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• W1982185609 abstract "Based on the kinetic model of substrate phage proteolysis, we have formulated a strategy for best manipulating the conditions in screening phage display libraries for protease substrates (Sharkov, N. A., Davis, R. M., Reidhaar-Olson, J. F., Navre, M., and Cai, D. (2001) J. Biol. Chem. 276, 10788–10793). This strategy is exploited in the present study with signal peptidase SpsB from Staphylococcus aureus. We demonstrate that highly active substrate phage clones can be isolated from a phage display library by systematically tuning the selection stringency in screening. Several of the selected clones exhibit superior reactivity over a control, the best clone, SIIIRIII-8, showing >100-fold improvement. Because no conserved sequence features were readily revealed that could allow delineation of the active and unreactive clones, the sequences identified in five of the active clones were tested as synthetic dodecamers, Ac-AGX 8GA-NH2. Using electrospray ionization mass spectrometry, we show that four of these peptides can be cleaved by SpsB and that Ala is the P1 residue exclusively and Ala or Leu the P3 residue, in keeping with the (−3, −1) rule for substrate recognition by signal peptidase. Our successful screening with SpsB demonstrated the general applicability of the screening strategy and allowed us to isolate the first peptide substrates for the enzyme. Based on the kinetic model of substrate phage proteolysis, we have formulated a strategy for best manipulating the conditions in screening phage display libraries for protease substrates (Sharkov, N. A., Davis, R. M., Reidhaar-Olson, J. F., Navre, M., and Cai, D. (2001) J. Biol. Chem. 276, 10788–10793). This strategy is exploited in the present study with signal peptidase SpsB from Staphylococcus aureus. We demonstrate that highly active substrate phage clones can be isolated from a phage display library by systematically tuning the selection stringency in screening. Several of the selected clones exhibit superior reactivity over a control, the best clone, SIIIRIII-8, showing >100-fold improvement. Because no conserved sequence features were readily revealed that could allow delineation of the active and unreactive clones, the sequences identified in five of the active clones were tested as synthetic dodecamers, Ac-AGX 8GA-NH2. Using electrospray ionization mass spectrometry, we show that four of these peptides can be cleaved by SpsB and that Ala is the P1 residue exclusively and Ala or Leu the P3 residue, in keeping with the (−3, −1) rule for substrate recognition by signal peptidase. Our successful screening with SpsB demonstrated the general applicability of the screening strategy and allowed us to isolate the first peptide substrates for the enzyme. Bacterial type I signal peptidase is responsible for cleaving the signal peptide from precursor proteins, and its activity is an integral part of the export and maturation of secreted proteins in vivo. The essential function of the enzyme to bacterial cell viability has been demonstrated using genetic approaches with both Gram-positive and Gram-negative organisms (1Cregg K.M. Wilding I. Black M.T. J. Bacteriol. 1996; 178: 5712-5718Crossref PubMed Google Scholar, 2Date T. J. Bacteriol. 1983; 154: 76-83Crossref PubMed Google Scholar, 3Zhang Y.B. Greenberg B. Lacks S.A. Gene (Amst.). 1997; 194: 249-255Crossref PubMed Scopus (34) Google Scholar), supporting the notion that the signal peptidase is potentially an antibacterial target (4Paetzel M. Dalbey R.E. Strynadka N.C. Pharmacol. Ther. 2000; 87: 27-49Crossref PubMed Scopus (124) Google Scholar). Drug discovery efforts with the enzyme, however, may be hampered by the lack of an effective in vitro assay employing a nonprotein substrate such as a peptide (4Paetzel M. Dalbey R.E. Strynadka N.C. Pharmacol. Ther. 2000; 87: 27-49Crossref PubMed Scopus (124) Google Scholar).Our current understanding is that signal peptides are highly variable in sequence (5von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1520) Google Scholar). Based on the studies carried out over the past 2 decades, it has been established that the recognition sites for signal peptidases lie between −6 and +1 in sequences encompassing the site of cleavage (6Talarico T.L. Barkocy-Gallagher G.A. Ray P.H. Bassford P.J., Jr. Biochem. Biophys. Res. Commun. 