Thioredoxin-(dithiol-)linked inactivation of elastase

Molecular Immunology 38 (2001) 759–763

Thioredoxin-(dithiol-)linked inactivation of elastase Gregorio del Val a,c , Frank E. Hagie Jr. b , Bob B. Buchanan a,∗ a

Department of Plant and Microbial Biology, University of California, 111 Koshland Hall, Berkeley, CA 94720, USA b Applied Phytologics Inc., 4110 N Freeway Blvd., Sacramento, CA 95834, USA c Torrey Mesa Research Institute, 3115 Merryfield Road, San Diego, CA 92121-1125, USA Received 17 July 2001; accepted 3 October 2001

Abstract Following inactivation by the alpha-1-antitrypsin (AAT) inhibitor, the protease elastase was reduced by thioredoxin, itself reduced by NADPH and NADP-thioredoxin reductase (NTR). Under these conditions, reduction of enzyme disulfide groups was accompanied by loss of more than 60% of the activity measured following dissociation of the enzyme–inhibitor complex with NaCl. The inhibitor was required (1) to prevent proteolysis of both reduced thioredoxin and NTR and (2) to assess the progress of the reduction reaction. At elevated temperature, elastase was also reduced by dithiols (dithiothreitol and lipoic acid) but not by monothiols (reduced glutathione, beta-mercaptoethanol). When reduced by dithiols under these conditions, the enzyme digested itself. Self-digestion was independent of the antitrypsin inhibitor and was proportional to temperature in the 37–50 ◦ C range. These findings open the door to a new mode of regulation of elastase and to possible new therapies for treating diseases associated with the enzyme. © 2002 Elsevier Science Ltd. All rights reserved.

1. Introduction Elastase, a 26 kDa disulfide protein belonging to the trypsin protease family, is widely distributed in mammalian tissues and is involved in inflammation responses (Bode et al., 1987). Human leukocyte elastase, which occurs in three major catalytically active forms, participates in a number of inflammatory diseases through the production of mediators of inflammation and through the destruction of keratin, elastin and different types of collagen (Janoff, 1972; Barrett, 1981). The enzyme is an aggravating factor in diseases of lung (pulmonary emphysema), articulation (rheumatoid arthritis) and skin (psoriasis). Because elastase is inhibited by a naturally occurring glycoprotein, serum—alpha-1-antitrypsin (AAT), it has been proposed to use the inhibitor to alleviate psoriasis, a chronic condition of skin (Wiedow et al., 1992). In view of the presence of four disulfide bonds, we have considered whether another treatment might be feasible—namely, inactivation of the enzyme by disulfide reduction. This approach seemed reasonable in view of reports that thioredoxin, a 12 kDa protein with a catalytically active disulfide group, is expressed at elevated levels with inAbbreviations: AAT, alpha-1-antitrypsin inhibitor; NTR, NADP-thioredoxin reductase; mBBr, monobromobimane. ∗ Corresponding author. Tel.: +1-510-642-3590; fax: +1-510-642-7356. E-mail address: [email protected] (B.B. Buchanan).

flammatory diseases such as arthritis (Maurice et al., 1999; Yoshida et al., 1999). It therefore seemed possible that thioredoxin might play a beneficial role in inflammatory responses involving elastase. A large number of disulfide proteins are inactivated or become susceptible to proteolysis when reduced with thioredoxin, itself reduced with NADPH and the associated flavin enzyme NADP-thioredoxin reductase (NTR) (Eqs. (1) and (2)) (Jiao et al., 1992; Lozano et al., 1994; del Val et al., 1999). NADPH + thioredoxin hoxidized −NTR S−S

→ thioredoxin hreduced + NADP

(1)

−SH HS−

thioredoxin hreduced + target proteinoxidized (−SH HS−)

→

(S−S) active indigestible

target proteinreduced (−SH HS−) inactive digestible

+ thioredoxin hoxidized (S−S)

(2)

In the present study, we have investigated whether reduced thioredoxin or a dithiol substitute can alter the catalytic activity in the presence or absence of AAT and heat. In addition to introducing a possible new mode of regulating the enzyme, the results demonstrate that a combination of the AAT inhibitor, thioredoxin (or a dithiol substitute) and heat effectively inactivate elastase in vitro. The findings raise the possibility that such a combination could be applied in the treatment of psoriasis.

