1 Department of Microbiology, University of Texas Southwestern, Dallas, TX, USA
2 Maurice and Gabriela Goldschleger Eye Research Institute, Tel-Aviv University Sackler Faculty of Medicine, Sheba Medical Center, Tel Hashomer, Israel
3 Department of Microbiology and Immunology, Medical College of Virginia Campus of Virginia Commonwealth University, Richmond, VA, USA
4 McGuire Veterans Affairs Medical Center, Richmond, VA, USA
Correspondence
Dennis E. Ohman
deohman{at}hsc.vcu.edu
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ABSTRACT |
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INTRODUCTION |
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Subtilisin E is an alkaline serine protease secreted by Bacillus subtilis and was the first demonstrated IMC-containing protease. Its 77 residue propeptide is required to guide its folding into an active enzyme (Ikemura et al., 1987; Zhu et al., 1989
). The
-lytic protease of Lysobacter enzymogenes is an extracellular serine proteinase that has a 166 residue propeptide that promotes folding of the enzyme into an active, secretion-competent, highly stable conformation (Fujishige et al., 1992
; Silen & Agard, 1989
; Silen et al., 1989
). No obvious sequence homology exists between the propeptides of these two proteases, but both probably function by lowering the kinetic barrier between an inactive molten-globule protein and conversion to a native proteinase (Baker et al., 1992
; Eder et al., 1993
). Many eukaryotic proteases exhibit a similar requirement for the propeptide to reduce the rate of nonproductive folding, including serine proteinases (e.g. Saccharomyces cerevisiae carboxypeptidase Y and Yarrowia lipolytica alkaline extracellular protease), aspartic proteinases (e.g. S. cerevisiae proteinase A and Rhizopus niveus proteinase I) and thiol proteinases (e.g. cathepsins L and B) (reviewed by Shinde & Inouye, 2000
). The diversity of proteases that depend on their propeptides for proper folding suggests a convergent evolution of this propeptide-mediated folding mechanism (Eder et al., 1993
).
The subtilisin family of proteinases is large, and its members are found in both prokaryotic and eukaryotic cells. The propeptide sequences of subtilisin proteases have two conserved domains, each consisting of hydrophobic residues flanked by charged amino acids (Shinde & Inouye, 1994). Random mutagenesis of the prosubtilisin propeptide identified three short hydrophobic sequences located within these conserved domains that are critical for production of active subtilisin (Kobayashi & Inouye, 1992
; Lerner et al., 1990
). A second-site suppressor mutation of a propeptide mutation has been mapped to the mature enzyme, suggesting that functional regions within the propeptide interact with mature subtilisin during the folding process (Kobayashi & Inouye, 1992
). The crystal structure of the subtilisin Epropeptide complex has been determined (Bryan et al., 1995
; Janknecht et al., 1991
), and this provided additional information on the nature of the interactions of specific residues within subtilisin and its propeptide.
Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that secretes a large number of toxic and degradative enzymes, including several proteolytic enzymes, that play important roles in pathogenesis. The most abundant of the secreted proteases of P. aeruginosa is elastase. This protease has been classified as a member of family M4 (clan MA), which includes a large group of thermolysin-like, neutral zinc-metalloproteases (TNPs) that are produced by both Gram-positive and Gram-negative bacteria (Hase & Finkelstein, 1990; Kessler & Ohman, 1998
; Wetmore et al., 1992
).
Elastase (also called LasB protease and pseudolysin) is initially synthesized as a precursor with a pre-pro-mature domain structure consisting of a signal peptide (23 residues), a propeptide (174 residues) and a carboxy-terminal catalytic domain (301 residues) (Kessler & Ohman, 1998). The propeptide is cleaved autocatalytically within the periplasm (McIver et al., 1991
). There it immediately forms an inactive complex with the processed enzyme (Kessler & Safrin, 1994
), and it is in this form that elastase is secreted into the extracellular environment (Kessler et al., 1998
). While the propeptide can be detected in the culture supernatant (Braun et al., 2000
; Kessler et al., 1998
), it is degraded shortly after secretion and only the mature moiety is stably found in the culture supernatant.
