Department of Biochemistry and Biotechnology, Martin-Luther Universität Halle-Wittenberg, Kurt-Mothes Strasse 3, 06120 Halle, Germany
1 To whom correspondence should be addressed. e-mail: arnold{at}biochemtech.uni-halle.de
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Abstract |
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Keywords: proline concept/proteolysis/ribonucleaseA/site-directed mutagenesis/stabilization
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Introduction |
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Proteases are ubiquitous in nature. Owing to the manifold processes in which they are involved (protein degradation, processing, activation, etc.) (Neurath, 1989), a huge number of proteases with very different specificities exist. The primary specificity of a protease describes the amino acid residue that is accepted in its S1 (or S1') position (Hubbard, 1998
), corresponding to the complementary positions P1 (or P1'), respectively, in the substrate protein. Moreover, the substrate specificity is characterized by the acceptance of amino acid side chains in additional subsite positions of the protease, Sn to Sn' (Schechter and Berger, 1967
). The conformation and flexibility of the peptide backbone of the substrate protein also play an important role in efficient cleavage (Hubbard et al., 1998
). Accordingly, so-called unspecific proteases accept various side chains in the P1 or P1' position of the substrate and make small demands on the substrate conformation. As a consequence, they are often able to degrade even natively folded proteins (Hubbard, 1998
).
Cleavage of peptide bonds is sterically impeded by embedding into elements of secondary structure (Hubbard et al., 1998), by glycosylation (Rudd et al., 1995
; Arnold et al., 1998
) or by the presence of disulfide bonds (Mansfeld et al., 1997
; So et al., 1997
). While such cases can often be found in nature, they are difficult to exploit for tailoring the proteolytic resistance of proteins by means of protein engineering. Here, prevention of proteolysis is possible by the replacement of amino acid residues at the cleavage site with residues that are not accepted as substrate. The substitution of positively charged residues by negatively charged residues or of non-polar residues by polar residues and vice versa to prevent proteolysis is denoted the charge concept (Frenken et al., 1993
). A clear disadvantage of this concept is that prevention of proteolysis by one protease may increase the susceptibility toward another protease. The second concept is based on the replacement of amino acid residues with proline. Proline is the only proteinogenic imino acid and is not accepted by most of the proteases at the potential cleavage site (Brömme et al., 1986
). In positions P2 or P4, however, proline can sometimes even promote the cleavage at P1P1' (Thompson and Blout, 1973a
). Another reason for a decreased proteolytic susceptibility of proline-containing protein regions may be the rigidity of the XaaPro peptide bond. Here, the reduced flexibility of the peptide backbone concerned (Jaenicke, 2000
) impedes the binding of the protease to the substrate. Substitution of amino acid residues by proline to increase the proteolytic resistance is termed the proline concept (Frenken et al., 1993
). However, owing to the restrictions of the peptidyl prolyl peptide bond, proline substitutions bear an increased risk of affecting the protein conformation and/or stability.
Bovine pancreatic ribonuclease A (RNase A, EC 3.1.27.5, Figure 1) is a small and compact protein. RNase A is characterized by high thermostability (Arnold and Ulbrich-Hofmann, 1997; Ramos and Baldwin, 2002
) and its structure is cross-linked by four disulfide bonds (Wlodawer et al., 1982
). Although RNase A resists proteolytic degradation by trypsin and thermolysin at room temperature (Arnold et al., 1996
), it is degraded efficiently by the unspecific proteases subtilisin Carlsberg (subtilisin C.) (Richards and Vithayathil, 1959
), subtilisin BPN' (Doscher and Hirs, 1967
; Neumann and Hofsteenge, 1994
), proteinase K (Rauber et al., 1978
) and elastase (Klee, 1965
) (Figure 1). The flexibility of the loop region around Ala20 is responsible for this proteolytic susceptibility resulting in the primary cleavage of the Ala20Ser21 peptide bond by subtilisin C. and proteinase K. Degradation by subtilisin BPN' is reported to start evenly at both Ala20Ser21 and Ser21Ser22 (Neumann and Hofsteenge, 1994
). Klee had postulated the primary cleavage of Ala19Ala20 by elastase (Klee, 1965
) (Figure 1). Based on these results, we had rationalized that replacement of Ala20 with Pro should prevent degradation of RNase A by these proteases (Markert et al., 2001
). Whereas for the mutant enzyme A20P-RNase A the rate constants of proteolysis (kp) by subtilisin C. and proteinase K in fact were decreased by two orders of magnitude, kp for elastase was surprisingly even slightly increased. Analysis of the primary proteolytic fragments identified Ser21Ser22 to be a primary cleavage site in A20P-RNase A as well as in wild-type RNase A (Markert et al., 2001
), explaining our results: in A20P-RNase A, proline occupies the P2 position for the cleavage of Ser21Ser22, where it should not interfere with the substrate binding to elastase (Thompson and Blout, 1973b
; Renaud et al., 1983
).