1993; 197: 1154-1166Crossref PubMed Scopus (5) Google Scholar, 7Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (74) Google Scholar, 8Kuhn A. Wickner W. J. Biol. Chem. 1985; 260: 15914-15918Abstract Full Text PDF PubMed Google Scholar, 9Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (732) Google Scholar, 10von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1598) Google Scholar, 11von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (458) Google Scholar, 12Carlos J.L. Paetzel M. Brubaker G. Karla A. Ashwell C.M. Lively M.O. Cao G. Bullinger P. Dalbey R.E. J. Biol. Chem. 2000; 275: 38813-38822Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Sequence conservation analyses of a large panel of naturally occurring signal peptides in bacteria and eukaryotes reveal that the predominant residue at the P1 site is Ala and that the predominant residues at the P3 site are large aliphatic residues (Leu, Ile, Val) as well as Ala and Ser, a consensus dubbed the (−3, −1) rule (9Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (732) Google Scholar, 10von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1598) Google Scholar, 11von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (458) Google Scholar). The (−3, −1) rule also holds for the cleavage of engineered preproteins in vivo as well as in vitro (6Talarico T.L. Barkocy-Gallagher G.A. Ray P.H. Bassford P.J., Jr. Biochem. Biophys. Res. Commun. 1993; 197: 1154-1166Crossref PubMed Scopus (5) Google Scholar, 7Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (74) Google Scholar, 8Kuhn A. Wickner W. J. Biol. Chem. 1985; 260: 15914-15918Abstract Full Text PDF PubMed Google Scholar, 12Carlos J.L. Paetzel M. Brubaker G. Karla A. Ashwell C.M. Lively M.O. Cao G. Bullinger P. Dalbey R.E. J. Biol. Chem. 2000; 275: 38813-38822Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The reaction of signal peptidases with synthetic peptides, on the other hand, is not as well explored as with protein substrates. For the signal peptidase LepB from Escherichia coli, the best characterized signal peptidase, Ala was found as the only residue permitted at the P1 site through single amino acid replacements of a peptide bearing the signal peptide sequence of theE. coli maltose-binding protein (13Kuo D.W. Chan H.K. Wilson C.J. Griffin P.R. Williams H. Knight W.B. Arch. Biochem. Biophys. 1993; 303: 274-280Crossref PubMed Scopus (59) Google Scholar).As demonstrated with the E. coli LepB enzyme, the catalytic efficiency of signal peptidase toward short peptide substrates is generally several orders of magnitude lower than toward polypeptides bearing the same protease recognition sequence (14Kuo D. Weidner J. Griffin P. Shah S.K. Knight W.B. Biochemistry. 1994; 33: 8347-8354Crossref PubMed Scopus (47) Google Scholar, 15Dev I.K. Ray P.H. Novak P. J. Biol. Chem. 1990; 265: 20069-20072Abstract Full Text PDF PubMed Google Scholar, 16Chatterjee S. Suciu D. Dalbey R.E. Kahn P.C. Inouye M. J. Mol. Biol. 1995; 245: 311-314Crossref PubMed Scopus (39) Google Scholar). Various approaches including computational designs have been attempted with limited success in search of more highly functional peptides to serve as substrates for the E. coli enzyme (17Schneider G. Wrede P. Biophys. J. 1994; 66: 335-344Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 18Wrede P. Landt O. Klages S. Fatemi A. Hahn U. Schneider G. Biochemistry. 1998; 37: 3588-3593Crossref PubMed Scopus (26) Google Scholar, 19Palzkill T., Le, Q.Q. Wong A. Botstein D. J. Bacteriol. 1994; 176: 563-568Crossref PubMed Google Scholar, 20Rosse G. Kueng E. Page M.G.P. Schauer-Vukasinovic V. Giller T. Lahm H.W. Hunziker P. Schlatter D. J. Combinatorial Chem. 2000; 2: 461-466Crossref PubMed Scopus (54) Google Scholar). For instance, peptide libraries were created by incorporating randomized sequences into the signal peptide of TEM-1 β-lactamase, varying six amino acid residues between −4 and +2 positions around the signal peptidase cleavage site (19Palzkill T., Le, Q.Q. Wong A. Botstein D. J. Bacteriol. 