[15] Pseudolysin and other pathogen endopeptidases of thermolysin family

242

[ 15]

METALLOPEPTIDASES

so it is not clear whether these metals might yield an active enzyme with, for example, 1% activity. That activity may seem small with respect to the native zinc enzyme activity, but it would still be orders of magnitude above a nonenzymatic reaction.

[15] P s e u d o l y s i n

and Other Pathogen Endopeptidases Thermolysin Family

By

KAZUYUKI

of

MORIHARA

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen which can cause fatal infection in vulnerable hosts. 1 Several products of the organism that are related to virulence have been identified and characterized. 2 Among them, elastase has long been thought to be of major significance? The elastase is now called pseudolysin (EC 3.4.24.26) by recommendation of the IUBMB. Pseudolysin is a Zn-metalloendopeptidase belonging to the thermolysin family (or family MH, see [13] in this volume)? Similar pathogenic endopeptidases are also produced by Vibrio cholerae,4 Legionella pneumophila, 5 and other bacteria. 6 This chapter mainly concerns pseudolysin, with some mention of related enzymes. Assay Methods

Principle Three assay methods are described, namely, casein digestion, elastin digestion, and hydrolysis of synthetic peptides. The first method is based on that of Kunitz, 7 which involves enzymatic digestion of casein under defined conditions. The digestion mixture is treated with trichloroacetic acid 1 R. D. Feigin and W. T. Shearer, J. Pediatr. 87, 677 (1975). 2 O. Pavlovskis and B. Wretlind, in "Medical Microbiology" (C. S. F. E a s m a n and J. Jeljaszewicz, eds.), Vol. i, p. 97. A c a d e m i c Press, London, 1982. 3 K. Morihara and J. Y. H o m m a , in "Bacterial E n z y m e s and Virulence" (I. A. Holder, ed.), p. 41. C R C Press, Boca Raton, Florida, 1985. 4 B. A. Booth, M. Boesman-Finkelstein, and R. A. Finkelstein, Infect. Immun. 42, 639 (1983). 5 L. A. Dreyfus and B. A. Iglewski, Infect. lmmun. 51, 736 (1986). 6 p. L. M~ikinen, D. B. Clewell, F. A n , and K. K. Makinen, J. Biol. Chem. 264, 3325 (1989). 7 M. Kunitz, J. Gen. Physiol. 30, 291 (1947).

METHODSIN ENZYMOLOGY.VOL. 248

Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.

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(TCA) to remove undigested casein. The extent of digestion is measured by the amount of unprecipitated products, which contain tyrosine, according to the method of Lowry et al. s The method is not specific for pseudolysin, but it is usually used for routine assay. The second method is based on the colorimetric procedure of Sachar et al., 9 in which orcein-elastin is used as substrate. Enzymatic digestion of elastin results in dissolution of insoluble elastin. The extent of digestion is then measured by the amount of colored soluble products. The third method is based on that of Feder, 1° which gives a precise measure of peptidase activity using a chromophoric substrate, furylacryloyl-Gly-Leu-NH2. The hydrolysis is assayed by following the decrease in absorbance at 345 nm that accompanies hydrolysis of the Gly-Leu bond in the substrate. Casein-Digestion Method Reagents