Elastase requires its propeptide for both proper folding and secretion and represents the first TNP family member for which the IMC function of its propeptide has been demonstrated (Braun et al., 1996; McIver et al., 1995
). It has since been shown that thermolysin from Bacillus thermoproteolyticus, the prototype of this family of Zn-dependent metalloendopeptidases, also has a long N-terminal propeptide that is processed autocatalytically (Marie-Claire et al., 1998
) and plays a role in the folding of thermolysin (O'Donohue & Beaumont, 1996
). TNPs are produced by a variety of bacterial species, and all appear to be synthesized as pre-proenzyme precursors with large amino-terminal propeptides, suggesting that their propeptides may function as IMCs as well. However, very little is known about the specific residues in the propeptide that are involved in IMC functions.
The propeptides of TNPs from Bacillus species contain conserved residues in the carboxy-terminal portion proximal to the mature processing site that are also found in the propeptide of P. aeruginosa elastase (Wetmore et al., 1992). In the present study, we compared the P. aeruginosa elastase propeptide sequence to several proteins in the database in order to identify the conserved amino acids that may represent critical residues required for chaperone function. Some of these residues were then chosen as targets for site-directed mutagenesis as an initial step toward testing the prediction that sequence conservation indicates a common and critical function. Using a native system designed to study lasB mutations in P. aeruginosa (McIver et al., 1995
), we constructed strains with single amino acid substitutions in the propeptide of the elastase precursor. These were then tested for their effects on the accumulation of stable, extracellular elastase.
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METHODS |
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DNA manipulations.
Most routine DNA manipulations were performed as described by Maniatis et al. (1982). DNA sequences were determined by the chain-termination technique with custom oligonucleotides. Sequence comparisons were performed using the basic local alignment search tool (BLAST) algorithm (Altschul et al., 1990
). Enzymes belonging to the TNP family were based upon significant homologies within the protease domain of their molecules (Hase & Finkelstein, 1990
).
Construction of lasB alleles encoding propeptide amino acid substitutions.
Oligonucleotide-directed site-specific mutagenesis was performed using the Altered Sites in vitro mutagenesis system (Promega) as described by the manufacturer. Single-base-pair substitutions encoding mutant LasB propeptide residues were introduced within the plasmid pKSM4 (McIver et al., 1991), which contained the wild-type lasB gene and regulatory region on a 2·5 kb EcoRIPstI fragment in pAlter-1 (Promega). Site-specific substitutions were verified by DNA sequence analysis before being cloned into the broad-host-range vector pLAFR3 (McIver et al., 1991
) to generate the pKSM66 series of plasmids (Table 1
). pKSM66 clones were mobilized into the P. aeruginosa lasB-deficient strain FRD740 by triparental mating as previously described (Goldberg & Ohman, 1984
). Plasmid pKSM3, with wild-type lasB in broad-host-range plasmid pLAFR3, has been previously described (McIver et al., 1995
).
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SDS-PAGE and immunoblotting.
Equivalent amounts of cells in the 18 h cultures were demonstrated by turbidity (OD600) and then centrifuged. Pellets were used for cell lysates, and supernatants were subjected to TCA precipitation to concentrate secreted proteins (McIver et al., 1995). All of the respective samples were suspended in SDS sample buffer (60 mM Tris/HCl, 2 % SDS, 10 %, v/v, glycerol, 0·1 mg ml1 bromophenol blue, 5 % 2-mercaptoethanol [pH 6·8]) and loaded onto a 12·5 % polyacrylamide gel for electrophoresis (SDS-PAGE). Proteins in polyacrylamide gels were electrotransferred to nitrocellulose in a Trans-Blot apparatus (Bio-Rad) for 2 h at 160 mA at 4 °C. Immunoblotting was performed as previously described (McIver et al., 1991
) using rabbit anti-denatured elastase IgG (Kessler & Safrin, 1988
) as the primary antibody, followed by a goat anti-rabbit horseradish-peroxidase conjugate (Sigma).