1994; 176: 563-568Crossref PubMed Google Scholar). Functional sequences were found to support the production of active TEM-1 but none better than the wild type. Reported more recently were combinatorial synthetic peptide libraries in which four positions, −4, −3, −2, and +2, were varied in the signal peptidase recognition sequence, and better than 10-fold improvements over the control were observed among the selected peptides (20Rosse G. Kueng E. Page M.G.P. Schauer-Vukasinovic V. Giller T. Lahm H.W. Hunziker P. Schlatter D. J. Combinatorial Chem. 2000; 2: 461-466Crossref PubMed Scopus (54) Google Scholar).One unsurpassed advantage of phage display over other combinatorial approaches is its capacity to generate a vast number of possible combinations. It is experimentally feasible to randomize up to eight amino acid residues in one library. Phage display has been successfully applied to proteases for selection and optimization of peptide substrates by way of optimizing the substrate phage (21Matthews D.J. Wells J.A. Science. 1993; 260: 1113-1117Crossref PubMed Scopus (316) Google Scholar, 22Ding L. Coombs G.S. Strandberg L. Navre M. Corey D.R. Madison E.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7627-7631Crossref PubMed Scopus (89) Google Scholar, 23Smith M.M. Shi L. Navre M. J. Biol. Chem. 1995; 270: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 24Coombs G.S. Bergstrom R.C. Pellequer J.L. Baker S.I. Navre M. Smith M.M. Tainer J.A. Madison E.L. Corey D.R. Chem. Biol. 1998; 5: 475-488Abstract Full Text PDF PubMed Scopus (110) Google Scholar). A good peptide substrate in turn would aid the development of protease assaysin vitro. Recently, we reported that the proteolysis of substrate phage is a single exponential process and provided the kinetic basis for how to control the rate of proteolysis to ensure the success of substrate phage selection (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The experimental design strategy we put forward is now exploited in the present study, where we applied it to the screening of an 8-mer phage display library with the type I signal peptidase SpsB from Staphylococcus aureus. By systematically tuning the screening stringency in the selection process, we discovered several active substrate phage clones. The sequences found in the most reactive clones were subsequently evaluated as synthetic peptides and characterized for their competency to serve as substrates for SpsB with respect to the substrate recognition and digestion kinetics.DISCUSSIONHaving an efficient in vitro assay not only can facilitate the biochemical study of an enzyme but is also imperative to inhibitor screens if the enzyme is a potential drug target. For an enzyme such as a protease, which catalyzes transformations to protein substrates under physiologic conditions, finding a peptide substrate is usually the first step toward achieving this goal, and phage display technology has been instrumental to the discovery of substrates for proteases.Substrate Phage ReactivityWe recently described the detailed kinetics for the proteolysis of substrate phage and provided a quantitative basis for the design of a phage library screening experiment (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). We further formulated a screening strategy for meeting the desired outputs through the control of screening stringency. In the present study, we methodically applied the strategy to the signal peptidase SpsB from S. aureus and succeeded in finding reactive substrate phage for the enzyme, validating our screening strategy. In the five independent screening experiments performed, the screening stringency was controlled by varying the length of time the phage library was incubated with the enzyme. The incubation was as short as 30 min to as long as 24 h, but only when it was extended to 24 h did we start to uncover a significant number of active clones (Table I). The overall number of active clones was quite small, but