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Materials and methods |
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RNase A from Sigma (St Louis, MO) was purified to homogeneity on a MONO S FPLC column (Pharmacia-Biotech, Uppsala, Sweden). Subtilisin C., subtilisin BPN' (both from Sigma), porcine elastase and proteinase K (both from Roche Diagnostics, Mannheim, Germany) were used without further purification.
Plasmid pET 26b(+) was obtained from Novagen (Bad Soden, Germany), oligonucleotides from MWG Biotech (Ebersberg, Germany) and the restriction enzymes SacI, NdeI and HindIII from New England Biolabs (Frankfurt/Main, Germany). The components for growth media were obtained from Difco Laboratories (Detroit, MI). Escherichia coli strains XL-1 Blue and BL21 (DE3) were supplied by Stratagene (Heidelberg, Germany). All other chemicals were of the purest grade commercially available.
Site-directed mutagenesis
The rnase A gene was cloned from the plasmid pBXR (delCardayré et al., 1995) (a gift from Professor R.T.Raines, University of Wisconsin, Madison, WI) into the vector pET 26b(+) by standard methods using the PCR (5'-TAA TAC GAC TCA CTA TAG GG-3' forward; 5'-CTA GTT ATT GCT CAG CGG TGG-3' reverse) and the restriction sites for NdeI and HindIII. Subsequently, the rnase A gene was modified by use of a QuikChange site-directed mutagenesis kit (Stratagene) to obtain the mutations S21P, S21K and A20P/S21P. A SacI restriction site (underlined) was introduced to facilitate the selection of positive clones. The nucleotides at the desired positions were replaced (bold face) using the following oligonucleotides: 5'-CCA GCA CTT CCG CCG CTC CGA GCT CCA ACT ACT GTA AC-3' forward, 5'-GTT ACA GTA GTT GGA GCT CGG AGC GGC GGA AGT GCT GG-3' reverse; 5'-CAC TTC CGC TGC CAA GAG CTC CAA CTA C-3' forward, 5'-GTA GTT GGA GCT CTT GGC AGC GGA AGT G-3' reverse; 5'-CCA GCA CTT CCG CCC CGC CGA GCT CCA ACT ACT GTA-3' forward, 5'-GTT ACA GTA GTT GGA GCT CGG CGG GGC GGA AGT GCT-3' reverse. The mutations were verified by DNA sequencing according to Sanger et al. (1977
) using a SequiThermExcel Long-Read DNA sequencing kit (Biozym, Hess Oldendorf, Germany) and a Li-COR 4000 DNA sequencer (MWG). The plasmid carrying the correct DNA sequence was transformed into the E.coli expression host strain BL21 (DE3). The mutation A20P was produced as described previously (Markert et al., 2001
).
