Characterization of the amino acids essential for the photo- and radioprotective effects of a Bowman–Birk protease inhibitor-derived nonapeptide

Klaus H. Dittmann,1, Nuri Gueven, Claus Mayer and Hans-Peter Rodemann,1

Section of Radiobiology and Molecular Environmental Research, Department of Radiotherapy, Eberhard-Karls-University,72076 Tübingen, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Bowman–Birk protease inhibitor has been reported to exert photo- and radioprotective activity. This effect was assigned to a cyclic nonapeptide sequence which is known to contain the amino acids responsible for the anti-chymotryptic activity of the BBI. The present study indicated that linearization of the nonapeptide resulted in a significant loss of anti-proteolytic activity, whereas the photo- and radioprotective capacity persisted. Substitution of the amino acids Leu or Ser of the nonapeptide, essential for the anti-proteolytic activity, with different amino acids, indicated that rather the hydrophobic features of the amino acids in this position than charge are critical to retain the photo- and radioprotective effect. These results suggest the existence of a bifunctional peptide sequence with anti-proteolytic and photo-/radioprotective capacity. However, the lack of correlation between the photo-/radioprotective activity and the anti-proteolytic activity within the peptides generated by modification of the linear nonapeptide argues for the existence of two closely colocalized domains within the nonapeptide responsible for photo-/radioprotection and protease inhibition.

Keywords: BBI-derived peptide/clonogenic survival/photoprotection/protease inhibitor/radioprotection


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The Bowman–Birk protease inhibitor (BBI) (Birk, 1985Go) is a soybean-derived polypeptide of 71 residues (Odani and Ikenaka, 1973Go). It is characterized by its anti-proteolytic activity against trypsin and chymotrypsin (Odani and Ikenaka, 1973Go). The two distinct protein domains responsible for the selective activity against trypsin and chymotrypsin show significant homology, which suggests their evolution from a common ancestor (Odani and Ikenaka, 1978Go).

In addition to the anti-proteolytic action of BBI, several publications report an anti-carcinogenic effect both in vitro (Billings et al., 1987aGo; Moy and Billings, 1994Go) and in vivo (St. Clair, 1991Go). However up to now, only fragmentary knowledge exists about the molecular mechanism of the anti-carcinogenic effect (Kennedy, 1994Go). As BBI has been primarily characterized as a protease inhibitor so far, all attempts to elucidate the anti-carcinogenic effect have been concentrated on the identification of the proteases interacting with BBI. Indeed Billings et al. (1987b, 1991) and Carew and Kennedy (1990) described an intracellular proteolytic activity which is inhibited by BBI; however, the function of this enzyme and its link to the anti-carcinogenic effect of BBI are unresolved so far.

Previous work from our laboratory demonstrated that, in addition to the anti-carcinogenic activity, BBI exerts photo- and radioprotective activity when cells are exposed to ionizing radiation (Dittmann et al., 1995Go, 1998aGo) or UVB (Gueven et al., 1998Go). Interestingly, this photo-/radioprotection could only be demonstrated for normal human skin fibroblasts, and not for transformed fibroblasts with mutated p53 (Dittmann et al., 1998aGo). In addition, we failed to detect the radioprotective effect of BBI in xeroderma pigmentosum fibroblasts defective for nucleotide excision repair (Dittmann et al., 2000Go). The molecular mechanism of the BBI-mediated photo-/radioprotection most likely involves the activation of DNA repair relevant genes prior to irradiation (Dittmann et al., 1998bGo). As a consequence, radiation-induced cell inactivation and cell differentiation are reduced.

Previous data (Gueven et al., 1998Go) suggest that the photo- and radioprotective effect of BBI is independent of its anti-proteolytic activity. This study demonstrated that a synthetic cyclic nonapeptide, which contains the sequence responsible for the anti-chymotryptic activity of BBI (Terada et al., 1980Go), is competent to mimic the photo- and radioprotective effect of the whole BBI molecule. In the present study, we characterized the amino acids essential for the photo- and radioprotective function. On the basis of this knowledge, the optimization of the peptide sequence can be approached with respect to the application of such peptides for use in photo-/radiation protection.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell cultures

In the present experiments the normal human fibroblast strains HSF1 and HSF6 (human skin, passage 5–10) were used. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (f.c.s.) (Gibco, Eggenstein, Germany). Cultures were propagated according to routine procedures (Rodemann et al., 1989Go).

