Degradation of Elastin by a Cysteine Proteinase from Staphylococcus ...

THE JOURNALO F BIOLOGICALCHEMISTRY

Vol. 263, No. 6, Issue of February 25, pp. 2664-2667,1988 Printed in U.S.A.

8 1988 by The American Society for Biochemistry and Molecular Biology, Inc

Degradation of Elastin by a Cysteine Proteinase from Staphylococcus aureus* (Received for publication, August 31, 1987)

Jan Potempa$, Adam Dubin$, Gabriela KorzusS, andJames TravisQTi From the $Institute of Molecular Biology, Jagiellonian University,31-120 Cracow, Poland and the §Department of Biochemistry, University ofGeorgia, Athens, Georgia 30602

Staphylococcus aureus is known to produce three elastinolytic activity stronger than that present in human very activeextracellular proteinases. One of these en- neutrophil elastase, as well as studies on its inhibition by zymes, a cysteine proteinase, after purification to ho- protein proteinase inhibitors. mogeneity was found to degrade insoluble bovine lung elastin at a rate comparable to human neutrophil elasMATERIALS ANDMETHODS tase. This enzyme had no detectable activity against a Purification of S. aureusProteinases-S.aureus strain V-8 (a range of synthetic substrates normally utilized by elastase, chymotrypsin, or trypsin-like proteinases. How- generous gift from Dr. S. 0. Arvidson, Karolinska Institute, Stokholm) was cultivated according to the procedure of Drapeau et al. (8). ever, it did hydrolyze the synthetic substrate carbo- The serine proteinase (commonly referred to as the V-8 serine probenzoxy-phenylalanyl-leucyl-glutamyl-p-nitroanilide teinase) andmetalloproteinase were isolated as described by Drapeau ( K , = 0.5 mM, kc,,= 0.16 s-’). The proteolytic activity (9) using ammonium sulfate fractionation, acetone precipitation, and of the cysteine proteinase was rapidly and efficiently DEAE-cellulose chromatography. The latter step resulted in the inhibited by az-macroglobulinand also by the cysteine- complete separation of the cysteine proteinase, as previously despecific inhibitor rat T-kininogen (Ki = 5.2 X 10” M). scribed (9),which wasobtained from the unadsorbed DEAE-cellulose Human kininogens, however, did not inhibit. Human fraction. This enzyme was purified to homogeneity in two further plasma apparently contains other inhibitors of this steps, including ( a ) chromatography on CM-Sephadex (1.5 X 15-cm enzyme, since plasma depleted of a,-macroglobulin re- column) equilibrated with 0.05 M sodium phosphate buffet at pH 6.0, with the enzyme being eluted at 0.1 M sodium chloride when a linear tained significant inhibitory capacity. The elastolytic gradient (400ml total volume) from 0 to 0.3 M was applied, followed activity of this S. aureus proteinase and its lack of by ( b ) gel filtration of active fractions on Sephadex G-75 (1.5 X 90control by human kininogens or cystatin C may explain cm column) equilibrated with the above buffer but also containing some of the connective tissue destruction seen in bac- 0.15 M sodium chloride. The purity and molecular weight of the S. terial infections due to this and related organisms such aureus cysteine proteinase was determined by NaDodS0,’-polyacrylas may occurin septicemia, septic arthritis, and otitis. amide gel electrophoresis (IO) with the latteralso being confirmed by

Many different species of bacteria produce extracellular elastin-degrading proteinases whose activities are often considerably higher than that of porcine pancreatic elastase (1). In some strains (e.g. Pseudomonas aeruginosa (2), Flauobacterium rnenningosepticum (3), and Bacteroides nodosus (4)) positive correlations between pathogenicity and elastase production have been found, indicativeof a degradative role for this type of enzyme. Staphylococcihavelong been known to be the etiologic agents ina large variety of diseases ( 5 ) ,with massive connective tissue damage being associated with theirpresence. They can also produce elastinolytic enzymes since this activity has been detected in the culture fluids of Staphylococcus aureus and ineven higherconcentrations in strains of Staphylococcus epidermidis ( 6 ) . However, none of the elastin-degrading enzymes in either of these bacteria have been purified to homogeneity or characterized. Recently (7), it was noted that the V-8 strain of S. aureus secreted three different proteinases, and each of these was partially characterized. In the present investigation, we describe an improved purification procedure for oneof these enzymes, a cysteine proteinasewith