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RESULTS |
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Effects of amino acid substitutions in the propeptide on extracellular elastolytic activity
To evaluate the hypothesis that conserved amino acids in the propeptide play a role in the production of active elastase, alleles of lasB that encoded proteins with substitutions in conserved residues of region ProM were constructed by oligonucleotide-directed mutagenesis of the wild-type lasB allele in pKSM4. Following verification by sequence analysis, each altered gene was cloned into a low-copy, broad-host-range plasmid (pLAFR3) and introduced into FRD740, a lasB-deleted strain of P. aeruginosa FRD2. To determine the consequences of the mutations on extracellular elastolytic activity, supernatants from 18 h standardized cultures were obtained from the strains expressing the mutant lasB alleles, and the rate of hydrolysis of elastin Congo red was used to measure elastolytic activity.
The culture supernatant from FRD740(pKSM3) expressing wild-type lasB from a plasmid exhibited high elastolytic activity (Table 1). FRD740(pLAFR3), the
lasB strain containing the cloning vector, produced low elastolytic activity, representing about 10 % of FRD740(pKSM3) levels, and this non-LasB value was subtracted from the other test reactions. This residual elastolytic activity in a
lasB background is predominantly due to the secreted LasA protease (Gustin et al., 1996
).
When the conserved, hydrophilic Asn68 residue in the ProM region was substituted with another amide residue (Gln) or an acidic residue (Asp), extracellular elastolytic activity was drastically reduced to about one-fifth of wild-type levels (Table 1). This indicated that Asn68 was a critical residue in the propeptide for production of active elastase. Likewise, when the conserved, charged Arg74 residue was substituted with another basic residue (Lys), elastolytic activity was reduced to about one-third, and introducing a hydrophobic residue (Val) reduced elastolytic activity almost completely. This suggests that a positive charge at position 74 was important for IMC function, but that there was some specificity for Arg. In contrast, when the conserved and hydrophobic Gly69 residue was substituted with hydrophilic Ser, extracellular elastolytic activity dropped only by half. Interestingly, a radical substitution at hydrophobic Gly81 with acidic Glu had a limited effect on activity. Changing the hydrophobic Val84 to another aliphatic residue (Ala) or an acidic residue (Glu) reduced extracellular elastolytic activity to about 20 or 87 %, respectively, suggesting that the size of the R group here was more important than hydrophobicity (Table 1
).
Single base pair substitutions were made in the ProC region to determine the effects of alterations to a highly conserved cluster of residues: Ile181-Asp182-Ala183. These mutant alleles, cloned into pLAFR3, were then introduced into the lasB mutant FRD740 and tested for elastase activity. Substitution of hydrophobic residue Ile181 with another hydrophobic residue (Val) had a minor effect, but introducing an acidic group (Glu) resulted in almost background elastolytic activity levels (Table 1
). Despite its conservation among TNP propeptides, substitutions at acidic residue Asp182 with another acidic residue (Glu) or an unionized amino acid (Ser) had no adverse effect on extracellular elastolytic activity. In contrast, substitutions at the adjacent hydrophobic Ala183 with an unionized hydrophilic residue (Cys) resulted in a drastic loss of extracellular elastolytic activity (Table 1
).
Effects of propeptide amino acid substitutions on elastase stability and secretion
The loss of extracellular elastolytic activity in the propeptide mutants above could be due to defects in the ability to fold the mature protease into a stable protein and/or in the ability to be secreted by the type II secretion machinery. These possibilities were addressed by immunoblot analysis in which the relative amounts of elastase antigen in cell extracts (Fig. 2a, c) and culture supernatants (Fig. 2b, d
) were evaluated. Expression of each mutant allele was compared to that of the wild-type lasB allele (lane 2) under the same conditions. A defect in the recognition of an altered proelastase by the type II secretory apparatus would be expected to cause abnormal accumulation of periplasmic proelastase (51 kDa) and/or periplasmic elastase (33 kDa).