BBI/nonapeptide pre-incubation, UVB exposure and clonogenic assay

Confluent normal skin fibroblasts were incubated either in control medium, in medium containing BBI (10 µM) (Sigma, Munich, Germany) or in medium containing synthetic peptides (20–80 µM) (Interactiva, Ulm, Germany), for 16 h. Subsequently, the media were removed and the cultures were irradiated with UVB (100 or 200 J/m2). UV light (312 nm) was generated by a UVB lamp (Bioblock Scientific, Illkirch, France) with a dose rate of 450 J/min2/min. Ionizing irradiation was performed as described (Dittmann et al., 1998aGo) with 4 MeV photons from a linear accelerator (Mevatron 60, Siemens, Erlangen, Germany) with a dose rate of 2 Gy/min at room temperature. Irradiated cells were trypsinized 6 h after exposure and plated at a density of 500 cells per 50 cm2 dish. To allow colony formation from single plated cells, cultures were incubated in DMEM supplemented with 20% f.c.s. for 14 days. Cultures were then fixed and stained as described (Dittmann et al., 1995Go). For counting the number of colonies with more than 50 cells, culture dishes were coded and scored by two persons independently.

Determination of the inhibitor constant for the linear nonapeptide

A 50 µl volume of a chymotrypsin solution (corresponding to 280, 28, 2.8 or 0.28 mU) were incubated with 10 µl of BBI in the concentration range 100–0.1 µM or with the linear nonapeptide P2 in the dose range 2–0.2 mM for 10 min at room temperature. Subsequently, 50 µl of protease substrate (AAPF-pNA, Bacchem, Heidelberg, Germany) (0.5 mg/ml) were added and substrate hydrolysis was quantified at 405 nm in an ELISA reader. Inhibitor constants (Ki) are given by the slope of the corresponding regression curves (Salvesen and Nagase, 1989Go).

Synthetic peptides

All peptides used were synthesized by Interactiva (Ulm, Germany) and are protected against terminal degradation by N-terminal acetylation and C-terminal amidation. The purity was >95% as determined by reversed-phase HPLC. For HPLC analysis peptides were applied to a reversed-phase C18 column (Nucleosil, diameter 4 mm, length 250 mm, particle diameter 5 µm, pore size 300 Å). The column was developed with a continuous linear gradient from buffer A (0.1% v/v trifluoroacetic acid in water, 10% acetonitrile) to buffer B (0.1% v/v trifluoroacetic acid in water, 50% acetonitrile) at a flow rate of 1 ml/min.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Comparison of the photo- and radioprotective effect of a synthetic peptide with the effect of BBI

As previously described (Gueven et al., 1998Go), the photo- and radioprotective activity of the Bowman–Birk protease inhibitor is represented by a cyclic nonapeptide corresponding to amino acids 41–49 of the original BBI molecule. As shown in Figure 1Go, we were able to demonstrate that the photo- and radioprotective activity towards UVB or ionizing radiation is also mediated by the linear version of the nonapeptide (P2). However, to mimic the photo- and radioprotective effect of 10 µM BBI the linear nonapeptide has to be applied at a concentration of at least 20 µM. Increasing the concentration for P2 up to 80 µM failed to enhance the photo- and radioprotective effect further. This was also observed with BBI (Figure 1Go).



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Fig. 1. Survival of normal fibroblasts after treatment with UVB and ionizing radiation in the presence of BBI or the linear nonapeptide P2. Confluent layers of normal skin fibroblasts (HSF1, HSF6) were treated either with BBI or P2 peptide in the concentration range 0–80 µM for 16 h. Cells were irradiated either with 100 J/m2 UVB or 4 Gy and clonogenic assays were performed. After 14 days, the colonies formed were fixed and stained. Each data point represents the mean value of three independent experiments ± SEM.

 
Determination of the inhibitor constant (Ki) for the linear nonapeptide P2 in comparison with the intact BBI

Since the nonapeptide is deduced from the chymotrypsin-inhibitory site of the BBI molecule, we performed assays to estimate the residual protease inhibitory activity of the linear nonapeptide P2. The kinetics of hydrolysis of a synthetic substrate for chymotrypsin in the presence of P2 or BBI (Figure 2Go) suggested that a concentration of 2x10–3 M P2 acts equivalent to BBI at a concentration of 1x10–8 M. The exact Ki values were deduced from the kinetics of substrate hydrolysis after linear regression analysis and are determined as 0.013 µM for BBI compared with 3450 µM for P2. Thus, the anti-proteolytic effect of P2 corresponds to 1/265 384 compared with the effect of BBI, whereas the photo- and radioprotective effect of P2 is only reduced to 50%.



