates including changes in cleavage site preferences. Together, these data support the hypothesis that amino acid changes in the active site of NEP can potentially give rise to therapeutically relevant forms of NEP.

Selection of sites for mutagenesis
Mutations were made at the Phe 563 and Ser 546 sites in a secreted form of human NEP (shNEP) expressed as a C-terminal hexahistidine fusion protein. The NEP crystal structure reveals that Phe 563 forms part of the S19 substrate binding pocket believed to impart the preference for hydrophobic/aromatic P19 residues at this position, Figure S1. Phe 563 is located in a coil region just prior to the helix containing the active site residues. Ser 546 is part of a ß-sheet lining the substrate-binding site [23] and is positioned to interact with the P2 or P3 residues of a bound substrate on the carboxyl side of the scissile bond. Based on the NEP crystal structure, the position of both Phe 563 and Ser 546 , and their conservation among species, we hypothesized that these residues contribute to substrate specificity.

Expression of mutant NEP
To test the contribution of Phe 563 and Ser 546 to catalysis we used degenerate oligonucleotides to construct NEP libraries in which we introduced amino acid substitutions at these positions. Substitutions made at Phe 563 included valine, leucine, methionine, isoleucine, serine, histidine, aspartic acid, arginine, glutamine, asparagine, and lysine. Substitutions made at Ser 546 included glutamate, lysine, threonine, glycine, arginine, and alanine. Individual mutant library members were transfected in HEK293 cells and analyzed for expression. Of the seventeen sequences examined, five mutants, NEP F563L , NEP F563V , NEP F563M , NEP F563I , and NEP S546E expressed at levels near to that of wildtype NEP and were selected for further purification and analysis. The low expression of other mutants appeared to be due to their cellular instability as they all produced similar amounts of mRNA, Figure S2, which did not correlate with protein expression nor did the poorly expressing mutants accumulate intracellularly. These results suggest that active-site residues Phe 563 and Ser 546 play a role in overall protein folding and/or stability and that the non-expressing mutants were likely degraded intracellularly.
The five expressing NEP mutants were purified by nickel affinity chromatography, and the amount of NEP present determined by Sypro ruby staining of SDS-PAGE gels. We initially compared Sypro Ruby and Western blot analysis for enzyme quantitation and obtained equivalent results with either method, Figure S3.
Reaction of NEP mutants with the synthetic substrate Glut-Ala-Ala-Phe-MNA Activity assays were first performed using the synthetic peptide Glut-Ala-Ala-Phe-MNA. This substrate is cleaved between the Ala-Phe peptide bond and thus any effects of mutations on the cleavage at this site will be reflected in the reaction kinetics. We demonstrated that Glut-Ala-Ala-Phe-MNA hydrolysis by NEP and each of the studied mutants was completely inhibited by the relatively specific inhibitor, phosphoramidon at 100 mM, and the highly specific inhibitor CGS 24592 [24] at 10 nM, thus demonstrating that hydrolysis was attributed to NEP or its variant and not a contaminating protein.
Kinetic constants for mutants determined with Glut-Ala-Ala-Phe-MNA as substrate are presented in Table 1. These kinetic constants were derived under first-order assay conditions monitored in a continuous mode. The wild-type enzyme and the NEP F563L mutant exhibited essentially the same specific activity of 46 and 44 pmoles/min/ng, respectively, while the NEP F563I , NEP F563V , NEP F563M , and NEP S546E mutant activities varied from ,25% to 45% of the wild-type enzyme, Table 1. K m values varied ,2.5 fold ranging from 51 to 118 mM, with V max /K m values varying three fold or less. Thus mutating Phe 563 and Ser 546 produce small but detectable affects on the cleavage of Glut-Ala-Ala-Phe-MNA confirming that these residues contribute to catalysis.