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As for mutations in the ProC region (Fig. 2c, d), the conservative Ile181Val substitution generally had little effect on elastase processing or secretion (lane 11). Substitutions at Asp182 (lanes 13 and 14) also had no detrimental affect. However, the Ile181Glu (lane 12) and Ala183Cys (lane 15) substitutions resulted in a minor accumulation of periplasmic proelastase, as well as accumulation of smaller degradation products, and both blocked the formation of a stable mature elastase protein in the supernatant. Thus, the ProC residues Ile181 and Ala183 apparently play an important role, possibly in self-processing, secretion or folding of a stable elastase outside of the cell.
Another interesting phenotype observed among some of the mutant precursor proteins was an overall reduction in total elastase antigen in the culture when compared to that of wild-type lasB expression. For example, in the ProM region, substitution of Asn68 with Gln (lane 3) or Asp (lane 4) did not cause intracellular accumulation, but still dramatically reduced extracellular elastase antigen and enzymic activity. This presumably represents propeptide mutations that caused folding defects, suggesting that Asn68Gln- and Asn68Asp-proelastases are susceptible to nonspecific degradation by other proteases of P. aeruginosa. Also, the Gly81Glu substitution (lane 8) showed less intracellular proelastase and less extracellular mature elastase than wild-type (lane 2), suggesting a folding defect. The Val84Ala substitution (lane 9) resulted in reduced extracellular elastase antigen without intracellular accumulation, although a Val84Glu substitution (lane 10) was readily tolerated. In the ProC region, substitutions at Asp182 with Glu or Ser had no adverse effect on secretion of stable elastase (lanes 13 and 14). Overall, these studies support the prediction that some conserved amino acid residues, or in some cases their charge or hydrophobic character, are essential for the elastase propeptide to function efficiently as an IMC for both protein folding and secretion.
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DISCUSSION |
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The IMC of subtilisin exhibits an interesting bias toward charged amino acids within its propeptide when compared to the mature protein; the propeptide has 36 % charged residues as compared to only 12 % in the mature protease (Shinde & Inouye, 1996). It has been postulated that the charged propeptide residues cover the hydrophobic surfaces of the mature protease in order to provide a more even charge distribution of the proenzyme in the cell (Shinde & Inouye, 1993
). However, this mechanism is unlikely for TNP subfamily molecules because both the prodomain and the protease domain exhibit the same proportion (2327 %) of charged amino acids. The prodomain of subtilisin contains two conserved motifs essential for IMC function, which are composed of predominately hydrophobic amino acids flanked by charged residues (Shinde & Inouye, 1993
). Mutations that increase the hydrophilicity of the core sequences are deleterious to subtilisin-folding reactions. In contrast, the propeptides of the TNP subfamily show no obvious homology to conserved motifs in the subtilisin propeptides, and the presence of hydrophobic amino acids flanked by charged residues is not as striking (data not shown). In addition, TNPs from Gram-negative bacteria must also cooperate with a second secretion pathway (e.g. the type II/Xcp system in P. aeruginosa) for export through the outer membrane (reviewed by Filloux et al., 1998
). Therefore, the proposed mechanisms used by subtilisin IMCs may be different from those used by the TNP IMCs to mediate chaperone function.
To begin to understand the mechanisms by which TNP propeptides act as IMCs, we first compared the propeptide sequences of several TNPs from Gram-negative and Gram-positive bacteria to identify conserved residues. Two conserved regions were observed; they were located in the middle of the propeptide and at the C-terminus and called ProM and ProC, respectively. A hydrophilicity plot of the pre-propeptide sequence of elastase (Fig. 3) showed that the ProM region was generally hydrophilic, suggesting that charge is important to its function and/or that this area may be surface-exposed. The ProC region was generally less hydrophilic than the ProM region, suggesting that it may not be surface exposed in proelastase.