* This research was sponsored in part by grants from the National Heart, Lung, and Blood Institute and by Grant 04.01.2.10 from the Polish Academy of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. ll To whom correspondence should be addressed.

gel filtration on Sephadex G-75. Assay of Enzyme Activity-The activity of papain (Sigma) was measured with Bz-Arg-pNA (final concentration 1 mM) in 0.1 M sodium phosphate buffer, pH 7.4, 2 mM cysteine, 5 mM EDTA. The reaction was monitored by following the change in A,,, at 25 “C during a IO-min time period. The proteinase activity of both chymotrypsin and theV-8 serine proteinase was measured with hide powder azure (Behring Diagnostics) (final substrate concentration 1%w/v) a t pH 7.8. S. aureus cysteine proteinase proteolytic activity was estimated with fluorescein isothiocyanate-labeled hemoglobin (11) or with hide powder azure as substrate in 0.1 M sodium phosphate buffer, pH 7.4, 2 mM cysteine, 5 mM EDTA. Elastin degradation was monitored by the elastin plate technique (12). Briefly, 10 ml 1%(w/v) agarose in 20 mM sodium phosphate, pH 7.4, 0.15 M NaC1, 2 mM cysteine, 5 mM EDTA containing suspended bovine ligamentum nuchae elastin (Worthington) (3 mg/ ml) was poured onto glass plates (6 X 10 cm). Wells were cut (3-mm diameter) and filled with enzyme solution (generally from 0.05 to 0.2 nmol of active proteinase).Plates were incubated at 25 “C in a humidified chamber and after 48-h zones of elastin digestion were measured. This method was also utilized in estimating the dependence of the elastinolytic activity of S. aureus cysteine proteinase on pH and NaCl concentration. Proteinuses and Proteinase Inhibitor Titration-Kininogens (high and low molecular weight) were purified by the procedure of Gounaris et al. (13). a,-Macroglobulin (a2M)purification and active site titration were performed as previously described (14). Rat al-cysteine proteinase inhibitor (rat T-kininogen) was kindly provided by Dr. A. Guzdek (Institute of Molecular Biology,Jagiellonian University, Cra-

’ The abbreviations used are: NaDodSO,, sodium dodecyl sulfate; a,M,ol,-macroglobulin; pNA, p-nitroanilide; CBZ, carbobenzoxy; Bz-, benzoxy-; Tos-, tosyl-; SUC-,succinyl-; OMeSuc-, methoxysuccinyl-; Ac-, acyetyl; OEt-, ethoxy-; E-64, L-trans-epoxysuccinylleucylamido-(4-guanidino)butane.