Substrate, 2% casein: the substrate solution is made by dissolving 2 g of casein (Hammrsten, Merck, Darmstadt, Germany) in 70 ml of 0.1 N NaOH; the solution is then adjusted to pH 7.4 by addition of 1 M KH2PO4, and the total volume is adjusted to 100 ml with distilled water Tris-HC1 buffer, 10 mM, pH 7.4, with 10 mM CaC12 Trichloroacetic acid (TCA), 10% (w/v) Na2CO3, 0.4 M Folin-Ciocalteu phenol reagent (Sigma, St. Louis, MO), diluted with an equal volume of distilled water Procedure. The casein solution and pseudolysin solution suitably diluted with 10 mM Tris-HC1 buffer are incubated in a water bath at 40° for at least 10 min prior to the assay. The reaction is carried out in 1 ml of 2% casein solution with 1 ml of pseudolysin solution (enzyme content, 0.4-4 /~g) at 40° for 10 min. The reaction is terminated by the addition of 2 ml of 10% TCA, and, after shaking, the reaction mixture is kept in the water bath at 40° for about 30 min. The precipitate is filtered through Whatman (Clifton, N J) No. 1 filter paper. One milliliter of the filtrate is mixed with 5 ml of 10% Na2CO3, followed by the addition of 1 ml of diluted phenol reagent. After 15 min, the absorbance at 670 nm is read for determination of the amount of liberated tyrosine. The blank is prepared by first mixing 8 0 . H. Lowry, N. J. Rosebrough, A. L. Farrand, and R. J. Randall, J. Biol. Chem. 193, 265 (1951). 9 L. A. Sachar, K. K. Winter, N. Sicher, and S. Frankel, Proc. Soc. Exp. Biol. Med. 90, 323 (1955). 10j. Feder, Biochem. Biophys. Res. Commun. 32, 326 (1968).

244

METALLOPEPTIDASES

[ 15]

the casein solution with TCA and then adding the enzyme solution to the casein-TCA mixture. The specific activity is expressed as milligrams of tyrosine released per minute (units) per milligram of enzyme. Purified psuedolysin exhibits approximately 20 units/mg. In variants of the above procedure, the TCA-soluble products can be assayed directly at 280 nm, 11 or a chromophoric substrate such as azocasein (Serva, Heidelberg, Germany) is used, 12 and the extent of digestion is determined by reading the absorbance of the TCA-soluble products at 370 nm. Elastin-Digestion M e t h o d Reagents

Orcein-elastin: Orcein (50 mg) is dissolved in 5 ml of 70% (v/v) ethanol containing i ml of concentrated HC1, and 1 g of elastin (Calbiochem, La Jolla, CA, C grade) is added. The suspension is stirred at room temperature for several hours. The insoluble product is collected by centrifugation (4000 rpm, 10 min) and washed with a large quantity of 70% ethanol until the supernatant becomes colorless. The precipitate is suspended in distilled water and lyophilized Tris-HC1 buffer, 30 raM, pH 8.0, with 2 mM CaC12 Sodium phosphate buffer, 0.7 M, pH 6.0 Procedure. The enzyme solution is dialyzed against 30 mM Tris-HC1 buffer containing 2 mM CaCI2 for 2 days (two buffer changes a day) in the cold to remove inhibitory ions prior to the assay, and the enzyme content is suitably adjusted by dilution with the same buffer or by concentration using an Amicon (Danvers, MA) miniconcentrator. The reaction mixture containing 3 ml of enzyme solution (25-100 /~g of purified pseudolysin) and 20 mg of orcein-elastin is shaken rapidly at 40° for 3 hr. The reaction is terminated by the addition of 2 ml of 0.7 M phosphate buffer, pH 6.0, and the mixture is then filtered. The absorbance of the filtrate is read at 590 nm. An increase in absorbance of 0.42 results from the complete hydrolysis of 20 mg orcein-elastin. Activity is expressed as milligrams of elastin dissolved per hour (units) per milligram of enzyme. Purified pseudolysin exhibits approximately 35 units/mg. Elastin-Congo Red (ICN Biochemicals, Costa Mesa, CA) can also be used as a chromophoric substrate in place of orcein-elastin. When native elastin is used as substrate, the protein content of the soluble product is assayed according to the method of Lowry et aL 8 11H. Matsubara, this series, Vol. 19, p. 642. 12E. Kessler,M. Safrin, N. Landshman,A. Chechick,and S. Blumberg, Infect. Immun. 38, 716 (1982).