Reaction of NEP mutants with physiological substrates
We extended the comparison of the various mutants by studying their reaction with three physiological substrates; leucine 5enkephalin (leu-ENK = Tyr-Gly-Gly-Phe-Leu), which is cleaved by NEP at the Gly-Phe bond, insulin B chain, which has multiple cleavage sites, and amyloid beta peptide 1-40 (Aß 1-40 ), which also has multiple cleavage sites. The rate of cleavage of these peptides was determined by following the disappearance of the parent peptide by reverse-phase high performance liquid chromatography (HPLC). Leu-ENK was cleaved at the Gly-Phe bond by all of the mutants, with rates varying from approximately 80% of the wild-type NEP rate (NEP F563L ) to less than 20% of the wild-type rate (NEP F563V ), Table 2. The specific activities of the various mutants with the physiological substrate leu-ENK showed a similar pattern as observed with Glut-Ala-Ala-Phe-MNA. NEP F563L had near wildtype levels of activity for both Glut-Ala-Ala-Phe-MNA and leu-ENK, while NEP F563M , NEP F563I , and NEP S546E all showed approximately 40-60% activity towards these substrates, and NEP F563V exhibited a 75-85% decrease in activity towards both. Thus the effects of these mutations can be attributed to those produced for cleaving N-terminal to single phenylalanine residue.
Insulin B chain and Aß 1-40 contain multiple cleavage sites. Cleavage at any one of these sites will result in peptide disappearance as determined by HPLC. Compared to wild-type NEP, mutants NEP F563V , NEP S546E , NEP F563M , and NEP F563I all exhibited reduced hydrolysis rates for insulin B chain (p values of 0.03 or lower), whereas with NEP F563L the hydrolysis rate was higher (p = 0.03). With Aß 1-40 as substrate all of the mutants showed reduced rates of hydrolysis (all exhibited p values,0.05).
When the relative cleavage rates for insulin B chain, Aß 1-40 , and leu-ENK were compared between NEP and the various mutants there was a discernable change in substrate preference. For example, NEP F563L hydrolyzed insulin B chain at a rate 1.4 times faster than NEP, but cleaved Aß 1-40 at nearly half the rate of the wild-type enzyme. Thus NEP F563L exhibits a greater than 2 fold preference for insulin B chain over Aß  . NEP F563V cleaved leu-ENK at 1/6 the rate of the wild-type enzyme and Aß 1-40 at 1/12 the wild type rate, Table 2, increasing the preference of this mutant for leu-ENK by two fold. Thus different mutations differentially altered NEP specificity.
To further demonstrate that the NEP mutants differ in their ability to discriminate between substrates, we measured rates of cleavage of a mixture of two substrates, leu-ENK and insulin B chain. The rate of cleavage of a substrate mixture is determined by both the k cat for that substrate as well by the affinity for the substrate. As can be seen in Table 3 all NEP mutants showed a decrease in the rate of hydrolysis of both leu-ENK and insulin B chain when present together. This is expected, given that each peptide acts as a competitive inhibitor of the other. NEP F563V , NEP F563L , and NEP F563M all showed approximately the same wild-type enzyme ratio of rates for leu-ENK/insulin B chain indicating no change in overall substrate preference. However, NEP F563I exhibited a shift in substrate preference toward leu-ENK, while the substrate preference for NEP S546E shifted more toward insulin B chain, even though the rate remained higher for leu-ENK. That is the ratio for leu-ENK/insulin B chain cleavage is 3 for NEP S546E compared to 6 for wild-type NEP. Thus these single amino acid substitutions had a measureable effect on substrate preference.

Identification of NEP cleavage sites in insulin B chain
We next looked in more detail on the effect of the NEP F563 and NEP S546 mutations on the hydrolysis rates at individual cleavage sites in insulin B chain. We first tested for a change in the affinity of insulin B chain for mutant NEPs by using insulin B chain as an alternate substrate (competitive) inhibitor of Glut-Ala-Ala-Phe-MNA hydrolysis. We found that there was no dramatic change in the K i for any of the mutants, with variations of two fold or less ( Table 1).