2664

Proteinase CysteineS. aurew

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cow, Poland). Human cystatin C was the generous gift of Dr. M. Abrahamson (University of Lund, Malmo, Sweden). Active site titration of both papain andS. aureus cysteine proteinase was performed with ~-trans-epoxysuccinyIleucylamido-(4-guanidino)butane (E-64) (Sigma) (15) in 0.1 M sodium phosphate buffer, Mr. IO' pH 7.4, 2 mM cysteine, 5 mM EDTA. Papain of known active site 94 concentration was used to titrate the inhibitory activity of kininogens as well as cystatin C. a2M of known inhibitory activity was used for 67 titration of the V-8 serine proteinase. Amidolytic and Esterolytic Assays-The substrates Bz-Arg-pNA, Tos-Gly-Pro-Arg-pNA, Tos-Gly-Pro-Lys-pNA, DL-Val-Leu-Arg43 pNA, D-Val-Leu-Lys-pNA, Bz-Tyr-pNA,Suc-Phe-pNA, Ac-PhepNA, Suc-Ala-Ala-Pro-Phe-pNA, Suc-Ala-Ala-Ala-pNA,OMeSucAla-Ala-Pro-Val-pNA (all from Sigma) and CBZ-Phe-Leu-Glu-pNA 30 (Behring Diagnostics) were used to test the specificity of S. aureus cysteine proteinase. Substrate stock solutions were prepared in dimethyl sulfoxide. Hydrolysis of substrates was determined following 21 the addition of 100 pl of enzyme (5 nmol of active S. aureus cysteine proteinase) to 0.1 M sodium phosphate buffer, pH 7.4,2 mM cysteine, 5 mM EDTA, containing the substrate being tested. Incubations of up to 12h were made a t 25 "C, and theA,,,, measured against control I"---& samples containing no enzyme. The esterolytic activityof S. aureus as substrate wasmeasured cysteine proteinase using Bz-Tyr-OEt A B C according to theprocedure of Hummel(l6). Kinetic constants for the hydrolysis of CBZ-Phe-Leu-Glu-pNA by the V-8 serine proteinase L _" . * and S. aureus cysteine proteinase were determined from the initial FIG.1. NaDodS0,-polyacrylamidegelelectrophoresis of rates of hydrolysis and were based on duplicate rate determinations at six substrateconcentrationsintherange of0.05-2.0mM. An purified S. aureus cysteine proteinase. A, protein standards; B extinction coefficient a t 8,800 at 410 nm for p-nitroaniline was used and C, purified proteinase, 0.5 and 1.S pg, respectively. in the calculations(17). S. aureus Proteinase Inhibitionby Human Plasm-Different volor plasma umes of either suitably diluted normal human plasma depleted of active a2M by methylamine treatment (18) were mixed with V-8 serine proteinase or S. aureus cysteine proteinase in either 0.1 M Tris-HC1, pH 7.8, or 0.1 M sodium phosphate, pH 7.4, 10 mM cysteine, 5 mM EDTA. Incubation was for 15 min a t room temperature. Residualproteolytic activity was then measuredwithhide powder azure as substrate. Determination of Equilibrium Dissociation Constants-Equilibrium dissociation constants ( K , )were determined by the method of Bieth (19,20). Increasing amounts of rat T-kininogen were added to a constant quantity of S. aureus cysteine proteinase (final concentration 120 nM) and the mixture incubatedfor 10 min at 25 "C. Fluorescein isothiocyanate-labeled hemoglobin was then added and residual enzyme activity measured. . d""

-._

RESULTS

Properties of S. aureus Cysteine Proteinme-S. aureus cysteine proteinase, as isolated from the culture supernatant of S. aureus V-8 strain gave a single band after NaDodS04polyacrylamide gel electrophoresis with M , of 13,000 (Fig. 1). This unusually low M , for a proteinase was confirmed by gel filtration on Sephadex G-75and is in agreement with previous literature data (21). The enzymeitself was found to be a typical cysteine proteinase, only being active in thepresence of reducing agents such as cysteine. Inactivation could be readily observed with heavy metal ions such as Hg2+ or Ag', as well as with iodoacetamide in a millimolar concentration range (data not shown). Inhibition by E-64, an irreversible inhibitor of cysteine proteinases (15), enabled us tocalculate its active concentration, and this was found to be approximately 50%. The enzyme was not active against typical synthetic substrates for trypsin, chymotrypsin, or elastase, disagreement in with theprevious finding of Arvidson et al. (21). In fact, of all of the substrates tested only CBZ-Phe-Leu-Glu-pNA which is a substrate for the V-8 serine proteinase was hydrolyzed. Kinetic data for thehydrolysis of this substrate by S. aureus cysteine proteinaseindicated a K , = 0.5 mM, a kat= 0.16 s-', and a &/K, = 320 M" s-'. In comparison to theV-8 serine proteinase which was found to have a K , = 0.286 mM, a kcat = 6.6 s-', and a k,,,/K,,, = 23,300 M-' s-', it is clear that the cysteine enzyme is much less efficient with this substrate. It