[ 151

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Hydrolysis o f Synthetic Substrate Reagents Furylacryloyl (FA)-Gly-Leu-NH2 (Sigma), 10 mM, in 50 m M Tris-HCl buffer, p H 7.5:30.68 mg of FA-Gly-Leu-NH2 is dissolved in 0.1 ml of spectrophotometrically pure dimethylformamide, to which approximately 7 ml of distilled water and 1.0 ml of 0.5 M Tris-HC1 buffer (pH 7.5) are added. After final adjustment to p H 7.5, the volume is brought to 10 ml. The stock solution is stored at - 2 0 °. Tris-HC1 buffer, 50 mM, pH 7.5, with 10 mM CaC12. Procedure. A solution of 1 mM FA-Gly-Leu-NH2 is prepared by a 1/10 dilution of the 10 m M stock solution with 50 m M Tris-HC1 buffer (pH 7.5). The reaction is initiated by adding 20 ~1 of enzyme solution containing pseudolysin (20-100 /zg) to 1 ml of 1 mM FA-GIy-Leu-NH2 (0.870 absorbance units) in a 1.5-ml cuvette of 1-cm light path, maintained at 25 °. The decrease in absorbance at 345 nm is plotted on a chart recorder. Under these conditions, the recorder trace is linear for approximately 3 min, during which time less than 10% of the substrate is hydrolyzed; the initial velocity is determined from that portion of the trace. Complete hydrolysis of a 1 m M solution results in an absorbance change of 0.345 at 345 nm. One unit of activity is defined as the amount of enzyme that gives an initial hydrolytic activity of 1 /xmol of FA-GIy-Leu-NH2 per minute. Purified pseudolysin has a specific activity of approximately 3 units/mg. The peptidase activity can also be assayed by the same general procedure using any of several other FA-tripeptides such as FA-Gly-Leu-Ala or FAAla-Leu-Ala. 13 The activity on the FA-tripeptides can be more than 50 times that on FA-Gly-Leu-NH2, as it is with simple peptides (see Table I, below). P r o d u c t i o n a n d Isolation of P s e u d o l y s i n Organism Most cultures of Pseudomonas aeruginosa isolated from human infections exhibit marked elastinolytic activity, TM but then show a greater or lesser reduction in elastinolytic activity by repeated transfer on nutrient agar over a 6-month period. 15 Therefore, it has been necessary to select a 13j. M. Saulnier, F. M. Curtil, M.-C. Duclos, and J. M. Wallach, Biochim. Biophys. Acta 99S, 285 (1989). 14W. Scharmann, ZentralbL BakterioL Hyg. Abt. 1: Orig. A 220, 435 (1972). 15S. Goto, M. Ogawa, T. Takita, Y. Kaneko, and S. Kuwahara, in "Abstract of Proc. 9th Meet. Japan Pseudomonas aeruginosa Society," p. 22. Tokyo, Japan, 1975.

246

METALLOPEPTIDASES

[ 15]

particularly suitable strain for elastase production, which continues to produce the enzyme even through many subcultures. The strain IFO 3455 is suitable for that purpose, and it has been used for industrial production of pseudolysin for over 10 years at the Nagase Biochemical Co. (Fukuchiyamashi, Kyoto, Japan).

Culture A nutrient medium (1% w/v meat extract, 1% peptone, 1% yeast extract, 0.5% glucose, and 0.5% NaCI, p H 7.0) is usually used for the production. 16 A 500-ml flask containing 100 ml of the medium is shaken (130 rpm, 7 cm amplitude) for 1 or 2 days at 30 °. A 5-liter jar fermenter containing 3 liters of medium and a small amount of antifoamant can also be used. Two shaking flasks (200 ml broth) of fresh culture are used as the inoculum, and the culture is grown with aeration at 2 liters/min and stirring at 400 rpm at 30 ° for 7 - 8 hr.