Analysis of NEP mutant cleavage of insulin B chain
As a representative of the NEP F563 and NEP S546 mutants, we compared the cleavage profile of NEP F563L and NEP S546E to that of NEP using time course experiments, Figure 2. By adjusting the amount of NEP mutant used, the rate of hydrolysis of insulin B chain by all NEP forms was virtually identical. The overall  cleavage profile at 30% substrate hydrolysis revealed that all of the product peaks observed with NEP are present with the mutant enzymes indicating that there were no unique or missing cleavage sites between NEP F563L , NEP S546E and NEP, Figure 2A. Since rates were based on peak areas measured at 214 nm, which in turn is dependent on both the number of peptide bonds and the number of aromatic residues within a given peptide, only the observed rates of change for a particular peptide product can be compared between enzyme forms. A comparison of the rate of change of different peaks within the same enzyme form or between enzyme forms is not valid under our conditions of analysis.
NEP mutations affect cleavage sites preferences in insulin B chain Table 5 and Figure 2B,C show the rates of product accumulation normalized to the amount of NEP protein present. Relative to wild-type NEP the overall rate of hydrolysis of insulin B chain is slightly increased in NEP F563L and slightly decreased in NEP S546E , Table 2. Thus one scenario is that all sites in insulin B chain would be cleaved at the same relative rate compared to wildtype NEP. Alternatively, the introduced mutations may differentially affect specific cleavage sites. The data in Table 5 clearly shows the latter scenario with differential effects of mutations on specific cleavages. NEP S546E cleaves insulin B chain at an overall rate 0.7 times that of NEP, however it is clear that cleavage at A 14 -L 15 is nearly identical between NEP and this mutant. Cleavage at H 5 -L 6 is well below the overall insulin B chain rate of 0.7 ( Figure 2B), while cleavages at H 10 -L 11 , Y 16 -L 17 , and G 23 -F 24 are all slightly slower than the expected 0.7 times the wild-type rate.
NEP F563L cleaves insulin B chain at a rate 1.4 times that of NEP. Similar to that seen with NEP S546E , NEP F563L products produced from single cleavage sites exhibit noticeably different rates compared to NEP, Figure 2C. Cleavage at A 14 -L 15 , H 5 -L 6 , and G 23 -F 24 are close to the expected 1.4 times faster that of NEP, but cleavage at H 10 -L 11 , L 11 -V 12 , and Y 16 -L 17 are slower than NEP, (0.8 times, 0.2 times, and ,0.7 times the NEP rate respectively, rather than the overall 1.4 times faster than the NEP rate).
Based on the data in Table 5 the elevated activity of NEP F563L towards insulin B chain can likely be attributed to an increased rate of cleavage at the primary cleavage site A 14 -L 15 . Although this cleavage involves a leucine residue, the finding that cleavage at Y 16 -L 17 is slower than with wild-type NEP shows the enhanced cleavage at A 14 -L 15 is not due to simply the F563L mutation  NEP mediated hydrolysis was carried out as described in Table 2. The reaction was stopped by adding 10 mL of 5% TFA when approximately half of the substrate had been hydrolyzed (180 min.). The acidified reaction mixture was subjected to HPLC as described in figure 1, individual peaks were collected and identified by mass spectral analysis. doi:10.1371/journal.pone.0032343.t004 producing enhanced reactivity toward leucine, but more likely an effect of neighboring residues.
Two other studies have identified NEP cleavage sites within Aß 1-40 [25,26]. These studies as well as the current study all detected cleavages at G 9 -Y 10 , F 19 -F 20 , and A 30 -I 31 . Cleavage at A 3 -F 4 was detected in this study as well as by Howell et al [25,26].
Cleavages at K 28 -G 29 and G 29 -A 30 were detected in this study as well as by Leissring et al. [25,26]. In the current study additional unreported cleavage sites at E 11 -V 12 , K 16 -L 17 , and L 17 -V 18 were detected. These were previously pointed out as potential cleavage sites, but were not observed [25,26]. Both Leissring and Howell identified cleavage at G33-L34, yet this cleavage was not found in this study. These differences likely reflect differences in the resolution of the Aß 1-40 cleavage products on the HPLC columns used and gradient conditions.