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The signal peptide of cytoplasmic pre-proelastase is removed during translocation across the inner membrane into the periplasmic space, where folding occurs (Kessler & Safrin, 1988). The propeptide of elastase is secreted as a complex with the mature enzyme, suggesting that the propeptide may play an important role in trafficking elastase to the secretory machinery (Kessler et al., 1998
). Some of the substitutions at conserved amino acids were shown in this study to primarily affect processing and/or secretion of an active elastase into the extracellular milieu. In the ProM region, substitution of polar Arg74 with a nonpolar Val residue resulted in a drastic accumulation of proelastase within the periplasm and no detectable extracellular elastase antigen. Even substituting this Arg residue with another charged residue (Lys) had a similar effect, though less drastic. The Arg74Val-propeptide may still possess chaperone activity such that proelastase was protected from nonspecific degradation, but the substitution prevented self-processing and caused a secretion defect, which led to the periplasmic accumulation of the unprocessed proenzyme. Since ProM is predicted to be surface-exposed, it is possible that Arg74 interacts directly with the components of the type II secretion machinery. At another ProM site, the Val84Glu substitution also resulted in increased intracellular accumulation of proelastase, indicating a secretion defect, although a substitution with Ala did not show the same effect.
With regard to the ProC region, earlier studies showed that carboxy-terminal deletions within the prodomain of a Bacillus cereus TNP, encompassing conserved residues corresponding to ProC of proelastase, adversely affect secretion (Wetmore et al., 1992). However, these deletions removed over 40 amino acids of the propeptide and involved residues proximal to the propeptide cleavage site. Here we showed that substituting only single amino acids at Ile181 or Ala183 within the ProC region of the elastase propeptide adversely affected accumulation of stable elastase protein in the supernatant. However, a conservative substitution of Val181 for Ile, so that it retained the hydrophobic nature of the region, had no effect on the activity of the elastase prodomain. Thus, the hydrophobic character here is apparently important to function. Another study of lasB expression, in a Pseudomonas putida heterologous system, also identified a role for Ala183, in that a substitution adversely affected the inhibitor function of the propeptide on elastase activity (Braun et al., 2000
). Since the Ile181 and Ala183 residues are relatively close to the site of propeptide removal, they may play a role in the rate of processing and thus mature protein stability. Interestingly, our two different substitutions at the conserved Asp182 had no adverse effect on processing, secretion or protein stability.
Several substitutions in the propeptide at conserved amino acids appeared to primarily affect the folding of elastase into a conformation that can resist degradation by the other proteases in the cell and/or culture supernatant. If the propeptide is a chaperone, then this was a predictable phenotype of mutants defective in proper protein folding. In the ProM region, two different substitutions at Asn68 both permitted elastase secretion, but the levels of total elastase antigen inside and outside the cell were reduced. This suggested that Asn68 was primarily involved in the proper folding of the mature enzyme, and that the defects led to an increased degradation of proelastase within the periplasm. The strain expressing the Gly81Glu substitution contained no detectable periplasmic proelastase, suggesting an increased degradation of misfolded proelastase proteins; the extracellular elastase antigen levels and activity were less than half that of wild-type. The Val84Ala phenotype was reduced extracellular elastase antigen levels and activity, but without intracellular accumulation, suggesting a folding defect that rendered the protein more susceptible to the high level of non-elastase protease activity in the supernatants of 18 h P. aeruginosa cultures. A comprehensive mutation analysis of the effects of mutations in the propeptide is in progress to identify all residues likely to be important for elastase folding and secretion. Ultimately, the crystal structure of proelastase, which has yet to be determined, will reveal how these critical residues in the propeptide interact with the mature domain to bring about proper folding and efficient secretion.
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ACKNOWLEDGEMENTS |
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Received 19 May 2004;
revised 27 July 2004;
accepted 10 August 2004.
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