0161-5890/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 1 6 1 - 5 8 9 0 ( 0 1 ) 0 0 1 1 2 - 2

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G. del Val et al. / Molecular Immunology 38 (2001) 759–763

2. Experimental procedures 2.1. Materials Porcine pancreatic elastase was purchased from Calbiochem Novabiochem. Thioredoxin and NTR were obtained from Escherichia coli overexpressing the respective gene and glutathione reductase was isolated from spinach leaves (del Val et al., 1999). Thioredoxin, NTR and glutathione reductase were obtained as described previously (del Val et al., 1999). Chemicals and biochemicals were obtained from commercial sources and were of the highest quality available. 2.2. Assay of elastase Elastase activity was determined spectrophotometrically by adding 10 ␮l of 0.25 ␮M elastase to 500 ␮l of reaction mixture containing 0.5 mM Elastase Substrate II and 100 mM Tris–HCl pH 8.0 (Rubin et al., 1994; Ashe and Zimmerman, 1997). Activity was measured by following absorbance change at 410 nm. As indicated the enzyme was preincubated with AAT prior to assay. All enzyme reactions were carried out at 25 ◦ C.

proteins (Wong et al., 1995). Finally, the gel was incubated overnight at room temperature in 20% methanol containing 5% acetic acid and 0.025% Coomassie brilliant blue R-250 to stain protein and was then destained with a solution of 20% methanol and 5% acetic acid until the protein bands were visible.

3. Results We observed that elastase was reduced by thioredoxin, but only in presence of AAT, i.e. when the associated protease activity was inhibited (Fig. 1, lane 9) so as to prevent proteolysis of the added NTR and thioredoxin during the reaction (Fig. 1, lane 8). Two well defined bands appeared in the presence of reduced thioredoxin and the 55 kDa inhibitor, corresponding to the AAT–elastase complex with partly digested enzyme-inhibitor as described before at 60 and 50 kDa (Carrell and Owen, 1985; Rubin et al., 1994). When treated with dithiothreitol alone at 25 ◦ C the enzyme was only marginally reduced thus revealing no cysteine labeling with mBBr (Fig. 1, lane 10). Interestingly, when boiled with dithiothreitol, the enzyme showed no cysteine-labeling as earlier.

2.3. Reduction and monobromobimane labeling of elastase Conditions for reducing elastase with the thioredoxin system were determined using a temperature of 37 or 25 ◦ C as indicated. Because the protein components of the thioredoxin and glutathione systems were digested by elastase, we found it preferable in some experiments to premix the enzyme with a two-fold molar excess of AAT. After incubation, the sulfhydryls newly generated by thioredoxin were labeled with the fluorescent probe monobromobimane (mBBr) and the extent of their reduction was determined following SDS–PAGE analysis (Wong et al., 1995). To measure the elastase activity after treatment with the inhibitor, NaCl was added to 1.0 M to dissociate the enzyme (Rubin et al., 1994) and activity was assayed as shown earlier. Protein was determined according to Bradford (1976) with bovine gamma globulin as standard. 2.4. SDS–PAGE Protein samples that had been labeled with mBBr were dissolved in SDS sample buffer free of reducing agents and were not heated. Gels (10–20% acrylamide gradient) were prepared according to Laemmli (1970) and subjected to electrophoresis at constant current. Each sample contained 2.5% 2-mercaptoethanol as specified by Laemmli. After electrophoresis, the gel was immersed in 20% methanol that contained 5% acetic acid to remove excess mBBr and then examined under 365 nm UV light to detect mBBr-labeled

Fig. 1. Reduction of elastase dithiols. The mBBr SDS–PAGE gel showing disulfide bond reduction in UV light. Additions were as indicated. Each lane contained 10 ␮g protein. Reduction of the porcine pancreas enzyme or AAT, 10 ␮g, was carried out in 30 mM PBS, pH 7.4 (10 mM Na2 HPO4 , 1.8 mM KH2 PO4 , 2.7 mM KCl and 137 mM NaCl) in a final volume of 100 ␮l. Additions with the NADP-thioredoxin (NTS): 1.25 mM NADPH, 1.7 ␮g of E. coli NTR, 1.7 ␮g of E. coli thioredoxin; NADP–glutathione system (NGS): 1.25 mM NADPH, 3 mM reduced glutathione and 1.5 ␮g glutathione reductase; dithiothreitol (DTT): 5 mM DDT. As indicated 53 ␮g AAT was added. The reaction was carried out for 30 min at 25 ◦ C and was stopped by the addition of 2 and 8 mM mBBr for the NTS, NGS and DTT reactions, respectively.