Expression, renaturation and purification of the mutant enzymes
Expression and renaturation of the RNase A mutant enzymes were performed according to the procedure described previously (Markert et al., 2001) with minor modifications. Terrific broth containing 50 µg/ml kanamycin was inoculated with a fresh overnight culture of E.coli strain BL21 (DE3) that had been transformed with a plasmid directing the expression of the corresponding RNase A mutant enzyme. Cultures were grown with shaking at 200 r.p.m. at 37°C (Innova 4300 incubator shaker; New Brunswick Scientific, Nurtingen, Germany) to an OD600 of 2. Gene expression was induced by addition of isopropyl-ß-D-thiogalactopyranoside to a final concentration of 1 mM and cells were grown for an additional 4 h before being harvested. Cell lysis was performed in 20 mM TrisHCl, pH 8.0, containing 10 mM EDTA by treatment with lysozyme (1.5 mg/g of cells) at 4°C for 30 min and homogenization with a Gaulin homogenizer. The inclusion bodies were isolated by centrifugation at 30 000 g and 4°C for 30 min and resolubilized in 20 mM TrisHCl, pH 8.3, containing 6 M guanidine hydrochloride (GdnHCl), 100 mM dithiothreitol and 10 mM EDTA with stirring at room temperature for 2 h. After adjusting the pH to 4.0 with acetic acid, the protein solution was dialyzed against 2x10 l of 20 mM acetic acid overnight at 4°C. Precipitates formed during dialysis were removed by centrifugation at 15 000 g and 4°C for 30 min. The protein solution obtained from 4 l of culture was added to 1 l of renaturation buffer with a final concentration of 100 mM TrisHCl, pH 8.5, 100 mM NaCl, 1 mM glutathione (reduced), 0.2 mM glutathione (oxidized), 10 mM EDTA and kept for 24 h at room temperature. The folded protein was purified on a MONO S column (Pharmacia) that had been equilibrated with 50 mM TrisHCl, pH 7.5, with a linear gradient of NaCl (0500 mM).
Determination of the protein concentration
The protein concentration of RNase A and its mutant enzymes was determined using a molar absorption coefficient = 9800 M1 cm1 at 278 nm (Sela and Anfinsen, 1957
).
For activity measurements, the concentration of the RNase stock solutions was determined using a BCA protein assay kit (Pierce, Bonn, Germany) with bovine serum albumin as calibration protein according to the instructions of the manufacturer. The absorption of the samples was measured at 560 nm after an incubation at 37°C for 30 min using an MR 7000 micro plate reader (Dynatech, Denkendorf, Germany).
RNase activity
Values of kcat/KM of RNase A and its mutant enzymes were determined at 25°C in 100 mM 2-(N-morpholino)ethanesulfonic acid (MES)NaOH, pH 6.0, containing 100 mM NaCl, 50 nM 6-FAMdArUdAdA6-TAMRA and 0.250.5 ng/ml RNase according to Kelemen et al. (Kelemen et al., 1999). The increase in fluorescence emission at 515 nm (bandwidth 10 nm), upon excitation at 490 nm (bandwidth 1 nm), was followed in a 1 cm fluorescence cuvette using a Fluoro-Max-2 spectrometer (Jobin Yvon, Grasbrunn, Germany).
Circular dichroism (CD) spectroscopy
CD spectra of RNase A and its mutant enzymes were recorded in 10 mM TrisHCl, pH 8.0, containing 12 mg/ml of RNase on a Model 62 ADS CD spectrometer (Aviv, Piscataway, NJ) at 25°C. A cuvette of pathlength 1 cm was used for CD spectroscopy in the near-UV region (250340 nm) and a cuvette of pathlength 0.01 cm for the far-UV region (200260 nm).
GdnHCl-induced transition curves
GdnHCl-induced transition curves of RNase A and its mutant enzymes were obtained by fluorescence spectroscopy on a Fluoro-Max-2 spectrometer (Jobin Yvon) at 25°C using a cuvette of pathlength 1 cm. The bandwidth was 1 nm for excitation at 278 nm and 10 nm for emission. The fluorescence signal was recorded at 303 nm and averaged over 40 s. Protein concentration was 50 µg/ml in 50 mM TrisHCl, pH 8.0, containing 06 M GdnHCl. The fluorescence signals were fitted according to Pace et al. (Pace et al., 1989) by non-linear regression. The fraction of native protein (fN) was calculated from the fitted signals. The values of
G° (free energy in the absence of denaturant), [GdnHCl]1/2 (transition midpoint) and m (sensitivity of the free energy to denaturant) were calculated using the linear relation
G =
G° m[GdnHCl](1)
Proteolysis
Proteolysis was carried out at 25 or 50°C with final concentrations of 0.2 mg/ml RNase A or its mutant enzymes and 0.1 mg/ml subtilisin C. or elastase or 0.2 mg/ml subtilisin BPN' or proteinase K, respectively. The reaction was started by addition of 15 µl of protease in 50 mM TrisHCl, pH 8.0, containing 10 mM CaCl2, to 135 µl of RNase in 50 mM TrisHCl, pH 8.0. After defined time intervals, samples of 10 µl were withdrawn, mixed immediately with 10 µl of 100 mM phenylmethanesulfonyl fluoride in 2-propanol, dried under nitrogen and analyzed by SDSPAGE.
