Partial purification and characterization of intracellular car☐ypeptidase ofCandida albicans

EXPERIMENTALMYCOLOGY

Partial

11,

115-121(1987)

Purification and Characterization Carboxypeptidase of Candida DAVID

Department of Bioscience and Biotechnology,

of ~ntrace~~u~a~ albicans

A. LOGAN Drexel University, Philadelphia,

Pennsylvania 19104

LOGAN, D. A. 1987. Partial purification and characterization of intracellular carboxypeptidase of Candida albicans. Experimental Mycology 11, 115- 121. Intracellular carboxypeptidase was partially purified from the yeast form of Cundidu nlbicans H-317 by acid dialysis, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Peptidase activity was measured with an enzyme-coupled calorimetric assay. Fractionation by native polyacrylamide gel electrophoresis and Sephacryl S-200 gel filtration indicated the presence of a single peak of carboxypeptidase, with the isoelectric point at pH 4.6 and a molecular weight of 100,000. The pH optimum and apparent K,,, using N-carbobenzoxy-L-phenylalanine-L-leucine (N-Cbz-L-Phe-L-Leu) as substrate were 6.5 and 2 x 10e4 M, respectively. The best substrates for the enzyme were N-CbzAla-X peptides, with N-Cbz-Ala-Leu giving the highest rate of hydrolysis. Substrates that gave 7% or less of the control (N-Cbz-Phe-Leu) rate of hydrolysis were Gly-Leu, Gly-Phe, Ile-Phe, Ile-Met, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. There was no detectable hydrolysis of the following N-Cbz peptides: Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, p-chloromercuribenzoate, benzyloxy-carbonyi-Lphenylalanine chloromethyl ketone, and tosyl-L-phenylalanine chloromethyl ketone, but was not affected by EDTA, tosyl-L-lysine chloromethyl ketone, pepstatin A, leupeptin, bestatin, or antipain. Although secretory proteinases are thought to play a role in the pathogenesis of this organism, the role of this intracellular carboxypeptidase has yet to be determined. D 1987 AC&IGC Press.

Inc.

INDEX

DESCRIPTORS:

Candidu albicans; intracellular

It is clear that proteinases perform an essential role in the control of cellular metabolism. Considerable information on the involvement of proteinases in cellular nutrition, protein turnover, and protection against toxic peptides has been acquired from studies on bacteria (Hermsdorf and Simmonds, 1980) and fungi (Wolf and Holzer, 1980). The importance of these enzymes is reflected in the many studies on proteolysis in yeast reviewed in the past few years (Wolf, 1980; Achstetter and Wolf, 1985). Researchers have exploited Saccharomyces cerevisiae as a model system mainly to study proteinase involvement in fungal metabolism. In contrast, aside from the metal ion-dependent aminopeptidase and dipeptidase (Logan et al., 1983), proteinase B (Farley et al., 1986), and the neu-

carboxypeptidase.

tral proteinase (Logan, 1986) of C~~~~~~ albicans, there is limited information available regarding the intracellular ~r~teolyt~~ system of this yeast. The majority of t attention has been focused on the secretory proteinases of C. albicans (Ruchel et al., 1982) which were first reported by Staib (1965). The idea that extracellular teinases may have a role in the ~at~o~e~esis of G. a2bican.s led to interest in these inducible enzymes; however, reports (Remold et al., 1968; gensen, 1971; Chattaway eC al., maine and Tellefson, 1981) have prevented a clear understanding of the relationship between secretion of proteinase and pathsgenicity. Research in my laboratory has focused attention on the intracellular proteinases of C. albicans. This report describes the puri115 0147-5975187 $3.00 Copyright 0 1987 by Academic Press, inc. All rights of reproduction in any form reserved.

116

DAVID

A. LOGAN

fication and characterization of an intracellular carboxypeptidase of this opportunistic pathogen. MATERIALS

AND

METHODS

Chemicals. The following chemicals were purchased from Sigma Chemical Company: N-carbobenzoxy peptides, Lamino acid oxidase (crude type l), horseradish peroxidase (type ll), O-dianisidinediHC1, and most of the proteinase inhibitors. Leupeptin and pepstatin A were purchased from United States Biochemical Corporation. Most of the polyacrylamide gel electrophoresis reagents were purchased from Bio-Rad Laboratories. Organism growth and cell extract preparation. The organism used throughout all the studies was Candida albicans H317, a clinical isolate from the Centers for Disease Control, Atlanta, Georgia. The yeast was maintained by monthly transfer on slants of YPG agar containing (%, w/v): yeast extract (Difco), 0.3; Bactopeptone (Difco), 1; glucose, 2; agar (Difco), 2. The growth medium used for all experiments was Bacto yeast nitrogen base without amino acids and ammonium sulfate (Difco). The medium was sterilized by filtration, and sterile solutions of glucose as carbon and energy source (final concentration l%, w/v) and ammonium sulfate as nitrogen source (final concentration 0.5%, w/v) were added aseptically. For growth, cells were taken from a YPG agar slant and inoculated into 100 ml fresh medium contained in a 500-ml flask. The culture was incubated at 25°C on a gyrotory shaker (250 rpm) for 24 h. A portion (50 ml) of this culture was inoculated into 5 liters of fresh medium and incubated at 25°C on a gyrotory shaker for 24 h. Under these growth conditions, the cells remained in the yeast form. The cells (24-h culture) were harvested by centrifugation at 5000g for 10 min (4°C). The cell pellet was resuspended with 0.1 M imidazole-HCl (pH 7.0) to give a final volume of 150 ml. Three milliliters of the