Purification Procedure 1. The culture broth is centrifuged at 5000 rpm for 20 min to remove bacterial cells, and the clear supernatant is used as the starting material, a6 The supernatant is brought to 60% saturation with solid ammonium sulfate and allowed to stand overnight at 4 °. The precipitate is then collected by centrifugation (5000 rpm, 20 min) and dissolved in distilled water. After a second centrifugation, a clear supernatant is obtained. Acetone is then added to the solution in the cold (below 4 °) until the concentration reaches 65% (v/v). The precipitate formed by acetone treatment is collected by centrifugation (4°), dissolved in 20 m M phosphate buffer at p H 8, and then dialyzed against the same buffer for 2 days at 4 °. The retentate is applied to a column of DEAE-cellulose (Serva) which was previously washed with 1 M N a O H and 1 M Na2HPO4, and then equilibrated with 20 m M phosphate at p H 8.0. Column chromatography is performed at 4 °. After washing the column with 20 m M phosphate buffer (pH 8.0), purified pseudolysin can be obtained by stepwise elution with the same buffer containing 50 m M NaC1. The total yield of activity is about 25%, and the specific activity increases about 300-fold with this procedure. Pseudolysin-containing fractions are pooled, concentrated by ultrafiltration, and stored at - 7 0 °. Pseudolysin remains stable and active (retaining >90% activity) for more than 1 year when stored under these conditions. Procedure 2. Pseudolysin can be purified without separation of the bacterial cells, 17 which may be useful for large-scale production. The cula6 K. Morihara, H. Tsuzuki, T. Oka, H. Inoue, and M. Ebata, J. Biol. Chem. 240, 3295 (1965). 17 K. Morihara, Jpn. Pat. Appl., Sho-40-8114 (1965).

[151

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247

ture broth is adjusted to pH 5.0 with acetic acid and then mixed with a 1/30 volume of wet resin (Amberlite CG 50), which has previously been equilibrated with 0.5 M acetate buffer (pH 5.0) containing 1 mM CaC12 and 0.2 mM ZnC12. The suspension is gently stirred for 1 hr at 4°, and the resin is collected by centrifugation and washed with distilled water several times. Crude pseudolysin can be eluted from the resin by the addition of 2 M sodium acetate (pH 10) containing 1 mM CaC12 and 0.2 mM ZnC12. The eluted enzyme solution is concentrated by ultrafiltration. The activity yield is 80-90%. Further purification can be achieved by affinity chromatography using Sepharose-e-aminocaproyl-D-phenylalanine methyl ester as the ligand. TM The column is equilibrated with 20 mM glycine containing 5 mM CaC12. The crude enzyme preparation for affinity chromatography (such as the product of fractionation or adsorption on the ion-exchange resin, as above) is dialyzed against the buffer used for equilibration of the column before being applied to the column. After the column has been further washed with the buffer, purified pseudolysin is eluted with 0.1 M Tris-HCl buffer (pH 8.0). The enzyme is readily crystallized by the addition of ammonium sulfate to give a concentrated solution of the enzyme prepared by either Procedure 1 or2.

Properties of Pseudolysin Enzymatic Properties

The enzymatic properties of pseudolysin have been studied, a6 The optimum pH for hydrolysis of either casein or elastin is pH 7 to 8, and the enzyme is stable at pH 6 to 10 (16 hr at 4°) or at 70° (10 min at pH 7.0) in the presence of Ca >. The enzymatic activity is inhibited by metal chelators (1 mM) such as EDTA and 1,10-phenanthroline but is not affected by other inhibitors of endopeptidases such as diisopropyl fluorophosphate, tosyl-L-phenylalanine chloromethyl ketone, and p-chloromercuribenzoate. The enzyme contains 1 g atom Zn 2+ per molecule, which is essential for activity.18 The metal can be removed, reversibly, by treatment with metal chelators in the presence of Ca2+. Pseudolysin extensively digests various protein substrates such as casein, hemoglobin, ovalbumin, and fibrin, when they have previously been denatured. The specificity for cleavage of peptide bonds by pseudolysin has been 18K. Morihara and H. Tsuzuki,Agric. Biol. Chem. 39, 1123 (1975).

