Proline versus charge concept for protein stabilization against proteolytic attack

Yvonne Markert, Jens Köditz, Renate Ulbrich-Hofmann and Ulrich Arnold1

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


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The virtue of the so-called ‘proline concept’ and the ‘charge concept’ for stabilizing protease-susceptible regions of a protein structure was compared on bovine pancreatic ribonuclease A. Alanine 20 and serine 21, both of which are located in a loop that is susceptible to the unspecific proteases subtilisin Carlsberg, subtilisin BPN', proteinase K and elastase, were replaced with proline or lysine by site-directed mutagenesis. The rate constant of proteolysis was decreased by up to three orders of magnitude for the proline mutants depending on the site of the mutation and the protease used. In contrast, substitution by lysine increased the proteolytic resistance by only one order of magnitude characterizing the ‘proline concept’ as superior to the ‘charge concept’. Although the four applied proteases are considered to be unspecific, the degree of stabilization of the ribonuclease molecule varied considerably, indicating the impact of individual differences in their substrate specificity on the proteolytic resistance and degradation pathway of the target protein.

Keywords: proline concept/proteolysis/ribonucleaseA/site-directed mutagenesis/stabilization


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
One of the limitations to the biotechnological or medical application of proteins is their susceptibility towards proteolytic attack. Although natively folded proteins usually are compact entities, they can contain loop regions that are flexible enough to be cleaved by proteases (Price and Johnson, 1990Go; Hubbard, 1998Go). Hence tailoring the protein structure to increase the proteolytic resistance is a desirable goal.

Proteases are ubiquitous in nature. Owing to the manifold processes in which they are involved (protein degradation, processing, activation, etc.) (Neurath, 1989Go), 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, 1998Go), 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, 1967Go). The conformation and flexibility of the peptide backbone of the substrate protein also play an important role in efficient cleavage (Hubbard et al., 1998Go). 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, 1998Go).

Cleavage of peptide bonds is sterically impeded by embedding into elements of secondary structure (Hubbard et al., 1998Go), by glycosylation (Rudd et al., 1995Go; Arnold et al., 1998Go) or by the presence of disulfide bonds (Mansfeld et al., 1997Go; So et al., 1997Go). 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., 1993Go). 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., 1986Go). In positions P2 or P4, however, proline can sometimes even promote the cleavage at P1–P1' (Thompson and Blout, 1973aGo). Another reason for a decreased proteolytic susceptibility of proline-containing protein regions may be the rigidity of the Xaa–Pro peptide bond. Here, the reduced flexibility of the peptide backbone concerned (Jaenicke, 2000Go) 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., 1993Go). 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, 1997Go; Ramos and Baldwin, 2002Go) and its structure is cross-linked by four disulfide bonds (Wlodawer et al., 1982Go). Although RNase A resists proteolytic degradation by trypsin and thermolysin at room temperature (Arnold et al., 1996Go), it is degraded efficiently by the unspecific proteases subtilisin Carlsberg (subtilisin C.) (Richards and Vithayathil, 1959Go), subtilisin BPN' (Doscher and Hirs, 1967Go; Neumann and Hofsteenge, 1994Go), proteinase K (Rauber et al., 1978Go) and elastase (Klee, 1965Go) (Figure 1). The flexibility of the loop region around Ala20 is responsible for this proteolytic susceptibility resulting in the primary cleavage of the Ala20–Ser21 peptide bond by subtilisin C. and proteinase K. Degradation by subtilisin BPN' is reported to start evenly at both Ala20–Ser21 and Ser21–Ser22 (Neumann and Hofsteenge, 1994Go). Klee had postulated the primary cleavage of Ala19–Ala20 by elastase (Klee, 1965Go) (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., 2001Go). 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 Ser21–Ser22 to be a primary cleavage site in A20P-RNase A as well as in wild-type RNase A (Markert et al., 2001Go), explaining our results: in A20P-RNase A, proline occupies the P2 position for the cleavage of Ser21–Ser22, where it should not interfere with the substrate binding to elastase (Thompson and Blout, 1973bGo; Renaud et al., 1983Go).