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Fig. 2. Hydrolysis of the synthetic chymotrypsin substrate AAPF-pNA in presence of BBI or the linear nonapeptide P2. A 50 µl volume of a chymotrypsin solution (corresponding to 0.28 mU) was incubated with 10 µl of BBI in the range 0.1–100 µM or with the linear nonapeptide P2 in the range 0.2–2 mM for 10 min at 20°C. Subsequently, 50 µl of protease substrate (AAPF-pNA, 0.5 mg/ml) were added and substrate hydrolysis was quantified at 405 nm in an ELISA reader. Inhibitor constants (Ki) are given by the slope of the corresponding regression curve (Salvesen and Nagase, 1989Go). Each determination was performed four times.

 
Modification of the photo- and radioprotective effect of the nonapeptide P2 by truncation

To characterize the active centre responsible for the photo- and radioprotective effect of the nonapeptide, a series of peptides with amino acid deletions or modifications were generated (Table IGo). As already mentioned, the cyclic structure of the nonapeptide is not essential for the photo- and radioprotective effect, since the effect of the cyclic peptide P1 did not differ significantly from the effect of the linear version P2 (P1 versus P2, p = 0.71) (Table IGo). Furthermore, truncation of the nonapeptide to a heptapeptide, which results in a loss of the terminal cysteines, did not affect the photo- and radioprotective effect (Table IGo, P3 versus P2). Even after the progressive truncation of P2 to a pentapeptide with the sequence Ala–Leu–Ser–Tyr–Pro, the peptide retained the photo- and radioprotective effect (P4 versus P2).


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Table I. Modification of the photo- and radioprotective effect of the nonapeptide P2 by manipulation of the peptide sequence
 
Modification of the photo- and radioprotective effect of the nonapeptide P2 by exchange of the amino acid Ser in position 4

Since it is reported that the amino acids Ser and Leu in positions 42 and 43 of the intact BBI molecule are obligatory for the anti-proteolytic effect towards chymotrypsin, we exchanged both consecutively. Alteration of Ser to amino acids with charged side groups, such as Arg or Glu, completely abolished the photo- and radioprotective effect (Table IGo, P9 versus P2 and P8 versus P2). The same effect was apparent after substitution of the Ser with the neutral amino acid Gly (Table IGo, P6 versus P2). The exchange of Ser to Thr, which like Ser is an amino acid with polar side chains, abrogated the photo- and radioprotective activity (Table IGo, P7 versus P2). On the other hand, the substitution of Ser with Val, a member of the amino acids with a hydrophobic side chain, did not abolish the photo- and radioprotective activity, although a significant reduction was observed (Table IGo, P5 versus P2).

Modification of the photo- and radioprotective effect of the nonapeptide P2 by exchange of the amino acid Leu in position 3

Exchange of the Leu in position 3 of the P2 peptide to Ile or Ala, both amino acids with hydrophobic side chains, resulted in a loss of photo- and radioprotective activity (Table IGo, P11 versus P2 or P12 versus P2). A loss is also apparent after substituting Leu with Arg, which belongs to the amino acids with positively charged side chains (Table IGo, P15 versus P2). Exchange of Leu by the negatively charged Glu or with Val, which belongs to the hydrophobic amino acids, maintains the photo- and radioprotective effect of the peptide, but at a reduced level (Table IGo, P14 versus P2 and P13 versus P2).

Modification of the photo- and radioprotective effect of the nonapeptide P2 by exchange of the amino acid Tyr in position 5

Whereas manipulations of the amino acids Ser or Leu are known to affect the anti-proteolytic properties, no information is available for the exchange of the Tyr in position 5. Substitution of Tyr with Gly (Tables I and IIGoGo, P16) resulted in a complete loss of photo- and radioprotective activity and a reduced anti-proteolytic activity compared with P2.


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Table II. Correlation between the relative photo-/radioprotective effect and anti-proteolytic effect of the peptides P2, P5, P6, P10 and P16
 
P2 modifications and the anti-chymotryptic effect of the resulting peptides

The exchange of the Ser in position 4 to Gly (P6) led to a reduction of the anti-proteolytic effect of the P2 peptide (Table IIGo), whereas the exchange of Ser to Val (P5) resulted in a distinct increase in its protease inhibitory activity (Table IIGo). In comparison with P2 both peptides exhibit, however, a reduced photo- and radioprotective effect. In addition, the exchange of Leu in position 3 to Gly (P10) did not result in an altered anti-proteolytic effect of P2, whereas the photo- and radioprotective effect was completely lost. Even for P16 (Y to G), which is associated with the lowest clonogenic survival after treatment with UVB, a residual anti-proteolytic activity was observed.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In addition to cyclic nonapeptide derived from BBI (Gueven et al., 1998Go), we demonstrated here that its linear analogue also acts photo- and radioprotectively. This observation suggests that the photo- and radioprotective effect of BBI-derived peptides, in contrast to the anti-proteolytic effect, is independent of circular structure, since it is reported that the open-chain analogue exerts a significant reduced inhibition towards chymotrypsin (Terada et al., 1980Go) owing to the loss of the ring structure (Gariani and Leatherbarrow, 1997Go). The observation that the ring structure is not essential for the photo- and radioprotective effect is confirmed by the detection of photo-/radioprotection with the heptapeptide, which lacks the terminal cysteine residues, essential for ring formation.