248

METALLOPEPTIDASES

[ 1 5]

TABLE I R A T E OF HYDROLYSIS OF SYNTHETIC SUBSTRATES BY PSEUDOLYSIN a

Substrate b

Rate (/zmol/min/mg enzyme)

Z-Ala-Gly + Leu-Ala Z-Ala + L e u - A l a Z - G l y + Leu-Ala Z-Gly + Leu-Gly Z-Gly + Leu-NH2

5280 1130 410 30 8

a The reaction mixture contained 2.5 m M peptide, 0.1 M Tris-HC1 (pH 7.0), 2.5 m M CaC12, and a suitable amount of enzyme, which was kept at 40 °. + shows the bond split.

studied using oxidized insulin B chain and various synthetic peptides. 18-2° The results indicate that pseudolysin cleaves on the amino side of hydrophobic or aromatic amino acid residues (i.e., with such residues in the PI' position). This is typical of endopeptidases belonging to the thermolysin family. There are some quantitative differences between pseudolysin and thermolysin, however. For example, with synthetic substrates of the general structure Z-Phe-Xaa-Ala, in which Z is benzyloxycarbonyl and the PheXaa bond was cleaved, the order of preference for Xaa was as follows: Phe (1130) > Leu (380), Tyr (300) > Val (240) > lie (130) for pseudolysin. For thermolysin the order was Leu (490) > Phe (310), Ile (310) > Val (190) >> Tyr (10) (the value in parentheses being micromoles peptide hydrolyzed/min/mg enzyme at pH 7.0 and 40°). The activity of pseudolysin against compounds based on Z-Gly-LeuNH2 was markedly accelerated when the amino acid residues at either P2' or P1 or both were replaced with Ala. Also, elongation of the peptide bond to the P2 position of peptide substrates results in a marked increase in peptidase activity (Table I). A fluorogenic substrate, 2-aminobenzoyl-AlaGly+Leu-Ala-4-nitrobenzylamide ( K m = 0.11 mM, kcat = 100 sec -1, at pH 7.2, 25°; + , cleavage site), has been synthesized for assay of pseudolysin, 21 in which the rate of hydrolysis can be conveniently assayed by the increase in fluorescence caused by separation of the fluorogenic and the quenching group (see [2] in this volume). Phosphoramidon (N-a-L-rhamnopyranosyloxy(hydroxyphosphinyl)-Lleucyl-L-tryptophan), an inhibitor of thermolysin, is also an efficient com19 K. Morihara and H. Tsuzuki, Arch. Biochem. Biophys. 114, 158 (1966). s0 K. Morihara and H. Tsuzuki, Arch. Biochem. Biophys. 1416, 297 (1971). 21 N. Nishino and J. C. Powers, J. BioL Chem. 255, 3482 (1980).

[ 15]

PSEt;DOI~VSIN

249

petitive inhibitor of pseudolysin with a Ki value of 40 nM. 22 Pseudolysin is also inhibited competitively21 by synthetic peptides containing hydroxamic acid [HONHCOCH(CHzC6Hs)-CO-Ala-GIy-NH2, Ki = 44 nM; HONHCOCH[CH2CH(CH3)2]CO-Ala-GIy-NH2, Ki = 57 nM], and thiol functional groups [HSCH2CH(CH2C6Hs)-CO-Ala-Gly-NH2, Ki = 64 nM]. These inhibitors contain a ligating group which interacts with the activesite Zn 2+ of pseudolysin. These inhibitors (which are also good competitive inhibitors of thermolysin) can be used as ligands for affinity chromatography of pseudolysin. Thermolysin is irreversibly inhibited by C 1 C H 2 C O - H O L e u - O C H 3 , but pseudolysin is not 21 ( H O L e u is N-hydroxyleucine). The tripeptide analog C1CH2CO-HOLeu-Ala-Gly-NH2 can inhibit pseudolysin irreversibly) k3/Ki = 0.092 M -1 sec-1), 3 as well as thermolysin (k3/Ki = 40 M -1 sec-l). 3 This may indicate that the binding pocket of pseudolysin is bigger than that of thermolysin. Structure