G. del Val et al. / Molecular Immunology 38 (2001) 759–763

Moreover, protein staining with Coomassie blue (data not shown) resembled mBBr in failing to show the presence of elastase after dithiothreitol treatment at 100 ◦ C (Fig. 1, lane 11). It was, therefore, difficult to obtain mBBr gels demonstrating the reduced enzyme in the absence of AAT at elevated temperature. Accordingly, mBBr labeling of reduced elastase was successful only in the presence of AAT either with the thioredoxin system or with dithiothreitol at 100 ◦ C. Under both of these conditions, the reduced enzyme formed a high molecular weight aggregate that just barely entered the gel (Fig. 1, lanes 9 and 12). The nature of this aggregate was not investigated. Finally, as found previously for other disulfide proteins (Jiao et al., 1992; Lozano et al., 1994; Buchanan et al., 1997; del Val et al., 1999), elastase was not significantly reduced by glutathione in either the absence or presence of AAT (Fig. 1, lanes 4–6; cf. lanes 2 and 6). In conjunction with the above experiments, elastase was assayed using 10 ␮l of a preincubated mixture containing enzyme and AAT. To permit measurement of activity, NaCl was added to the reaction mixture to dissociate the treated enzyme from the inhibitor as described by Rubin et al., 1994 (Fig. 2). The NADP–thioredoxin system and dithiothreitol were added to the preincubation mixture as indicated. Activity measurements revealed that, when reduced under these conditions, elastase lost more than 60% of its activity. Dithiothreitol showed an effect similar to thioredoxin in the presence of the inhibitor, but as seen in the following paragraphs the inhibitor was not required for inactivation of elastase under these conditions. The difficulty in obtaining mBBr and Coomassie-stained gels with the reduced enzyme in the absence of AAT prompted experiments to determine whether reduced elastase disappeared when the temperature was being elevated. To this end, we reduced the enzyme as described earlier, but in the absence of AAT: (1) with 5 mM dithiothreitol at temperatures ranging from 25 to 50 ◦ C (Fig. 3A), and (2) at 37 ◦ C with dithiothreitol concentrations ranging from

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Fig. 2. Effect of thioredoxin and DDT on elastase activity. The enzyme was bound with AAT, reduced with thioredoxin (NTS) or dithiothreitol (DTT) for 30 min at 25 ◦ C, dissociated from the inhibitor with NaCl and then assayed for activity. NADP–thioredoxin system (NTS). Elastase was assayed using 10 ␮l of a preincubated mixture containing in 100 ␮l (final volume) 13.7 ␮M enzyme and 22.8 ␮M AAT that had been treated with 1 M NaCl to dissociate the enzyme–inhibitor complex. Additions were made to the preincubation mixture as indicated.

0.05 to 10 mM (Fig. 3B). The results showed that elastase progressively lost activity with temperature: thus 95, 70, 25 and 0% of the original enzyme activity remained after reduction at 25, 37, 45 and 50 ◦ C, respectively. At 37 ◦ C, the loss of activity was proportional to dithiothreitol concentration in the 0.25–5 mM range: at 0.25 and 5 mM activity was reduced by 20 and >90%, respectively. The data show that, under appropriate conditions, dithiothreitol alone inhibited elastase. These results contrast with the recent report that dithiothreitol enhanced elastase inhibition in the presence of the inhibitor endopin (Hwang et al., 2000). The reduction of the endopin was required under their conditions.

Fig. 3. Effect of varying (A) temperature and (B) dithiothreitol (DTT) concentration on elastase activity. As indicated, elastase was assayed (A) in 5 mM dithiothreitol at varying temperatures, and (B) with varying dithiothreitol concentrations at 37 ◦ C.

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Fig. 4. Self-digestion of elastase following reduction by dithiothreitol (DTT) at elevated temperature. (A) Coomassie blue SDS–PAGE gel showing the effect of time on the enzyme incubated at 50 ◦ C with 5 mM DTT. (B) Densitometric scan of the gel shown in (A). Elastase, 60 ␮g, was incubated at 50 ◦ C in 100 ␮l PBS buffer with or without the addition of 5 mM DTT. Aliquots of 10 ␮l were collected at 0.5, 1, 2, 8, 15 and 60 min and the reaction was stopped by addition of 10 ␮l of SDS–PAGE loading buffer containing no reductant. Samples were then subjected to 15% SDS–PAGE without having been previously boiled and analyzed for protein with Coomassie blue staining as described earlier. The extent of elastase self-digestion was integrated with an imaging gel system.