Analysis of specific cleavage sites in Aß 1-40
We next compared the Aß 1-40 cleavage profile of the NEP F563L and NEP S546E mutants to that of wild-type NEP, Figure 4. Since NEP, NEP F563L and NEP S546E cleave Aß 1-40 at different rates, the amount of the mutant enzymes used in the reaction was as before adjusted in order to analyze products formed at the same fraction of degradation. Interestingly, although all wild-type peaks were present in the NEP S563E mutant, the peak corresponding to Aß [1][2][3][4][5][6][7][8][9] was not present among the hydrolysis products of the NEP F563L mutant.
Similar to the analysis conducted with insulin B chain, the linear rate of product accumulation was determined for each peak and normalized to the amount of protein in the reaction, Table 7. Unlike the hydrolysis of insulin B chain, the hydrolysis of Aß 1-40 , even at the earliest time points, produced products resulting from multiple cleavage events. Out of the 12 identified product peaks, only 3 could be produced by a single cleavage. Rates for all cleavages were calculated and are given in Figure 4, B-D, but only the three putative primary cleavage sites can be compared. NEP S546E hydrolyzes Aß 1-40 at an overall rate 0.5 times that of wild-type NEP. Two of the three products resulting from a possible single cleavage by NEP S546E exhibit a higher than predicted rate of cleavage. Cleavage at K 16 -L 17 producing Aß 1-16 is identical rather than half the wild-type rate while cleavage at L 17 -V 18 producing Aß 1-17 was approximately 70% rather than 50% of the NEP rate. The remaining single cleavage site at G 9 -Y 10 shows a slower rate being about 30% that of wild-type enzyme. Thus it would appear that all three of these cleavages contribute to the overall rate and together produce an average rate 0.5 times that of NEP.
Cleavage at L 17 -V 18 is approximately three times faster for NEP S546E compared to NEP F563L . Thus cleavage at L 17 -V 18 for mutant NEP S563E is 70% of the wild-type NEP whereas cleavage at this bond for the NEP F563L is 25% of NEP. In order to account for this finding there must either be an undetected cleavage that is significantly reduced in NEP S546E or more likely that NEP F563L exhibits a unique cleavage pattern that yields these products.
The rate of appearance of Aß 1-16 is linear over the entire 360minute time course for all three enzymes. Aß 1-17 , on the other hand, shows a linear increase with both NEP F563L and NEP S546E , but is non-linear with wild-type NEP showing very little increase after 150 min. This is consistent with Aß 1-17 being further metabolized by NEP, most likely by being cleaved at G 9 -Y 10 giving rise to Aß 1-9 and Aß 10-17 , both of which show higher peak areas in NEP than with either mutant. In contrast the absence of an obvious reduction of Aß 1-9 and Aß 10-17 in the reaction of NEP F563L and NEP S546E suggests these mutants cleave the G 9 -Y 10 bond at a much slower rate.
Although NEP F563L did not produce a discrete Aß 1-9 peak, it appears to cleave the G 9 -Y 10 bond as evidenced by the presence of the products Aß 10-16 and Aß 10-17 . Since Aß 1-9 is absent in the NEP F563L profile but Aß 10-16 and Aß 10-17 are observed, the cleavage by NEP F563L at G 9 -Y 10 is likely dependent on the cleavage at A 3 -F 4 . Aß 1-9 is not further degraded by hydrolysis at the A 3 -F 4 site with both NEP and NEP S546E as evidenced by its linear increase as a function of time. If hydrolysis occurred at the A 3 -F 4 bond of the Aß 1-9 product, one would expect either no time dependent increase or a decrease in the Aß 1-9 peak.
The finding that most of the observed products of Aß 1-40 cleavage result from multiple cleavages, even for early time points, would suggest that NEP is processive in its cleavage of Aß 1-40 and can make several cleavages before product release. Whether the Time course assays were carried out by incubation of NEP with insulin B chain using conditions as described in Table 2. At 0, 30, 60, 90, 120, and 180 min., aliquots of 100 mL were removed followed by the addition of 10 mL of 5% TFA to stop further hydrolysis. Each reaction mixture was subjected to HPLC analysis as in Table 4 and peak areas measured. The rate of accumulation for each peak was calculated from the linear phase of the reaction. doi:10.1371/journal.pone.0032343.t005 enzyme does this with both C-terminal and N-terminal products is not clear and how the products are reoriented in the active site for additional cleavages is also unclear. However, the alternative explanation for observing products derived from multiple cleavages requires an extremely high affinity of the product peptide to be bound and cleaved in the presence of a large amount of unreached Aß 1-40 . The processive model is consistent with the overall structure of the enzyme, which has only a small opening leading to the large, enclosed chamber that borders the active site. Once a peptide diffuses through the narrow opening, it is likely that it and product peptides are retained in the enclosed chamber sufficiently long enough for multiple active site binding events to occur. Of the eleven substitutions made at Phe 563 only four produced enzyme of sufficient stability to be studied. The four residues that did produce stable forms of NEP all represented conservative change to hydrophobic residues, whereas the other non-conservative or semi-conservative changes produced unstable enzyme forms. This suggests position 563 likely serves as an important anchor residue in the folding of the enzyme, and interaction of a hydrophobic residue at this position with other hydrophobic and aromatic residues is required. Changes at Ser 546 produced more variable results in terms of enzyme expression, and this position is therefore likely less critical in folding.
Amino acid substitutions at both Phe 563 and Ser 546 affected the cleavage pattern of NEP. Phe 563 forms part of the S19 substrate binding pocket and helps define the specificity of NEP for hydrophobic/aromatic P19 residues, thus changing this amino acid would likely affect cleavage specificity. Ser 546 is positioned to contribute to the S2/S3 binding site, although this registration is more speculative, since subsites N terminal to the scissile bond are not defined by available structures with bound inhibitors. Although selectivity is less stringent at these positions, we have previously obtained evidence that residues N terminal to the scissile bond, particularly the S1 subsite, also contribute to substrate specificity [27] and this study certainly supports a role for the P2/P3 peptide positions in selectivity.
Of the two mutants studied in detail no changes in the peptide bonds that were cleaved were observed, but the relative rates of cleavage were affected by substitution at both Phe 563 and Ser 546 . In general substitution of leucine for phenylalanine at the S91 site either had no effect or increased the rate at which cleavage occurred with a P91 leucine or phenylalanine residue, but significantly decreased the rate when valine occupied the P91 position. Since the NEP F563L mutation substitutes a smaller residue in the hydrophobic S19 subsite, this result can be rationalized on the basis that the relatively small valine at P19 leaves an unfavorable gap upon substrate binding. Substitution of glutamate for serine at the S2/S3 position produced marked differential changes in cleavage rates, but these changes were complex and not easily rationalized. This result extends our earlier results indicating that residues N terminal to the scissile bond play an important role in selectivity. It is possible given the complex nature of the observed rate changes that positioning of the N terminal side of the substrate peptide may vary in a sequence dependent manner.
Taken together this study shows that a single amino acid substitution within the active site of NEP can cause changes in cleavage site preference, which strongly supports the notion that it may be possible to alter the NEP active site to generate substrate specific variants that will be useful therapeutically.

