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Keywords:

  • Protein folding;
  • Alpha-amylase;
  • Levansucrase;
  • Teichoic acid;
  • Cell wall translocation;
  • Bacillus subtilis

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

In order to characterize the dynamics of the interaction between the emergent membrane translocated exoprotein and the components of Bacillus subtilis cell wall, we examined the kinetics of the in vitro refolding of levansucrase and α-amylase, at pH 7 and 37°C, in the presence of polyphosphates (polyP) of various chain lengths (2≤n≤65). These soluble anionic polymers are considered here to mimic the role of teichoic acids. Even in the absence of calcium, levansucrase rapidly refolded in the presence of polyP of n≥16. In contrast, polyP modulate indirectly the rate of α-amylase refolding via their affinity for calcium. These differential effects might explain that the rate of the cell wall translocation of α-amylase secretion was found to be half that of levansucrase.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

It is now widely accepted that proteins are translocated under a non-native state [1–5]. In B. subtilis, proteins emerging on the trans side of the membrane encounter the cell wall, a highly cross-linked semi-porous macromolecule composed of peptidoglycan and teichoic acids. The subsequent translocation of the extracellular proteins through the cell wall requires a rapid and efficient folding into their native conformation in order to prevent the proteolytic action of cell wall associated proteases [6,7] and to minimize interactions with cell wall components. It was proposed that PrsA [8], a lipoprotein linked to the outer surface of the cytoplasmic membrane, assists the folding. Other possible cofactors of folding are metal ions such as calcium or iron which are concentrated at the membrane wall interface [9,10] and mediate the in vitro refolding of several proteins [11–13].

In the present work, we explore the hypothesis that structural components of cell wall could per se facilitate the folding of proteins en route to be secreted. We focused our attention on the possible role of teichoic acids. These phosphorylated polymers, which account for almost 50% of the cell wall dry-weight [14], are linear polyanionic molecules that usually contain between 10 and 50 glycerophosphate units, displaying affinity for metal ions such as Ca2+ and possessing a KA of 4.5×104 M−1[15]. This compound is not commercially available and is difficult to obtain in an homogeneous and pure unmodified state from cell wall extracts [16]. Allowing for that, we chose inorganic short chain length polyphosphates (polyP) as models to mimic the properties of teichoic acids and their effects on the refolding of α-amylase and levansucrase, two B. subtilis exocellular proteins of which the in vitro reversible unfolding-folding transition at pH 7 and 37°C are well characterized [11,12].

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Materials

PolyP (sodium salts) of various chain lengths were purchased from Sigma.

2.2Purification and assays of proteins

Levansucrase (stock solution 30 mg ml−1) was purified from the culture supernatant of induced B. subtilis QB112 (degU32(Hy), sacA321) according to the published procedure [17].

α-Amylase (stock solution 30 mg ml−1) was prepared from the culture supernatant of induced B. subtilis GM96101 (degU32(Hy), sacA321, ΔsacB, sacRamyE) [18], according to the published procedure [19].

2.3Fluorescence measurements

Changes in intrinsic fluorescence and fluorescence spectra were recorded with a F2000 Hitachi thermoregulated spectrofluorimeter.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Unfolding-refolding transition of levansucrase and α-amylase in the presence of polyP and in the absence of calcium

We showed previously [12,20] from in vitro studies that the unfolding-refolding transition of the two proteins was mediated by calcium at pH 7 and 37°C. In the absence of this metal, the proteins remained in an unfolded state after dilution of denaturing agents. We studied the effect of the presence of 1 mM polyP of various chain lengths under such conditions. Refolding was monitored by measuring changes in the intrinsic fluorescence and the disappearance of proteolytic sensitivity of the refolded state. We observed that levansucrase rapidly and fully refolded in the presence of polyP of n≥16. In contrast, these polymers did not promote α-amylase refolding. The effect of polyP45 (Fig. 1) illustrated this study.

image

Figure 1. Unfolding-refolding transition of levansucrase and α-amylase mediated by polyP45 in the absence of calcium. The transition was monitored by fluorescence measurements (A) or by protease sensitivity (B). 4 μl levansucrase or α-amylase stock solution was mixed with 16 μl of 6 M Gdn-HCl in buffer A (0.1 M sodium phosphate pH 7, containing 1 mM EDTA). After 10 min incubation at 37°C, 10 μl denaturing mixture was diluted into 2 ml buffer A in the absence (a) or in the presence (b) of 1 mM polyP45. A: Traces of changes in fluorescence were recorded at 343 nm (excitation wavelength was 283 nm). The dead-time for mixing was approximately 8 s. B: Protease sensitivity changes were estimated from 100 μl aliquots withdrawn at 30 s and 720 s after initiation of refolding and mixed with 1 μl of subtilisin 1 mg ml−1. Time 0 of refolding was obtained by mixing 1 μl denaturing mixture with 200 μl refolding buffer containing 10 μg ml−1 subtilisin. The samples were submitted to SDS-PAGE analysis. The gel was stained with Coomassie brilliant blue.