SDSPAGE and determination of the rate constants of proteolysis (kp)
Electrophoresis was carried out on a Midget Electrophoresis Unit (Hoefer, San Francisco, CA) according to Laemmli (Laemmli, 1970) using 5 and 15% acrylamide for stacking and separating gels. The gels were stained with Coomassie Brillant Blue G250. After staining, the gels were evaluated using a CD 60 densitometer (Desaga, Heidelberg, Germany) at 595 nm.
Values of kp were calculated from the decrease in the peak areas of the intact RNase band as a function of time of proteolysis, which followed pseudo-first-order kinetics. The determination of kp was performed three times.
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Results and discussion |
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Starting from the result that A20P-RNase A is resistant to proteolysis by subtilisin C. and proteinase K but not by elastase and the identification of the Ser21Ser22 peptide bond as a primary cleavage site for elastase in RNase A (Markert et al., 2001), we replaced Ser21 with proline or by lysine. Molecular modeling using the program WHAT IF (Vriend, 1990
) suggested that a substitution in position 21 will distort the RNase A structure even less than in position 20 because the side chain of Ser21 does not interact with the rest of the protein molecule. In the resulting mutant enzymes, S21P- or S21K-RNase A, proline or lysine will occupy the P1' position for the 2021 peptide bond or the P1 position for 2122 bond, thereby disfavoring a cleavage by subtilisin C., subtilisin BPN' and proteinase K or by elastase and subtilisin BPN', respectively (Brömme et al., 1986
; Bigler et al., 1993
; Hubbard et al., 1998
) (Figures 1 and 2). For the cleavage of the 1920 peptide bond by elastase as proposed by Klee (1965
), amino acid residue 21 corresponds to the P2' position (Figure 2) where proline and lysine should be accepted (Renaud et al., 1983
) because substrates bind mainly to the subsites (S5)S4S3S2S1S1' of elastase (Thompson and Blout, 1973a
,b). Thus, we also produced an RNase A mutant enzyme with both Ala20 and Ser21 replaced with proline, A20P/S21P-RNase A, where the occupation of P1' and P1 by proline should prevent the cleavage of both 1920 and 2122 bonds.
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The mutant enzymes A20P-, S21P-, A20P/S21P- and S21K-RNase A were obtained by renaturation from inclusion bodies after recombinant expression. Enzymatic activity provides a sensitive measure of the impact of modifications on the native structure of an enzyme (Knowles, 1987). The kcat/KM values of the mutant enzymes resemble that of wild-type RNase A with those of A20P/S21P- and A20P-RNase A being slightly decreased (Table I), indicating that the mutant enzymes are properly folded. In addition, near-UV CD spectra characterizing the tertiary structure and far-UV CD spectra characterizing the secondary structure indicate that the amino acid substitutions have no dramatic effect on the conformation of the protein molecule (not shown).
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The efficiency of the amino acid substitutions to prevent proteolytic degradation of RNase A by the unspecific proteases subtilisin C., subtilisin BPN', proteinase K and elastase was quantified from the amount of non-degraded RNase A by SDSPAGE as exemplified for the degradation with elastase in Figures 4 and 5. By subsequent densitometric evaluation, pseudo-first-order rate constants of proteolysis (kp) were determined (Table II).
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Subtilisin BPN' also degrades native wild-type RNase A expeditiously, however, starting at both Ala20Ser21 and Ser21Ser22 (Doscher and Hirs, 1967). Consequently, replacement of Ala20 with proline stabilizes the RNase A molecule by only one order of magnitude because proline occupies the P2 position for the cleavage of the 2122 peptide bond where it is tolerated (Grøn et al., 1992
). For the 2021 peptide bond, on the other hand, proline occupies the P1 position, thereby impeding the cleavage. In S21P-RNase A in addition to A20P/S21P-RNase A, Pro21 occupies both the P1 (2122) and P1' (2021) positions and effectively prevents proteolytic cleavage of both peptide bonds. In marked contrast, the replacement of Ser21 with lysine does not result in a decrease in kp (Table II) revealing that the charge concept does not work for protecting RNase A against subtilisin BPN'.