Mutagenesis and production of expression vectors
NEP variants were constructed as gene segment cassette modules using degenerative oligonucleotide primers to introduce sequence diversity by PCR. Individual mutation cassettes were inserted into the pCDNA-shNEP-CHis (SacII+Pst1) expression vector, a re-engineered pCDNA-3.1 vector with a silent SacII mutation introduced 59 to the active site region within a secreted form of the human NEP (shNEP) coding sequence and a Cterminal hexahistidine affinity tag. Two silent mutations were made, using the QuickchangeH II site directed mutagenesis kit (Stratagene) to eliminate additional PstI sites and facilitate cassette subcloning of the shNEP gene. A 3 kb fragment of lambda ''stuffer'' DNA was inserted between the SacII and Pst1 sites to allow gene segment cassette subcloning while eliminating wild-type sequences from being selected [27,28]. Non-polar substitutions at Phe 563 were initially cloned into and sequenced using the pBPG1 vector for expression in yeast [29]. However, low enzyme yields from yeast led to the removal of the identified clones from the pBPG1 vector and subcloning into the pDNA-shNEP-CHis-(SacII-3kbstuffer-PstI) vector. PCR based mutagenesis was used to create NEP variants using degenerative primers containing appropriate restriction sites. For making polar substitutions at Phe 563 the 59 primer contained a PstI restriction site and the 39 primer contained an NcoI restriction site to facilitate movement from the pBPG1 vector to the pCDNA-shNEP-CHis-(SacII-3kbstuffer-PstI) vector. The degenerative primers (IDTDNA, Corralville, IA) used were: 59-gattcggcttgtacagcatatgtgg-39 (S546 reverse primer) 59-gggggctgcagaatgccggctgggaagactatctgatttcttcctgaTBYgtaaaatgcattgactaccgcg-39 (S546 forward primer) 59-cccattctgcagccccccVtStttagtgcccagcagtccaac-39 (F563 forward primer for non polar residues) 59-cccattctgcagccccccVDStttagtgcccagcagtccaac-39 (F563 forward primer for polar residues) 59-tccaccagtcaacgaggtctc-39 (F563 reverse primer) NEP mediated hydrolysis was carried out as described in Table 2. The reaction was stopped by adding 10 mL of 5% TFA when approximately half of the substrate had been hydrolyzed (360 min.). The acidified reaction mixture was subjected to HPLC analysis as described in Table 4. Each peak was isolated and subjected to mass spectral analysis. doi:10.1371/journal.pone.0032343.t006 where V = A, C, or G; S = C or G; D = A, G, or T; B = C, G, or T; Y = C or T.