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Fig. 1. Tertiary structure of RNase A and positions of the primary cleavage sites. The model was taken from the Brookhaven Protein Data Bank (5rsa) and drawn with Swiss PDB-Viewer v3.7b1. Arrows indicate cleavage sites by subtilisin C. (Richards and Vithayathil, 1959Go), subtilisin BPN' (Doscher and Hirs, 1967Go), proteinase K (Rauber et al., 1978Go) and elastase: according to [1] Klee (Klee, 1965Go) and [2] Markert et al. (Markert et al., 2001Go).

 
In this study, we compared the proline concept and the charge concept for protein stabilization to generate RNase A variants that are even more proteolytically stable than A20P-RNase A. Specifically, we replaced Ser21 with proline or lysine and created a double mutant, A20P/S21P-RNase A. In S21P-RNase A, proline occupies P1 for cleavage at Ser21–Ser22 and P1' for Ala20–Ser21 and therefore should prevent cleavage at both positions. By replacement of Ser21 with lysine we introduced a positively charged amino acid residue in P1 or P1', thereby disfavoring the respective cleavage according to the charge concept. The proteolytic degradation of the mutant enzymes and their catalytic activity and conformational stability were compared with those of wild-type RNase A.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Proteins and chemicals

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., 1995Go) (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. (1977Go) 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., 2001Go).

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., 2001Go) 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 Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, 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 Tris–HCl, pH 7.5, with a linear gradient of NaCl (0–500 mM).

Determination of the protein concentration

The protein concentration of RNase A and its mutant enzymes was determined using a molar absorption coefficient {epsilon} = 9800 M–1 cm–1 at 278 nm (Sela and Anfinsen, 1957Go).

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-FAM–dArUdAdA–6-TAMRA and 0.25–0.5 ng/ml RNase according to Kelemen et al. (Kelemen et al., 1999Go). 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 Tris–HCl, pH 8.0, containing 1–2 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 (250–340 nm) and a cuvette of pathlength 0.01 cm for the far-UV region (200–260 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 Tris–HCl, pH 8.0, containing 0–6 M GdnHCl. The fluorescence signals were fitted according to Pace et al. (Pace et al., 1989Go) by non-linear regression. The fraction of native protein (fN) was calculated from the fitted signals. The values of {Delta}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

{Delta}G = {Delta}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 Tris–HCl, pH 8.0, containing 10 mM CaCl2, to 135 µl of RNase in 50 mM Tris–HCl, 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 SDS–PAGE.

SDS–PAGE 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, 1970Go) 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.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Design of mutant enzymes

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 Ser21–Ser22 peptide bond as a primary cleavage site for elastase in RNase A (Markert et al., 2001Go), we replaced Ser21 with proline or by lysine. Molecular modeling using the program WHAT IF (Vriend, 1990Go) 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 20–21 peptide bond or the P1 position for 21–22 bond, thereby disfavoring a cleavage by subtilisin C., subtilisin BPN' and proteinase K or by elastase and subtilisin BPN', respectively (Brömme et al., 1986Go; Bigler et al., 1993Go; Hubbard et al., 1998Go) (Figures 1 and 2). For the cleavage of the 19–20 peptide bond by elastase as proposed by Klee (1965Go), amino acid residue 21 corresponds to the P2' position (Figure 2) where proline and lysine should be accepted (Renaud et al., 1983Go) because substrates bind mainly to the subsites (S5)S4S3S2S1S1' of elastase (Thompson and Blout, 1973aGo,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 19–20 and 21–22 bonds.



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Fig. 2. Positions of different subsite binding modes of RNase A sequence 18–22. The arrows indicate the position of cleavage with occupation of P2–P4', P3–P3' and P4–P2', respectively.

 
Characterization of the mutant enzymes

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, 1987Go). 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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Table I. Activity and thermodynamic parameters of RNase A and its mutant enzymes at 25°C
 
Transition curves (Figure 3) were constructed from the fluorescence emission signals at 25°C as a function of the concentration of GdnHCl and were used to determine the thermodynamic stability of wild-type RNase A and the mutant enzymes (Table I). Whereas the proline mutant enzymes revealed a stability similar to that of wild-type RNase A, replacement of Ser21 with lysine resulted in a >50% decrease in stability, mainly due to a decreased m-value (Table I). The occurrence of a native baseline (Figure 3) proves that S21K-RNase A is properly folded. Furthermore, the rate constant of thermal unfolding is indistinguishable from that of wild-type RNase A (not shown) and negligible at 25°C.