The situation for the proteolytic degradation of RNase A and its mutant enzymes by proteinase K is comparable to that with subtilisin C. (Table II), as deducible from the same primary cleavage site, Ala20Ser21, in wild-type RNase A (Rauber et al., 1978). Interestingly, the decrease in kp for the mutant enzymes containing proline in position 21 is less pronounced than that for subtilisin C., pointing to a different substrate specificity of proteinase K.
The most impressive result was found for the proteolytic degradation of RNase A and its mutant enzymes with elastase (Table II, Figures 4 and 5). Whereas replacing Ala20 with proline did not result in stabilization of the RNase A molecule against proteolytic attack by elastase, replacement of Ser21 in both S21P- and A20P/S21P-RNase A with proline dramatically reduced kp (Table II). Even at 45°C no proteolysis could be detected (not shown). In the degradation of the Pro21 RNase A mutant enzymes at 50°C (Figure 5), no distinct fragmentation could be observed. Proteolytic degradation under these conditions is probably due to the global unfolding of the protein molecule (Arnold and Ulbrich-Hofmann, 2000) and not to local changes of the accessibility near Pro21. The nearly total blockage of proteolytic degradation of the Pro21 RNase A mutant enzymes by elastase results in a revised interpretation of the degradation pathway of RNase A by elastase. From the amino acid composition analysis of elastase-digested RNase A, named RNase E, Klee concluded that the degradation starts at the Ala19Ala20 peptide bond followed by a quick release of Ala20 (Klee, 1965
). This interpretation, however, does not explain the lack of a serine in the amino acid composition of the RNase E preparation and the cleavage of the N-terminal Ala contradicts findings that elastase requires the binding of the substrate to the (S5)S4S3S2S1S1'(S2'S3') subsites (Thompson and Blout, 1973a
,b; Atlas, 1974
). Our results had identified Ser21Ser22 to be a primary cleavage site for elastase (Markert et al., 2001
). Provided that Ala19Ala20 is another primary cleavage site, the degradation of S21P-RNase A by elastase should be possible via this peptide bond owing to the minor relevance of the P2' position for the binding of the substrate molecule to elastase (Atlas, 1974
; Renaud et al., 1983
). The mutant enzymes with Pro21, however, are particularly stable against elastase. Consequently, we conclude that Ser21Ser22 is the only primary cleavage site in RNase A for elastase. This proteolysis event is followed by the release of Ser21 and Ala20, which meets the requirements for the substrate binding (Thompson and Blout, 1973b
) and explains the lack of serine and alanine in RNase E and also the detection of the Lys1Ala19 fragment of RNase A.
The decrease in kp for S21K-RNase A is more pronounced for elastase in comparison with the other unspecific proteases, but again much less than for S21P-RNase A (Table II), confirming the lower virtue of the charge concept for RNase A stabilization against elastase also.
While most proteases refuse proline at a potential cleavage site, the replacement of an amino acid residue according to the charge concept bears the risk of creating a substrate peptide bond for another protease. With studies on proteolysis of wild-type, A20P- and S21K-RNase A by trypsin, which cleaves C-terminally to lysine and arginine, we found that the introduction of lysine and, hence, of a potential cleavage site for trypsin indeed resulted in a more than 10 000-fold increase in kp for S21K-RNase A whereas the substitution of Ala20 by proline had no effect (not shown).
Conclusions
The decrease in the rate constants of proteolysis by single mutations in the RNase A molecule exemplifies the virtue of amino acid substitutions for stabilizing proteins against proteolytic attack. Based on the differences in the degree of stabilization of RNase A, the proline concept was found to be superior to the charge concept. The large differences in the stabilization effect against the various proteases indicate that the so-called unspecific proteases actually vary strongly in their degree of non-specificity. A rational design of the position of the amino acid substitution with respect to the primary cleavage site of the protease allows both efficient protection against proteolytic attack and the disclosure of degradation pathways.
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Acknowledgements |
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Received May 12, 2003; revised October 23, 2003; accepted October 30, 2003