The Mr of pseudolysin is 33,000, and the isoelectric point is 5.9.16,23 The sequence of 301 amino acid residues has been deduced from the nucleotide sequence of the pseudolysin gene. 23'24 Considerable homology can be seen between the amino acid sequence of pseudolysin and that of thermolysin, as shown below. The three-dimensional structure of pseudolysin has been solved at 1.6 /~ resolutionY The overall tertiary structures of pseudolysin and thermolysin are very similar, both having an upper and a lower domain separated by a cleft (Fig. 1). The secondary structure of pseudolysin is summarized in Fig. 2, where a comparison is made with that of thermolysin. 26 The sequence alignment of pseudolysin and thermolysin implied by the structural superposition is also shown in Fig. 2. The pattern of similarity between the proteins is reflected in conserved amino acid residues. In the aminoterminal domain, residues 1-135 (1-137 in thermolysin), 19% of the residues are identical. In the region encompassing the two-active site helices, residues 136-180 (138-182 in thermolysin), the identity in sequence is 48%. In the remainder of the two proteins, residues 181-301 (183-316 in thermolysin), 29% of the residues are identical. In total, 28% of the residues are identical. 22K. Morihara and H. Tsuzuki, Jpn. J. Exp. Med. 48, 81 (1978). 23R. A. Bever and B. H. Iglewski, J. Bacteriol. 170, 4309 (1988). 24j. Fukushima, S. Yamarnoto, K. Morihara, Y. Atsumi, H. Takeuchi, S. Kawamoto, and K. Okuda, J. Bacteriol. 171, 1698 (1989). 25M. M. Thayer, K. M. Flaherty, and D. B. McKay,J. Biol. Chem. 266, 2864 (1991). 26B. W. Manhews, J. N. Jansonius, P. M. Colman, B. P. Schoenborn, and D. Duporque, Nature (London) New Biol. 238, 37 (1972).

250

[ 151

METALLOPEPTIDASES

C FIG. 1. Schematic drawings of pseudolysin (left) and thermolysin (right), oriented to show the active-site clefts. Helices are drawn as cylinders, strands as ribbons; the active-site zinc is shown as a sphere. (From Thayer et al., 25 with permission.)

The area of greatest similarity (residues 136-180 in pseudolysin) spans the active-site cleft of pseudolysin which includes the three Zn 2+ ligands of His-140, His-144, and Glu-164; the active site Glu-141; and the three amino acids Tyr-155, His-223, and Asp-221, which are thought to comprise the substrate-binding region of pseudolysin. All these amino acids are conserved in thermolysin. Site-directed mutagenesis of pseudolysin at Glu14127 and His-22327'28 indicated that both amino acid residues are key ones for catalysis. The m a j o r difference between the two enzymes in tertiary structure is that the substrate-binding cleft is m o r e open in pseudolysin than in thermolysin. This m a y account for the difference in specificity against amino acid residues at the P I ' position, and in the effects of inhibitors as mentioned above. O t h e r differences include the presence of four cysteine residues in pseudolysin at positions 30, 58, 270, and 297, whereas thermolysin contains no cysteine. The crystal structure shows that in pseudolysin the cysteines 27S. Kawamoto, Y. Shibano, J. Fukushima, N. Ishii, K. Morihara, and K. Okuda, Infect. I m m u n . 61, 1400 (1993). 28K. McIver, E. Kessler, and D. E. Ohman, J. Bacteriol. 173, 7781 (1991).

[ 151

PSEUDOLYSlN

251

THERM PSI~UD

ITGTSTVGVGRG_( .... V L G D Q K N ..... ) I NT(TY STY) Y Y L Q D ( N T R G D G ) IFTYDAKlrRTT(LPGS. ) LW ..... A E A G G P G ( G N Q K I G K Y T Y O S D Y G P ) L I V ( . NDR. ) CEMDD( . . GN. • ) V I T V D M N S S T D ( D S K T T ) P F

THERM PSEUD

AD(,. A D N Q F F A S Y D . . ) A P A V D A H Y Y A G V T Y D Y Y K N V H N R RF(ACPTNTYKQVNGAY)SPLNDAHFFGGVVFKLYRDWFGT(

•~

'e"

=.* ==*

, .