FIG. 2. Comparison of the elastinolytic activity of various proteinases. An elastin-agarose plate was prepared in 20 mM sodium phosphate buffer, pH 7.4,0.15 M NaCI, 2 mM cysteine, 5 mM EDTA and incubated with enzyme for 48 h a t 25 "C. A, leucocyte elastase (0.1 nmol of active enzyme); B, papain (0.1 nmol); C, porcine pancreatic elastase (0.05 nmol); D, S. aureus cysteine proteinase (0.05 nmol); E, S. aureus cysteineproteinase (0.1 nmol); F, V-8 serine proteinase (0.2 nmol) + S. aureus metalloproteinase (0.2 nmol).

should also be pointed out that substrate hydrolysis by S. aureus cysteine proteinase is certainly not due to contamination by the V-8 proteinase, since the cysteine proteinase has no amidolytic activity in the absence of reducing agents or after treatmentwith E-64. Elastinolytic Actiuity of S. aureus Cysteine ProteinmeUnder physiological conditions it was found that S. aureus cysteine proteinase exhibited elastinolytic activity equal to that of human neutrophil elastase (Fig. 2). However, unlike the latter enzyme this activity was unaffected by changes in NaCl concentration between 0.1 and 1.0 M. The pHoptimum for elastin hydrolysis was rather narrow, with a maximum near pH 6.5 (Fig. 3). Thiscontrasted markedlywith the generally broad pHrange for the hydrolysis of other proteins (21). That this was the only proteinase in S. aureus culture filtrates capable of digesting elastin was proven by incubation

2666

S. aureus Cysteine Proteinase

-

200 -

N

E E 160

U

U W

5

-

!d 5

120

"1

c3

W

80-

W

52

cr

0

40

-

I T I A 4 3 20 4.0

5.0

6,O

7,O

8,O

PH FIG. 3. Measurement of pH optimum for the degradation of elastin by S. uureus cysteine proteinase. 0.2 nmol ofenzyme was incubated on elastin-agarose plates prepared in 0.05 M citrate-phosphate buffers, pH 4.0-7.0 (O),and 0.05 M sodium phosphate buffers, pH 6.5-8.0 (A), both containing 2 mM cysteine and 5 mM EDTA. Elastolysis was monitored after 48-h incubation at room temperature by measurement of the diameter of digested zones.

METHYLAMINETREATEDPLASMA

[PI]

e

m W

a 100

200

300

400

INHIBITORCONCENTRATION

500

[nM]

FIG. 5. Inhibition of S. uureus cysteine proteinaseby rat Tkininogen. Increasing amounts of inhibitor were added to enzyme and, after IO-minincubation, residual enzyme activity measured. The inset shows a replot of the curve accordingto Bieth (19), yielding an apparent K, = 5.2 X 10" M.

reduced using methylamine-treated plasma other inhibitors of this enzyme must also be present in plasma. Two likely candidates are the cysteine proteinase inhibitors (kininogens) and cystatin C which arepresentinnormalplasma at a concentration of 7.8 and 0.1 @I, respectively (22). However, neither were found to inactivateS. aureus cysteine proteinase, even at 20-fold higher molar concentrations. Highly purified rat T-kininogen did, however, inhibit S. aureus cysteine proteinase (Fig. 5) ( K , = 5.2 X M). DISCUSSION

2

4

6

8

NORMAL PLASMA [PI] FIG. 4. Inhibition of S. aureus cysteine proteinaseand V-8 serine proteinase activity by human plasma. Normal or methylamine-treated plasma was added to either enzyme. After 15-min incubation, residual proteolytic activity was measuredwith hide powder azure as substrate. A, V-8 serine proteinase activity after incubation with normal plasma; A, V-8 serine proteinase activity after incubation with methylamine-treated plasma; 0, S. uureus cysteine proteinase activity after incubation with normal plasma; 0, S. aureus cysteine proteinase activity after incubation with methylaminetreated plasma.