The question arose as to how dithiothreitol achieved the striking inhibitory effect summarized in Fig. 3. An analysis of enzyme levels revealed that elastase underwent self-digestion when reduced by dithiothreitol at 50 ◦ C (Fig. 4A). This process, which was strictly dependent on dithiothreitol, was relatively slow, requiring 60 min to reach completion (Fig. 4B). It seems possible that reduction perturbed the enzyme surface and uncovered proteolytic sites such that the oxidized (active) elastase degraded its reduced counterpart. The extent of elastase self-digestibility linked to reduction was not investigated with thioredoxin in combination with AAT. To determine reductant specificity, we incubated the enzyme at 50 ◦ C with a series of monothiols (beta-mercaptoethanol, reduced glutathione) and dithiols (lipoic acid, dithioerythritol, a dithiothreitol isomer). The results (not shown) demonstrated that dithiothreitol and dithioerythritol were the most effective (>90% inhibition) while lipoic acid worked less well (30% inhibition). Dithiothreitol effected maximal inactivation at 5 mM and lipoic acid at 10 mM. Significantly, the monothiols, beta-mercaptoethanol and glutathione, showed little effect (respective inhibitions of 20 and 5%). These limited inhibitory effects were in accord with the inability of glutathione to reduce the enzyme noted earlier (Fig. 1, lane 6).

4. Discussion Earlier work from our laboratory has shown that thioredoxin regulates protease activity in seeds either by (1) reductive inactivation of specific disulfide protein inhibitors (Jiao et al., 1993), or (2) reductive activation of the disulfide protease itself (Besse et al., 1996). The present study adds a new dimension in demonstrating that thioredoxin (or

a dithiol substitute) can act directly on a protease to effect its inactivation. A protease inhibitor was required to protect thioredoxin and NTR in the reduction of elastase. It remains to be seen whether such reductive inactivation takes place physiologically as has been observed, e.g. with chloroplast glucose 6-phosphate dehydrogenase (Schürmann and Jacquot, 2000). In addition to uncovering a new type of protease regulation, the present work has potential relevance for the clinical treatment of elastase-related diseases. For example, psoriasis, which is linked to oxidative stress and inflammation (Fuchs et al., 2001), has been reported to induce elevated levels of NTR (Schallreuther and Pittelkow, 1987). It seems possible that reduced thioredoxin (or a dithiol substitute) applied jointly with a trypsin inhibitor and heat could enhance the therapy due to the inhibitor alone. Under these conditions, the elastase that is rendered inactive by the inhibitor would self-digest and thereby be permanently inactivated at the inflammation site. Alternatively, it is possible that a therapeutic effect could be achieved with a dithiol such as lipoic acid applied with heat in the absence of an inhibitor. Finally, in view of its reported ability to inhibit another serine protease (trypsin), it is possible that oxidized thioredoxin could be beneficial for psoriasis and related diseases through the inhibition of elastase activity (Lunn et al., 1986). Each of these avenues seems worthy of pursuit in the treatment of psoriasis and possibly other diseases that involve elastase such as pulmonary emphysema and rheumatoid arthritis.

Acknowledgements GdV gratefully acknowledges support from a fellowship from the Swiss National Science Foundation.