it increased as we relaxed the screening stringency, confirming our prediction that a relaxed screening condition would facilitate the enrichment of reactive substrates (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). We recognize that adopting such a condition is especially important for an enzyme like signal peptidase, which appears to have a low catalytic efficiency. The single-exponential kinetic process, which we described previously for the reaction of stromelysin with its substrate phage (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar), was shown here for the signal peptidase. Furthermore, the reaction of the signal peptidase with substrate phage obeyed pseudo-first-order kinetics (Fig.2), suggesting that the kinetic model developed using stromelysin (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar) should be generally applicable to other enzymatic systems.Although the criterion we used to define the reactivity of a phage clone was somewhat arbitrary (Table I), the fraction of active clones identified out of the total number of colonies screened was apparently small, implying that only a small population of potential substrates for SpsB was present in the phage display library. The activity of the best clones was not especially high (Table II), and it proved difficult to achieve further enrichment probably due to the low abundance of potential substrates in the library. It is conceivable that the segment displayed on phage needs to be over eight residues long for it to be an efficient substrate for SpsB. In such a case, a phage display library of eight randomized residues would not be a rich source of substrate for SpsB.Peptide SubstrateWhile we were successful in finding active substrate phage for SpsB, the sequences of the phage clones did not readily allow the differentiation of the active from the unreactive. We found no plausible hints in the sequences that would allow prediction of the site of cleavage and therefore turned to synthetic peptides. The synthetic peptides were dodecamers, containing eight residues found in the variable region in addition to two flanking residues on each side from the constant region of the library. Using an LC/MS method to identify the proteolytic products, we found that all substrates were cleaved at the peptide bond at the C terminus of an Ala residue, which is consistent with Ala being the preferred P1 residue for signal peptidase, and that the cleavage pattern generally obeyed the (−3, −1) rule for substrate recognition by signal peptidase (9Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (732) Google Scholar, 10von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1598) Google Scholar, 11von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (458) Google Scholar). It is interesting that peptide 4 is cleaved at an Ala that is common to all phage clones and not in the variable region as seen with other peptides. It is very curious that peptide 1, derived from the most reactive phage clone, is completely resistant to cleavage by SpsB. One Ala residue found in the variable region of peptide 1 is N-terminally linked to a Pro. If peptide 1 were to be cleaved at this Ala by SpsB, it would place the Pro at the −2 position. The fact that we did not detect the digestion of peptide 1 at such a site is consistent with the current understanding that Pro is not allowed in the −3 to +1 region in the substrate for bacterial signal peptidase (5von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1520) Google Scholar, 11von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (458) Google Scholar). In light of how peptide 4 (and hence substrate phage clone SIRIII-30) was cleaved, it is conceivable that the digestion of substrate phage clone SIIIRIII-8 could also occur outside of the variable region.It is clear that the reactivity of the synthetic peptides correlated poorly with that of the corresponding phage (Table III), even if the results with peptide 1 were not taken into account. This finding reinforces our previous argument that such a correlation is not expected, since synthetic peptide and substrate phage are two