of a mixture of the V-8 serine proteinase and metalloproteinasewithelastin (Fig. 2 F ) . Even after 48-h incubation there was no evidence for degradation of this substrate. Inhibition of Proteinase Activity by Plasma ZnhibitorsHumanplasma readily inhibitedboth S. aureus cysteine proteinase and the V-8 serine proteinase (Fig. 4). This inhibition would appear to be primarily due to a2M since methylamine-treated plasmawas ineffective in reducing V-8 serine proteinase activity and was required in significantly higher quantities in order to reduce the activityof S. aureus cysteine proteinase.This was furtherconfirmed by using purified native or methylamine-treated alM (data not shown). Since the activity of S. aureus cysteine proteinase could still be

Several pathogenic bacteria have been reported to secrete elastin-degrading enzymes (23). S. epidermis culture filtrates (6,24, 25), in particular, have been found to contain readily detectable elastinolytic activity, and it has beensuggested thatthis couldplayamajor role inthe development of perifollicularmacula (26, 27). Sincetreatment of culture filtrates with iodoacetic acid abolished most of the elastindegrading activity, it was also suggested that it might be due to a cysteine proteinase(s) (26); however, since the enzyme was never purified or characterized this could not be confirmed. The studies reportedhere would, however, tend to be supportive since the enzyme we have isolated fromthe related organism S. aureus is a cysteine proteinase which has the ability todegrade elastin. Other members of the cysteine proteinase class, such as the plant enzymes papainand bromelain (1) and the human enzyme cathepsin L (28), have previously beenshown to degrade elastin. However, although microbial proteinases with elastinolytic activity have been investigated in some detail (29, 30), virtually all that were tested were of the serine or metalloenzyme class, active at alkaline pH and in low ionic strength. Indeed, cysteineproteinases withelastinolytic activity have been largely unrecognized, with only a fungal proteinase from Nunizzia fulve having been partially characterized (31). The elastinolytic activity of S. aureus cysteine proteinase is notaffected by changes in NaCl concentration (from 0.1 to 1.0 M) and in this respect is similar to the metalloenzymes from Streptomycesfradiae (1) and Flavobacterum (32). Al-