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3.2Unfolding-refolding transition of levansucrase and α-amylase in the presence of both polyP and calcium

The effects of 1 mM polyP of various chain lengths on the unfolding-refolding transition of each protein, occurring in the presence of 0.5 mM Ca2+, at pH 7 and 37°C, are shown in Fig. 2. The rate constants of levansucrase refolding increased with the chain length of polyP, whereas an opposite result was obtained for α-amylase. PolyP of n>5 totally inhibited the refolding of this latter protein. The variation in the folding rate constant of each protein versus the concentration of polyP65 in the folding mixture was studied (Fig. 3). We noted that both proteins refolded at an equivalent rate in the absence of polyP65. A gradual increase of polyP65 concentration decreased the folding rate constant of α-amylase, whereas it had the opposite effect on the folding rate of levansucrase.

image

Figure 2. Rate constants of unfolding-refolding transition of levansucrase and α-amylase in the presence of polyP of various chain lengths and a fixed concentration of calcium. Levansucrase and α-amylase were unfolded in 5 M Gdn-HCl as described in Fig. 1. The refolding at 37°C was initiated by diluting the denaturing mixture 200 times in 0.1 M. Sodium phosphate pH 7, 0.5 mM CaCl2, 1 mM polyP of chain length indicated. The apparent first order rate constants of refolding were determined from the regression adjustment, by the least square method, of changes in the intensity of fluorescence at 343 nm as a function of time [11,12]. Levansucrase (◯); α-amylase (•).

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image

Figure 3. Kinetics of unfolding-refolding transition of levansucrase and α-amylase with respect to polyP65 concentration and a fixed concentration of calcium. Levansucrase and α-amylase were unfolded in 5 M Gdn-HCl as described before. The refolding was initiated by diluting the denaturing mixture 200 times in 0.1 M sodium phosphate, pH 7, 0.5 mM CaCl2 containing 0–100 μM polyP65. The rate constants of refolding were determined from changes in the fluorescence intensity at 343 nm as a function of time. Levansucrase (◯); α-amylase (•).

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3.3How does polyP inhibit α-amylase refolding?

Two hypotheses can account for the inhibition of α-amylase refolding: either polyP stabilize the unfolded form of the protein through electrostatic interactions or polyP, via their high affinity to calcium (KA=6.3×106 M−1 for polyP60[21]), decrease the amount of free calcium needed to promote protein refolding. To test the second hypothesis, we studied whether inhibition of refolding by polyP could be abolished by the addition of a large excess of calcium. This hypothesis seems to be correct, as shown in Fig. 4.

image

Figure 4. Effect of the addition of a large quantity of calcium on the inhibition of α-amylase refolding by polyP65. α-Amylase was unfolded in 5 M Gdn-HCl as described above. 5 μl of the denaturing mixture was diluted in 1 ml 0.2 M sodium acetate pH 7, 0.5 mM CaCl2, containing 1 mM polyP65 preincubated at 37°C. The changes in intensity of fluorescence with respect to time were recorded. After 2 min, 25 μl 0.1 M CaCl2 (25 mM final concentration) was added (arrow) and changes in the intensity of fluorescence of the protein recorded.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

PolyP of n>16 act as cofactors of levansucrase folding even in the absence of calcium, α-amylase, in contrast, needs free calcium to promote its folding. If polyP mimic in vitro, at least partially, the role played by teichoic acids in the cell wall environment, one would expect that levansucrase emerging from the translocase complex fold more rapidly than α-amylase. This could explain the difference in the release kinetics of the two proteins, t1/2 of 1 min and 2 min, for levansucrase and α-amylase, respectively. Furthermore, we show that a high affinity of calcium for polyP inhibits α-amylase folding mediated by this metal ion. This might be an indication that modulation of teichoic acid anionic charge via alanylation [22] controls calcium affinity in such a way that free calcium remains available for an efficient folding of exoproteins that have properties in common with α-amylase. On the other hand, we cannot rule out the possibility that the polyP themselves contribute to the efficiency of levansucrase secretion. Although no counterpart of E. coli polyP kinase was found in B. subtilis[23], it is reasonable to suggest that different enzymic pathways might promote the synthesis of short chain length polyP in this bacterium [24].

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was supported in part by the European Commission (Biotechnology program, Contract BIO4-CT96-0097) and was carried out within the framework of the European Bacillus Secretion Group. We thank A. Kropfinger for revision of the English text.

References

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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