different substrate entities, subjected to different kinetic rules (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). Substrate phage is selected under single-turnover conditions, and any improvement in its reactivity would strictly affect the rate of substrate and enzyme binding. On the other hand, the activity of the peptide is usually assayed under steady-state conditions, and the substrate binding rate constant may be a small contributing factor to its overall catalytic efficiency.In general, peptides are known to be inefficient substrates for a signal peptidase such as the E. coli enzyme LepB (14Kuo D. Weidner J. Griffin P. Shah S.K. Knight W.B. Biochemistry. 1994; 33: 8347-8354Crossref PubMed Scopus (47) Google Scholar, 15Dev I.K. Ray P.H. Novak P. J. Biol. Chem. 1990; 265: 20069-20072Abstract Full Text PDF PubMed Google Scholar). The catalytic efficiencies of the peptide substrates for SpsB characterized in this study are not fundamentally different from those found for LepB (15Dev I.K. Ray P.H. Novak P. J. Biol. Chem. 1990; 265: 20069-20072Abstract Full Text PDF PubMed Google Scholar). For instance, K m for the most active peptide 4 (and most likely for peptide 2 as well) is still on the order of millimolar. Previous studies have shown that remarkable improvements in k cat/K m can be achieved when the peptide is modified so that it forms micelles, which enhance the enzyme and substrate interaction (26Stein R.L. Barbosa M.D. Bruckner R. Biochemistry. 2000; 39: 7973-7983Crossref PubMed Scopus (24) Google Scholar); bothk cat and K m values were found to be improved to a level approaching that of a preprotein substrate (16Chatterjee S. Suciu D. Dalbey R.E. Kahn P.C. Inouye M. J. Mol. Biol. 1995; 245: 311-314Crossref PubMed Scopus (39) Google Scholar). It would be interesting to incorporate the various SpsB substrate sequences from this study into a micelle-forming peptide or a preprotein construct and to test what effect it would have on the peptide reactivity. One interesting observation with peptide 4 is that only one residue, an Ala at P1, is present on the P side of the substrate sequence. It is not known what is the minimal requirement for the sequence on the P′ side of the substrate molecule. In a previous attempt to define the minimal substrate, it was found that the E. coli enzyme could cleave peptides with a sequence encompassing −2 and +5 or −7 and +2 residues (15Dev I.K. Ray P.H. Novak P. J. Biol. Chem. 1990; 265: 20069-20072Abstract Full Text PDF PubMed Google Scholar). The crystal structure of β-lactam-acylated LepB has been solved (27Paetzel M. Dalbey R.E. Strynadka N.C. Nature. 1998; 396: 186-190Crossref PubMed Scopus (2) Google Scholar); however, it is not clear from the structure what kind of interactions would exist in the active site for P′ residues on the substrate sequence. Evidently, more experiments are needed to probe the contribution of P′ residues to support the activity of a substrate like peptide 4.ConclusionsThis study shows that through systematic tuning of the selection stringency in screening phage display libraries, the isolation of highly reactive substrate phage clones for an enzyme like signal peptidase, which appears inefficient catalytically toward simple peptide substrates, can be achieved. It demonstrates the general applicability of the kinetic model of substrate phage proteolysis and the phage display library screening strategy that we described earlier (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). These experiments resulted in the isolation of the first peptide substrates for signal peptidase SpsB from S. aureus and provided the first insight into the recognition of substrate cleavage site by the enzyme. Bacterial type I signal peptidase is responsible for cleaving the signal peptide from precursor proteins, and its activity is an integral part of the export and maturation of secreted proteins in vivo. The essential function of the enzyme to bacterial cell viability has been demonstrated using genetic approaches with both Gram-positive and Gram-negative organisms (1Cregg K.M. Wilding I. Black M.T. J. Bacteriol. 