S. aureus Cystebine Proteinase

2667

7. Arvidson, S. 0.(1983) in Staphylococci and Staphylococcal Infecthough maximum activity on elastin hasbeen found to occur tions (Easmon, C. S. F., and Adlam, C . , eds) pp. 745-808, at pH 6.5, significant elastinolysis still occurs under physioAcademic Press Inc., Ltd., London logical conditions which is equal to that found with the human 8. Drapeau, G. R., Boily, Y., and Houmard, J. (1972) J. Biol. Chem. neutrophil enzyme. Thus, there is strong potential for the 247,6720-6726 contribution of such an enzyme to the pathological degrada9. Drapeau, G. R. (1978) J. Bacterid. 136,607-613 tion of connective tissue (33). Fortunately, it seems that the 10. Laemmli, U. K. (1970) Nature 2 2 7 , 680-685 existence of the cysteine proteinase among coagulase-positive 11. Twing, S. S. (1983) Anal. Biochem. 1 4 3 , 30-34 staphylococci is limited only to the laboratory strain V-8 of 12. Janoff, A,, and Carp. H. (1977) Am. Reu. Respir, Dis. 116,65-72 13. Gounaris, A. D., Brown, M. A., and Barrett, A. J. (1984) Biochem. S. aureus (7); however, itcannot be excluded that under J. 221,445-452 pathological conditions, synthesis of this enzyme might be 14. Dubin, A., Potempa, J., and Silberring, J. (1984) Biochem. Znt. 8 , induced by elastin, as has been found to occur for S. epider589-596 15. Barrett, A. J., and Kirschke, H. (1981) Methods Enzymol. 80, midis elastase (24, 25). 535-561 Human plasma has been found to strongly inactivate the proteolytic activity of both the V-8 serine proteinase and S. 16. Hummel, B.C. W. (1959) Can. J. Biochem. Physiol. 3 7 , 13931399 aureus cysteine proteinase, withazM being primarily respon- 17. Nakajima, K., Powers, J. C., Ashe, B. M., and Zimmerman, M. sible for this effect. Whereas plasma depleted of active a2M (1979) J. Biol. Chem. 2 5 4 , 4027-4032 did not inhibit the serine proteinase, some residual inhibitory 18. Howard, J. B., Swenson, R., and Eccleston, E. (1983) Ann. N . Y. activity against the cysteine proteinase was found.The nature Acad. Sci. 4 2 1 , 160-166 of this activity in plasma is not known, for neither of the 19. Bieth, J. (1974) in Bayer Symp. 5,463-469 other known cysteine proteinase inhibitors found in plasma, 20. Bieth, J. G. (1980) Bull. Eur. Physiopathol. Respir. 1 6 , (suppl.) 183-195 kininogens and cystatin C , were able to inactivate S. aureus 21. Arvidson, S., Holme, T., and Lindholm, B. (1973) Biochim. Biocysteine proteinase. In contrast, rat T-kininogen,which disphys. Acta 3 0 2 , 135-148 plays a similar inhibition profile to human kininogens (34), 22. Abrahamson, M., Barrett, A. J., Salvesen, G., and Grubb, A. readily inhibited S. aureus cysteine proteinase. This obser(1986) J. Biol. Chem. 2 6 1 , 11282-11289 vation and thelack of any detectable inhibitionof rat cathep- 23. Werb, Z., Banda, M. J., McKerrow, J. H., and Sandhaus, R. A. (1982) J. Inuest. Dermatol. 7 9 , 154-159 sin H by rat T-kininogen (34) show clear differences in the 24. Hartman, D. P., and Murphy, R. A. (1977) Infect. Zmmun. 1 5 , inhibition spectra of human kininogens and rat T-kininogen. 59-65 T o date,nocorrelationhas been reported between the 25. Janda, J. M. (1986) J. Clin. Microbiol. 2 4 , 945-946 pathogenicity of S. aureus strains and proteolytic activity 26. Varadi, D. P., and Saqueton, A. C. (1968) Nature 218,468-470 (35), although recent evidence has suggested that staphylo- 27. Varadi, D. P., and Saqueton, A. C. (1970) Br. J. Dermntol. 8 3 , 143-150 coccal proteinases may contributetothe development of influenza-derived pneumonia (36, 37). However, the elastin- 28. Mason, R. W., Johnson, D. A., Barrett, A. J., and Chapman, H. A. (1986) Biochem. J. 2 3 3 , 925-927 olytic activity of the s. aureus cysteine proteinase, as reported29. Bieth, J. G. (1986) in Regulution of Matrix Accumulation (Mehere, makes it tempting to speculate on the possible particicham, R. P., ed) pp. 217-320, Academic Press, Inc., Orlando, pation of this enzyme during tissue invasion and destruction, FL as has been found to occur in S. aureus-derived ulcerations, 30. Mandl, I., Keller, S., and Cohen, S. (1962) Proc. SOC. Exp.Biol. Med. 109,923-925 and as has alsobeen suggested to occurby the metalloelastase released in Pseudomonas aeruginosa-derived infections (38). 31. Rippon, J. W., and Varadi, D. P. (1968) J. Inuest. Dermatol. 5 0 , 54-58 Alternatively, Staphylococcal proteinases may facilitate the 32. Mandl, I., and Cohen, B. B. (1960) Arch. Biochem. Biophys. 9 1 , destructive action of endogenous proteinases such as human 47-53 neutrophil elastase and cathepsinG through the inactivation 33. Janoff, A. (1985) Annu. Reu. Med. 3 6 , 207-216 34. Sueyoshi, T., Enjyoji, K., Shimada, T., Kato, H., Iwanaga, S., of the controlling inhibitors of these enzymes, as reported Bando, Y., Kominami, E., andKatanuma, N. (1985) FEBS earlier (39).

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