There is clear evidence that P2 has to be administered at twice the concentration of BBI to achieve comparable photo-/radioprotection. One explanation for the reduced effect of the P2 nonapeptide could be the reduced stability of the short peptide compared with the BBI molecule. However, the observation that the photo- and radioprotective effect of the linear nonapeptide does not significantly differ from the effect of the cyclic version, which clearly shows increased stability (Terada et al., 1980Go), favours an argumentation independent of the stability. Interestingly, our earlier data (Gueven et al., 1998Go) indicated that after cleavage of the BBI molecule into two fragments which carry the chymotrypsin inhibitory site and the trypsin inhibitory sequence, both fragments act radioprotectively. Since both sites are structurally related (Odani and Ikenaka, 1978Go), it is not surprising that both fragments act radioprotectively. As a consequence, this may explain the observation that the photo- and radioprotective potential of BBI is twice that of the nonapeptide P2, which exerts only one active site.

The colocalization of the active sites for the anti-proteolytic and the photo- and radioprotective effect inside the nonapeptide sequence would in principle argue for an involvement of a protease in the regulation of cellular photo-/radiation sensitivity. However, the presented determinations of the inhibitor constants for BBI and the linear P2 showed a significantly reduced anti-proteolytic activity for P2 (3450 µM for P2 versus 0.013 µM for BBI). For cyclic P2 (P1), which represents the smallest BBI-derived unit with anti-proteolytic activity (Ando et al., 1987Go), a Ki value of 52 µM is reported (Terada et al., 1980Go). Consequently, linearization of the peptide is associated with a further reduction of anti-chymotryptic activity to 1/66, which suggests that the linear P2 acts more as a protease substrate than as a protease inhibitor. Furthermore, the lack of correlation between anti-proteolytic and photo- and radioprotective activities of the peptides P5, P6, P10 and P16 presented in Table IIGo contradicts an involvement of a protease for the photo- and radioprotective effect.

All substitutions performed for the amino acids Ser or Leu, which represent the active centre for the anti-proteolytic effect (Ando et al., 1987Go), led concomitantly to a reduced photo- and radioprotective effect. Independent of the replacement of Ser by amino acids negatively or positively charged, a reconstitution of the photo- and radioprotective effect was not observed. Even insertion of the polar Thr failed to reconstitute the effect. Reconstitution of the photo- and radioprotective capacity of the peptide was only possible by substitution of Ser with Val, which is also a hydrophobic amino acid. These results indicate that the hydrophobicity of the amino acid replacing Ser is essential for the photo- and radioprotective effect. Similar results were obtained for the exchange of the Leu residue. Thus, it can be summarized that the nonapeptide sequence is able to exert photo-/radioprotective and anti-proteolytic activity. However, different features of the sequence are essential for these two effects, which suggest a bifunctional domain. This hypothesis is strengthened by the data for the anti-proteolytic activity obtained after substitution of the amino acid Ser of the P2 peptide with Val (Table IIGo). The resulting peptide (P5) shows an increased anti-proteolytic activity compared with the native P2 peptide (x2.5) (Table IIGo), whereas the photo- and radioprotective effect is significantly reduced (x0.36). The same is true for peptide P10; despite showing comparable anti-proteolytic activity to P2, the photo- and radioprotective effect was completely lost.

Based on the data presented here, peptide design for the development of highly photo- and radioprotective molecules with optimized physico-chemical properties can be approached. In forthcoming studies the photo- and radioprotective mechanism of the P2 peptide will be elucidated and the question will be answered of whether this peptide is able to improve nucleotide excision repair as we observed after BBI treatment (Dittmann et al., 2000Go).


    Notes
 
1 To whom correspondence should be addressed.E-mail: klaus.dittmann{at}uni-tuebingen.de;hans-peter.rodemann{at}uni-tuebingen.de Back


    Acknowledgments
 
The financial support of the Deutsche Forschungsgemeinschaft (Di 402/6-2) and the Fortüne-Program (Medical Faculty of the University of Tübingen) is gratefully acknowledged


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received June 5, 2000; revised December 5, 2000; accepted December 20, 2000.