1996; 178: 5712-5718Crossref PubMed Google Scholar, 2Date T. J. Bacteriol. 1983; 154: 76-83Crossref PubMed Google Scholar, 3Zhang Y.B. Greenberg B. Lacks S.A. Gene (Amst.). 1997; 194: 249-255Crossref PubMed Scopus (34) Google Scholar), supporting the notion that the signal peptidase is potentially an antibacterial target (4Paetzel M. Dalbey R.E. Strynadka N.C. Pharmacol. Ther. 2000; 87: 27-49Crossref PubMed Scopus (124) Google Scholar). Drug discovery efforts with the enzyme, however, may be hampered by the lack of an effective in vitro assay employing a nonprotein substrate such as a peptide (4Paetzel M. Dalbey R.E. Strynadka N.C. Pharmacol. Ther. 2000; 87: 27-49Crossref PubMed Scopus (124) Google Scholar). Our current understanding is that signal peptides are highly variable in sequence (5von Heijne G. J. Mol. Biol. 1985; 184: 99-105Crossref PubMed Scopus (1520) Google Scholar). Based on the studies carried out over the past 2 decades, it has been established that the recognition sites for signal peptidases lie between −6 and +1 in sequences encompassing the site of cleavage (6Talarico T.L. Barkocy-Gallagher G.A. Ray P.H. Bassford P.J., Jr. Biochem. Biophys. Res. Commun. 1993; 197: 1154-1166Crossref PubMed Scopus (5) Google Scholar, 7Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (74) Google Scholar, 8Kuhn A. Wickner W. J. Biol. Chem. 1985; 260: 15914-15918Abstract Full Text PDF PubMed Google Scholar, 9Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (732) Google Scholar, 10von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1598) Google Scholar, 11von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (458) Google Scholar, 12Carlos J.L. Paetzel M. Brubaker G. Karla A. Ashwell C.M. Lively M.O. Cao G. Bullinger P. Dalbey R.E. J. Biol. Chem. 2000; 275: 38813-38822Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Sequence conservation analyses of a large panel of naturally occurring signal peptides in bacteria and eukaryotes reveal that the predominant residue at the P1 site is Ala and that the predominant residues at the P3 site are large aliphatic residues (Leu, Ile, Val) as well as Ala and Ser, a consensus dubbed the (−3, −1) rule (9Perlman D. Halvorson H.O. J. Mol. Biol. 1983; 167: 391-409Crossref PubMed Scopus (732) Google Scholar, 10von Heijne G. Eur. J. Biochem. 1983; 133: 17-21Crossref PubMed Scopus (1598) Google Scholar, 11von Heijne G. J. Mol. Biol. 1984; 173: 243-251Crossref PubMed Scopus (458) Google Scholar). The (−3, −1) rule also holds for the cleavage of engineered preproteins in vivo as well as in vitro (6Talarico T.L. Barkocy-Gallagher G.A. Ray P.H. Bassford P.J., Jr. Biochem. Biophys. Res. Commun. 1993; 197: 1154-1166Crossref PubMed Scopus (5) Google Scholar, 7Shen L.M. Lee J.I. Cheng S.Y. Jutte H. Kuhn A. Dalbey R.E. Biochemistry. 1991; 30: 11775-11781Crossref PubMed Scopus (74) Google Scholar, 8Kuhn A. Wickner W. J. Biol. Chem. 1985; 260: 15914-15918Abstract Full Text PDF PubMed Google Scholar, 12Carlos J.L. Paetzel M. Brubaker G. Karla A. Ashwell C.M. Lively M.O. Cao G. Bullinger P. Dalbey R.E. J. Biol. Chem. 2000; 275: 38813-38822Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). The reaction of signal peptidases with synthetic peptides, on the other hand, is not as well explored as with protein substrates. For the signal peptidase LepB from Escherichia coli, the best characterized signal peptidase, Ala was found as the only residue permitted at the P1 site through single amino acid replacements of a peptide bearing the signal peptide sequence of theE. coli maltose-binding protein (13Kuo D.W. Chan H.K. Wilson C.J. Griffin P.R. Williams H. Knight W.B. Arch. Biochem. Biophys. 1993; 303: 274-280Crossref PubMed Scopus (59) Google Scholar). As demonstrated with the E. coli LepB enzyme, the catalytic efficiency of signal peptidase toward short peptide substrates is generally several orders of magnitude lower than toward polypeptides bearing the same protease recognition sequence (14Kuo D. Weidner J. Griffin P. Shah S.K. Knight W.B. Biochemistry. 