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Fig. 3. Transition curves of RNase A and its mutant enzymes. Transition curves of GdnHCl-induced unfolding of wild type- (inverted triangles), A20P- (circles), S21P- (triangles), A20P/S21P- (squares) and S21K-RNase A (diamonds) were determined by fluorescence spectroscopy in 50 mM Tris–HCl, pH 8.0, at 25°C as described in Materials and methods.

 
Proteolytic susceptibility

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 SDS–PAGE 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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Fig. 4. SDS–PAGE of the proteolytic degradation of wild-type RNase A and its mutant enzymes by elastase. Wild-type (a), A20P- (b), S21P- (c), A20P/S21P- (d) and S21K-RNase A (e) were incubated with elastase at a ratio of 1:1 (w/w) at 25°C for (each from left to right): (a, b) 5 s, 10 s, 20 s, 30 s, 40 s, 50 s, 1 min, 1.5 min, 2 min, 3 min, 4 min, 5 min and 10 min; (c, d) 10 s, 5 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 12 h, 24 h, 30 h and 48 h; (e) 10 s, 5 min, 30 min, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h. Lane 1 in each gel shows molecular weight marker proteins {alpha}-lactalbumin (14.4 kDa), soybean trypsin inhibitor (21 kDa), carbonic anhydrase (30 kDa) and ovalbumin (43 kDa).

 


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Fig. 5. SDS–PAGE of the proteolytic degradation of S21P- and A20P/S21P-RNase A by elastase at 50°C. S21P- (a) and A20P/S21P-RNase A (b) were incubated with elastase at a ratio of 1:1 (w/w) at 50°C for (each from left to right) 10 s, 30 s, 1 min, 2 min, 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 1 h and 2 h. Lane 1 in each gel shows molecular weight marker proteins {alpha}-lactalbumin (14.4 kDa), soybean trypsin inhibitor (21 kDa), carbonic anhydrase (30 kDa) and ovalbumin (43 kDa).

 

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Table II. Rate constants of proteolysis at 25 or 50°C
 
Subtilisin C. is known to degrade native wild-type RNase A efficiently via the main primary cleavage of the Ala20–Ser21 peptide bond (Richards and Vithayathil, 1959Go), being reflected in a high kp value (Table II). In the mutant enzymes A20P-, S21P- and A20P/S21P-RNase A, proline occupies the P1 and/or P1' position and impedes the proteolytic degradation dramatically, by more than three orders of magnitude. In contrast, the substitution by lysine is much less effective and reduces kp <10-fold (Table II).

Subtilisin BPN' also degrades native wild-type RNase A expeditiously, however, starting at both Ala20–Ser21 and Ser21–Ser22 (Doscher and Hirs, 1967Go). 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 21–22 peptide bond where it is tolerated (Grøn et al., 1992Go). For the 20–21 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 (21–22) and P1' (20–21) 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, Ala20–Ser21, in wild-type RNase A (Rauber et al., 1978Go). 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, 2000Go) 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 Ala19–Ala20 peptide bond followed by a quick release of Ala20 (Klee, 1965Go). 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, 1973aGo,b; Atlas, 1974Go). Our results had identified Ser21–Ser22 to be a primary cleavage site for elastase (Markert et al., 2001Go). Provided that Ala19–Ala20 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, 1974Go; Renaud et al., 1983Go). The mutant enzymes with Pro21, however, are particularly stable against elastase. Consequently, we conclude that Ser21–Ser22 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, 1973bGo) and explains the lack of serine and alanine in RNase E and also the detection of the Lys1–Ala19 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.


    Acknowledgements
 
The authors thank Professor R.T.Raines (University of Wisconsin, Madison, WI) for the gift of the plasmid pBXR, Professor Dr Gerrit Vriend (University of Nijmegen, The Netherlands) for molecular modeling, Mrs M.Sonntag for excellent technical assistance and Mrs C.Ebeling for initial studies. Y.M. and J.K. were supported by the Max-Buchner-Forschungsstiftung, Frankfurt, Germany.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
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Received May 12, 2003; revised October 23, 2003; accepted October 30, 2003