,

==~

='*

--

(LSYDGNNA) AIRSSVH~SQGYNN~FW~SEM .SPLTH.. ) K L Y M K Y H Y G R S V E N A Y W D G T A M

=='b

55 54

==~--

120 120

THERM PSEUD

V Y G D G ( D G Q ) T F I P L S (GG) I D V V A H E L T H A V T D Y T A G L I Y Q N E S G A I N E A I SDI F G T L V E P Y A N (KNP) DWE L F G D G ( AT. ) M F Y P L V (S . ) L D V A A H E V S H G F T E Q N S G L I Y R G Q S G G M N E A F S D M A G E A A E F Y M R (GKN) D F L

187 185

THERM

I G E D V ( Y T P G I S G ) D S L R S M S D P A K Y G (D,) P D H Y S K R ( Y T G T Q D N G G ) V H I N S C l I N K A A Y L I S Q ( G G T H Y G IGYDI ( . KKGS.. ) G A L R Y M D Q P S R D G ( R S ) I D N A S Q Y ( . . Y N O I D . . ) V H H S S G V Y N R A F Y L L A N ( .... SP

252 240

V S V V G ) IGRD~LGKIFYRALTQ~L~PTSI{I~SQLRAAAVQSA~I)L(YOSTSQE) VASVKQAFDAVGVI~ . . . . . O .... ) W D T R K A F E V F V D A N R Y Y W T A T S N Y N H G A C G V I R S A Q N R (.. NYS.. ) A A D V T R A F S T V G V T C P S A L • u • u : •

316 301

PSEUD

THERH PSEUD

FIG. 2. Sequence alignment of thermolysin (THERM) and pseudolysin (PSEUD) implied by superposition of the structures. /3 strands are denoted by arrows, helices by cylinders; identical residues are highlighted with bars between the sequences. For segments of sequence included in parentheses, the structures do not superimpose; hence, sequences in these regions are not aligned. (From Thayer et al., 25 with permission.)

form disulfide bonds with their nearest neighbors. In addition, thermolysin has four calcium binding sites, whereas pseudolysin has only one.

Pathogenic Activity It is now clear that pseudolysin has the potential to contribute in many ways to pathogenesis by P. aeruginosa. 3,29-3l The evidence comes from studies with purified pseudolysin, as well as comparative studies using protease-deficient strains. Nonetheless, pseudolysin is relatively nontoxic, with an LDs0 (50% lethal dose) for mice in the range of 60-400/xg depending on the route of inoculation. Of primary interest, however, is the fact that 29 L. W. Heck, P. A. Alarcon, R. M. Kulahavy, K. Morihara, M. W. Russell, and J. F. Mestecky, J. I m m u n o l . 144, 2253 (1990). 30 L. W. Heck, K. Morihara, W. B. McRae, and E. J. Miller, Infect. l m m u n . 51, 115 (1986). 31 D. R. Galloway, Mol. Microbiol. 5, 2315 (1991).

252

METALLOPEPTIDASES

[ 15]