Expression and purification
NEP protein was expressed in HEK293T cells transfected with the pcDNA vector described above. Cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 10% FBS and 44 mM NaHCO 3 added as a supplement. For transfections, Polyfector (BamaGen Bioscience) and plasmid DNA were incubated at room temperature for 20 min in serum free DMEM media and then added to HEK293T cells in the DMEM media. The media was replaced with serum free DMEM 12-14 hours post transfection, and collected 72-96 hours post transfection. To the media was added 1 M Tris-HCl, pH 7.4 to a final concentration of 50 mM and the secreted enzyme was then purified on a His-Select Affinity Agarose Column (Sigma). The affinity purification step yielded enzyme with the purity dependent on the level of NEP expression. Activities measured in this study were attributed to NEP, since a mock transfection and purification resulted in no activity toward any of the substrates tested and NEP inhibitors eliminated all activity. We estimated the amount of NEP protein present by running the purified preparations on 8% or 10% SDS-PAGE gels along with purified NEP as a standard. The gels were stained with Sypro Ruby dye scanned on a Typhoon 9400 Imager, and quantified with Image Quant 5.2 software. In preliminary experiments the gel was transferred to a polyvinylidene fluoride (PVDF) and subject to Western blot analysis using goat anti-mNEP at 1:1000 (R&D systems) as the primary antibody and anti-goat IRDye800 at 1:20,000 (Rockland) as the secondary antibody. Probed membranes were imaged using an Odyssey infrared imager and Odyssey 2.1 software. Intensities of each band were analyzed with Image Quant 5.2 software. Data was analyzed using Prism4 software.

Activity assays
NEP activity was routinely assayed using the fluorogenic peptide glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamide (Glut-Ala-Ala-Phe-MNA, Sigma) [30]. Reactions of 400 ml contained 100 mM Glut-Ala-Ala-Phe-MNA, 1 mg of aminopeptidase [31] and 15 to 100 ng of NEP or mutant NEP depending on their activity in 20 mM MES buffer, pH 6.5. Activity was monitored with a Spectra Max Gemini XS plate reader using an excitation wavelength of 340 nm and an emission wavelength of 425 nm. Reaction specificity was determined using the NEP inhibitors  phosphoramidon and CGS 24592 [24], the latter being a highly specific and potent inhibitor.

Kinetic Analysis
Kinetic constants for NEP and its mutants were obtained using the assay conditions noted above, but with Glut-Ala-Ala-Phe-MNA varied from 20 to 500 mM. Typically 12 data points were obtained. The data were fit to the Michaelis-Menten equation using Prism4 software. The K i for insulin B chain was obtained by measuring the rate of Glut-Ala-Ala-Phe-MNA hydrolysis in the presence of varying concentrations of insulin B chain from 1 to 40 mM. Data were fit to a Dixon plot (1/rate versus [insulin B chain]) using Prism4 software and the ID 50 obtained as the -x intercept, where ID 50 corresponds to the concentration of insulin producing 50% inhibition. The actual Ki was obtained from the equation: ID 50 = K i (1+ [Glut-Ala-Ala-Phe-MNA]/K mGlut-Ala-Ala-Phe-MNA ) [32,33].

HPLC assays
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 mM insulin B chain (Sigma Aldrich), 24 mM Aß 1-40 (Anaspec), or 64 mM leu-ENK (Sigma) in 100 mL of 20 mM MES, pH 6.5, at 37uC. 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.