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References Ashe, B.M., Zimmerman, M., 1997. Specific inhibition of human granulocytes elastase by cys-unsaturated fatty acids and activation by the corresponding alcohols. Arch. Biochem. Biophys. 75, 194–199. Barrett, A.J., 1981. Leukocyte elastase. Methods Enzymol. 80, 581–588. Besse, I., Wong, J.H., Kobrehel, K., Buchanan, B.B., 1996. Thiocalsin: a thioredoxin-linked substrate specific protease dependent on calcium. Proc. Natl. Acad. Sci. U.S.A. 93, 3169–3175. Bode, W., Meyer, E., Powers, J., 1987. Human leukocyte and porcine elastase: X-ray crystal structures, mechanism substrate specificity and mechanism-based inhibitors. Biochemistry 28, 1951–1963. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal. Biochem. 72, 248–254. Buchanan, B.B., Adamidi, C., Lozano, R.M., Yee, B.C., Momma, M., Kobrehel, K., Ermel, R., Frick, O.L., 1997. Thioredoxin-linked mitigation of allergic responses to wheat. Proc. Natl. Acad. Sci. U.S.A. 94 (10), 5372–5377. Carrell, R.W., Owen, M.C., 1985. Plakalbumin, a1-antitrypsin, antithrombin and the mechanism of inflammatory thrombosis. Nature 317, 730–732. del Val, G., Yee, B.C., Lozano, R., Buchanan, B.B., Ermel, R.W., Frick, O.L., 1999. Thioredoxin treatment increases digestibility and lowers allergenicity of milk. J. Allergy Clin. Immunol. 103, 690–697. Fuchs, J., Zollner, T.M., Kaufmann, R., Podda, M., 2001. Redox-modulated pathways in inflammatory skin diseases. Free Radic. Biol. Med. 30, 337–353. Hwang, S.R., Steineckert, B., Hook, V.Y.H., 2000. Expression and mutagenesis of the novel serpin endopin 2 demonstrates a requirement for cyteine-374 for dithiothreitol-sensitive inhibition of elastase. Biochemistry 39, 8944–8952. Janoff, A., 1972. Neutrophil proteases in inflammation. Annu. Rev. Med. 23, 177–190. Jiao, J., Yee, B.C., Kobrehel, K., Buchanan, B.B., 1992. Effect of thioredoxin-linked reduction on the activity and stability of the Kunitz and Bowman-Birk soybean trypsin inhibitor proteins. J. Agric. Food Chem. 40, 2333–2336.

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Jiao, J., Yee, B.C., Wong, J.H., Kobrehel, K., Buchanan, B.B., 1993. Thioredoxin-linked changes in regulatory properties of barley alpha-amylase subtilisin inhibitor protein. Plant Physiol. Biochem. 31, 799–804. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lozano, R.M., Yee, B.C., Buchanan, B.B., 1994. Thioredoxin-linked reductive inactivation of venom neurotoxins. Arch. Biochem. Biophys. 309, 356–362. Lunn, C.A., Pak, P., Van Savage, J., Pigiet, V., 1986. The catalytic active site of thioredoxin: conformation and homology with bovine pancreatic trypsin inhibitor. Biochim. Biophys. Acta 871, 257–267. Maurice, M.M., Nakamura, H., Gringhuis, S., Okamoto, T., Yoshida, S., Kullmann, F., Lechner, S., van der Voort, E.A., Leow, A., Versendaal, J., Muller-Ladner, U., Yodoi, J., Tak, P.P., Breedveld, F.C., Verweij, C.L., 1999. Expression of the thioredoxin–thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis. Arthritis Rheum. 11, 2430–2439. Rubin, H., Plotnick, M., Wang, Z.M., Liu, X.Z., Zhong, Q., Schechter, N.M., Cooperman, B.S., 1994. Conversion of alpha(1)antichymotrypsin into a human neutrophil elastase inhibitordemonstration of variants with different association rate constants, stoichiometries of inhibition, and complex stabilities. Biochemistry 33, 7627–7633. Schallreuther, K.U., Pittelkow, M.R., 1987. Andralin inhibits elevated levels of thioredoxin reductase in psoriasis. A new mode of action for the drug. Arch. Dermatol. 123, 1494–1498. Schürmann, P., Jacquot, J.P., 2000. Plant thioredoxin systems revisited. Ann. Rev. Plant Phys. Plant Mol. Biol. 51, 371–400. Wiedow, O., Wiese, F., Streit, V., Kalm, C., Christophers, E., 1992. Lesional elastase activity in psoriasis contact dermatitis and atopic dermatitis. J. Invest. Dermatol. 99, 306–309. Wong, J.H., Kobrehel, K., Buchanan, B.B., 1995. Thioredoxin and seed proteins. Methods Enzymol. 252, 228–240. Yoshida, S., Katoh, T., Tetsuka, T., Uno, K., Matsui, N., Okamoto, T., 1999. Involvement of thioredoxin in rheumatoid arthritis: its costimulatory roles in the TNF-alpha-induced production of IL-6 and IL-8 from cultured synovial fibroblasts. J. Immunol. 163, 351–358.