purified pseudolysin is capable of degrading several molecules of biological significance to the host. These include various complement components, laminin, fibrin, human collagens, human al-proteinase inhibitor, and immunoglobulins of the IgG, IgA, and secretory IgA classes. Furthermore, it is now clear that pseudolysin contributes to the activation of Hageman factor and inactivates human "),-interferon. At the tissue level, it has been shown that pseudolysin is capable of damaging intact pulmonary and corneal tissue and can also destroy epithelial cell junctions. Thus, it seems clear that pseudolysin could easily destroy the host defense mechanism in a number of ways. This may be explained by an "aggressin activity" of pseudolysin, seen when the virulence of protease-deficient strains is increased by the addition of a minute amount of pseudolysin to the experimental animal model of P s e u d o m o n a s infection. 32'33 Although there seems to be little question that the proteolytic activity of pseudolysin contributes to the pathogenesis associated with P s e u d o m o n a s infections, the biochemical basis of elastin degradation remains unresolved. Elastin degradation is of particular significance in pathogenesis since several tissues are composed of elastin and require elastic properties for carrying out their function in the body. For example, lung tissue is composed of approximately 28% elastin and relies on that protein component for expansion and contraction. Furthermore, vascular tissue also contains elastin, which is an important factor in resilience. Consequently, the ability of pseudolysin to destroy elastin is likely to be particularly damaging. Evidence for the destruction of vascular tissue is cited in several studies that suggest that elastase may be responsible for internal organ (including lung) hemorrhages. O t h e r P a t h o g e n E n d o p e p t i d a s e s of T h e r m o l y s i n Family The sequences of the structural genes encoding the Zn-metalloendopeptidases from Vibrio cholerae, 34 Legionella p n e u m o p h i l a , 35 and Enterococcus (formerly Streptococcus) faecalis 36 have been determined, from which the amino acid sequences of the three enzymes were deduced. The sequences show extensive amino acid identity with pseudolysin, especially in the enzymes from V. cholerae (identity, 62%) and L. p n e u m o p h i l a (identity, 52%). The structural identity between each of the three enzymes and pseudolysin 32K. Kawaharajo and J. Y. Homma, Jpn. J. Exp. Med. 45, 515 (1975). 33I. A. Holder and C. G. Haidaris, Can. J. Microbiol. 25, 593 (1979). 34C. C. H/ise and R. A. Finkelstein, Z Bacteriol. 173, 331l (1991). 35W. J. Black, F. D. Quinn, and L. S. Tompkins, J. Bacteriol. 172, 2608 (1990). 36y. A. Su, M. C. Sulavik, P. He, K. K. M~ikinen, P.-L. M~ikinen, S. Fiedler, R. Wirth, and D. B. Clewell, Infect. Immun. 59, 415 (1991).

[ 1 6]

NEPRILYSIN

253

is most pronounced in the regions forming the enzymatic active site of pseudolysin. The residues constituting the active site of pseudolysin have been mentioned above, and all the residues are identical in each of the three enzymes from Vibrio, Legionella, and Enterococcus, except Tyr-215 in the E. faecalis enzyme, coccolysin (EC 3.4.24.30). It is therefore considered that the mechanism of catalysis by these enzymes is the same as that of thermolysin or pseudolysin (see [13] in this volume). It is suggested that each of the three metalloendopeptidases from V. cholerae, 37 L. p n e u m o p h ila, 38 and E. faecalis 39 play a role in the pathogenesis which occurs by infection with the respective bacteria. 37B. A. Booth, T. J. Dyer, and R. A. Finkelstein, in "Advances in Research on Cholera and Related Diarrheas" (R. B. Sack and Y. Zinnaka, eds.), p. 19. KTK Scientific Publ., Tokyo, 1990. 38L. A. Dreyfus and B. H. Iglewski, Infect. lmmun. 51, 736 (1986). 39P.-L. M~ikinen, D. B. Clewell, F. An, and K. K. M~ikinen, J. Biol. Chem. 264, 3325 (1989).

[16l Neprilysin: Assay Methods, and Characterization

Purification,

B y CHINGWEN LI and L o u i s B. HERSH

Introduction Neprilysin (neutral endopeptidase 24.11, E C 3.4.24.11) is a plasma m e m brane-bound zinc-containing enzyme which degrades and inactivates a number of bioactive peptides. Neprilysin is one of the m a n y zinc metallopeptidases (see [13] in this volume). The enzyme was first isolated from porcine kidney brush border as an endopeptidase hydrolyzing the B chain of insulin. 1,2 It is an ectoenzyme composed of a 23-amino acid N-terminal cytoplasmic domain, a 28-amino acid membrane-spanning domain, and an approximately 700-amino acid extracellular domain which contains the active site. The enzyme was rediscovered as an enkephalin degrading peptidase in rat brain and given the trivial name "enkephalinase". 3'4 Neprilysin was subsequently shown to be the protein described as CD10 or " C A L L A " 1M. A. Kerr and A. J. Kenny, Biochem. J. 137, 477 (1974). 2 M. A. Kerr and A. J. Kenny, Biochem. Z 137, 489 (1974). 3 B. Malfroy, J. P. Swerts, A. Guyon, B. Roques, and J. C. Schwartz, Nature (London) 276, 523 (1978). 4 S. Sullivan, H. Akil, and J. D. Barchas, Commun. Psychopharmacol. 2, 525 (1978).

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