Synthesis and analysis of NEP cDNA
In order to compare NEP transcript levels, RNA from 96-hour post-transfected HEK cells was collected using a QIAshredder column (Qiagen) and an RNeasy Mini Kit (Qiagen). Using 5 mg of the harvested RNA, cDNAs were produced with a Superscript First Strand Synthesis kit (Invitrogen) using the oligo(dT) primer included in the kit. Using NEP specific primers (59-aaagtaaacaactgaaga-3 and 59-tcctgaaattgcctggac-39) and primers for b-actin (59taggagccagagcagtaatc-39 and 59-tgtttgagaccttcaacacc-39) for controls, relative levels of cDNA were measured by comparing product formation at 20, 25, 30, and 35 cycles in PCR reaction comparing under standard conditions using 50uC annealing temperature.

Statistical Analysis
Statistical analysis comparing wild-type NEP and its mutants was performed with Prism 4 software using a two-tailed paired ttest with a 95% confidence interval. Figure S1 Sites Mutated in NEP. The active site region of the NEP-phosphoramidon complex [23] is shown with the protein in a ribbon and surface representation and the bound ligand in a stick representation. The mutated residue positions are in red with side chains shown. The zinc ion cofactor is represented by a yellow sphere. Phosphoramidon residues equivalent to substrate peptide positions P1-P29 are indicated. The approximate position of substrate P2 and P3 residues is shown by the blue ovals. Purple arcs indicate contact between the P19 residue and F563. (TIF) Figure S2 NEP and mutant NEPs produce similar levels of mRNA. Varying PCR cycles were used to estimate the relative amount of NEP mRNA of high and low expressing mutants. Total RNA was harvested from HEK293T cells 96 hrs post transfection and an equal amount of RNA was used as a template for firststrand synthesis to produce a cDNA library using an oligo(dT) universal primer. The cDNA libraries were then used as templates for PCR using primers specific for NEP (experimental) and b-Actin (control). Samples from PCR cycles 20, 25, and 30 were used to estimate NEP transcript levels. The NEP F563K mutant product band intensity relative to NEP was 0.9, 1.0, and 1.3 at cycles 20, 25, and 30 respectively. NEP F563V and NEP F563L were at a level approximately half of the wild-type NEP transcript. In contrast, NEP F563L exhibited the same activity as wild-type enzyme while NEP F563V displayed ,25% of the wild-type activity, while the activity for NEP F563K was undetectable (,1% relative to wild-type enzyme) under our assay conditions ( Table 1). (TIF) Figure S3 Determination of the concentration of NEP mutants. Purified NEP samples were subjected to SDS-PAGE on 8% polyacrylamide gels and stained for protein with Sypro Ruby dye (A). The gel contained 100, 250, and 500 ng of purified NEP, which was used to construct a standard curve (C) from which the concentration of each NEP form was calculated. Samples of purified NEP, NEP F563L , and NEP S536E were run at 15 ml and 30 ml. Intensities of each NEP band were fit to the standard curve (C) to give 8.960, 12.763.7, and 11.661.1 ng/mL for NEP, NEP F563L , and NEP S536E , respectively (E, solid bars). Similarly a Time course assays were carried out by incubation of NEP with 24 mM Aß 1-40 using reaction conditions as described in Table 5. At 0, 60, 150, 240, and 360 min., aliquots of 100 mL were removed followed by the addition of 10 mL of 5% TFA to stop further hydrolysis. Each reaction mixture was subjected to HPLC analysis as in Table 5 and peak areas measured. The rate of accumulation for each peak was calculated from the linear phase of the reaction. doi:10.1371/journal.pone.0032343.t007

Supporting Information
Western blot derived from a 10% SDS-PAGE was run containing 10, 50, and 100 ng of purified NEP from which a standard curve was derived (D). NEP, NEP F563L , and NEP S536E were run at 3.75 ml and 7.50 ml. Intensities of each NEP band were fit to the standard curve (D) to give 11.061.4, 13.360.6, and 13.162.2 ng/ mL for NEP, NEP F563L , and NEP S546E , respectively (E, empty bars). Note -the difference in size between the NEP standard and the NEP experimental samples is due to differences in glycosylation between NEP isolated from CHO cells and HEK cells, respectively. (TIF)