1994; 33: 8347-8354Crossref PubMed Scopus (47) Google Scholar, 15Dev I.K. Ray P.H. Novak P. J. Biol. Chem. 1990; 265: 20069-20072Abstract Full Text PDF PubMed Google Scholar, 16Chatterjee S. Suciu D. Dalbey R.E. Kahn P.C. Inouye M. J. Mol. Biol. 1995; 245: 311-314Crossref PubMed Scopus (39) Google Scholar). Various approaches including computational designs have been attempted with limited success in search of more highly functional peptides to serve as substrates for the E. coli enzyme (17Schneider G. Wrede P. Biophys. J. 1994; 66: 335-344Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 18Wrede P. Landt O. Klages S. Fatemi A. Hahn U. Schneider G. Biochemistry. 1998; 37: 3588-3593Crossref PubMed Scopus (26) Google Scholar, 19Palzkill T., Le, Q.Q. Wong A. Botstein D. J. Bacteriol. 1994; 176: 563-568Crossref PubMed Google Scholar, 20Rosse G. Kueng E. Page M.G.P. Schauer-Vukasinovic V. Giller T. Lahm H.W. Hunziker P. Schlatter D. J. Combinatorial Chem. 2000; 2: 461-466Crossref PubMed Scopus (54) Google Scholar). For instance, peptide libraries were created by incorporating randomized sequences into the signal peptide of TEM-1 β-lactamase, varying six amino acid residues between −4 and +2 positions around the signal peptidase cleavage site (19Palzkill T., Le, Q.Q. Wong A. Botstein D. J. Bacteriol. 1994; 176: 563-568Crossref PubMed Google Scholar). Functional sequences were found to support the production of active TEM-1 but none better than the wild type. Reported more recently were combinatorial synthetic peptide libraries in which four positions, −4, −3, −2, and +2, were varied in the signal peptidase recognition sequence, and better than 10-fold improvements over the control were observed among the selected peptides (20Rosse G. Kueng E. Page M.G.P. Schauer-Vukasinovic V. Giller T. Lahm H.W. Hunziker P. Schlatter D. J. Combinatorial Chem. 2000; 2: 461-466Crossref PubMed Scopus (54) Google Scholar). One unsurpassed advantage of phage display over other combinatorial approaches is its capacity to generate a vast number of possible combinations. It is experimentally feasible to randomize up to eight amino acid residues in one library. Phage display has been successfully applied to proteases for selection and optimization of peptide substrates by way of optimizing the substrate phage (21Matthews D.J. Wells J.A. Science. 1993; 260: 1113-1117Crossref PubMed Scopus (316) Google Scholar, 22Ding L. Coombs G.S. Strandberg L. Navre M. Corey D.R. Madison E.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7627-7631Crossref PubMed Scopus (89) Google Scholar, 23Smith M.M. Shi L. Navre M. J. Biol. Chem. 1995; 270: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 24Coombs G.S. Bergstrom R.C. Pellequer J.L. Baker S.I. Navre M. Smith M.M. Tainer J.A. Madison E.L. Corey D.R. Chem. Biol. 1998; 5: 475-488Abstract Full Text PDF PubMed Scopus (110) Google Scholar). A good peptide substrate in turn would aid the development of protease assaysin vitro. Recently, we reported that the proteolysis of substrate phage is a single exponential process and provided the kinetic basis for how to control the rate of proteolysis to ensure the success of substrate phage selection (25Sharkov N.A. Davis R.M. Reidhaar-Olson J.F. Navre M. Cai D. J. Biol. Chem. 2001; 276: 10788-10793Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). The experimental design strategy we put forward is now exploited in the present study, where we applied it to the screening of an 8-mer phage display library with the type I signal peptidase SpsB from Staphylococcus aureus. By systematically tuning the screening stringency in the selection process, we discovered several active substrate phage clones. The sequences found in the most reactive clones were subsequently evaluated as synthetic peptides and characterized for their competency to serve as subst" @default.
• W1982185609 created "2016-06-24" @default.
• W1982185609 creator A5040403700 @default.
• W1982185609 creator A5080594879 @default.
• W1982185609 date "2002-02-01" @default.
• W1982185609 modified "2023-09-29" @default.
• W1982185609 title "Discovery of Substrate for Type I Signal Peptidase SpsB fromStaphylococcus aureus" @default.
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