cell suspension and 9 g of glass beads (0.45-0.50 mm diameter) were added into each of fifty 20 x 150-mm culture tubes. The cells were disrupted by homogenizing 2 min at top speed with a Vortex mixer (Scientific Industries, Inc.). Glass beads, unbroken cells, and cell debris were removed by centrifugation for 30 min at 20,OOOg. The cell extract was dialyzed against 100 vol of buffer (0.1 M imidazoleHCl, pH 5.3) for 24 h in the cold with a buffer change after the first 3 h. Insoluble material was removed by centrifuging for 20 min at lO,OOOg, and solid ammonium sulfate (Schwarz/Mann Chem. Co.) was added to the supernatant with constant stirring for 1 h at 4°C. The pellet of the 80% (w/v) fraction was dissolved in a small amount of buffer and dialyzed overnight against gel filtration column buffer. Glycerol was added to a final concentration of 20% (v/v) and the extract was stored at - 20°C. Protein determination. Protein was determined by the method of Bradford (1976) using the Pierce-Coomassie protein assay reagent (Pierce Chem. Co.) or by the method of Lowry et al. (1951). The former method was used primarily for samples containing low levels of protein. The standard for both assays was bovine albumin fraction V. Quantitative peptidase activity. The method of Fujita et al. (1972) for quantitating hydrolysis of the N-carbobenzoxy peptides was modified as follows. The reaction mixture consisted of 280 p,l TrisHCl buffer (0.1 M, pH 6.5), lo-20 ~1 crude extract (l-2 mg protein/ml), and 80-90 ~1 distilled water. The preparations were incubated in the buffer for 10 min at 37°C. After the reaction was started by the addition of 18 ~1 of peptide (10 mg/ml, incubated at the same temperature) it was stopped by boiling. It was determined that the substrate was not rate limiting under the assay conditions. The amount of amino acid released was determined as reported

PEPTIDASE

OF Candida

elsewhere (Logan et al., 1983), except that the amino acid oxidase reagent was allowed to react for 60 min. One unit of enzyme activity is defined as the amount of enzyme releasing one nanomole of amino acid per minute. Specific activity is expressed as units per milligram of protein. Native polyacrylamide gel electrophoresislisoelectric focusing. Native polyacrylamide gels (0.5 x 11 cm) were prepared using a modification of Davis’ (1964) method. Gels were used immediately after polymerization. Peptidase activity could be measured following polyacrylamide gel (native) electrophoresis by eluting the enzymes from the gel (Strongin et al., 1976). Gels were sliced into 3-mm segments and each segment was placed into a separate tube containing 280 ~1 of 0.1 M Tris-HCl (pH 7.5) and 18 ~1 N-Cbz-Phe-Leu’ (10 mg/ml). The tubes were incubated at 37°C for 60 min and the amino acid released was determined as described above. The isoelectric pH for carboxypeptidase was determined using the system developed by United States Biochemical Corporation. The p1 marker protein kit consisted of acetylated cytochromes c (~1 = 4.1,4.9, .3, 9.7, and 10.6) which were visible ut staining. The migrations of the standards were recorded, the gels were into 2-mm segments, and enzyme acwas determined as stated above. Gel fiftrationlion-exchange chromatography. Gel filtration was performed using a 1.6 x g&cm column containing Sephacryl S-200 (wet bead diameter, 40-105 km) s equilibrated with 0.1 M imidazoleHCI, 8.05 M NaCl (pH 5.5). The downward flow elution rate during the fractionation was controlled by using a peristaltic pump 1 Abbreviations used: N-Cbz-Phe-Leu, N-carbobenzoxy-L-phenylalanine-L-Ieucine; PMSF, phenylmethylsulfonyl fluoride; PCMB, p-chloromercuribenzoate; ZPCK, benzyloxy-carbonyl+phenylalanine chloromethyl ketone; TPCK, tosyl-L-phenylalanine chloromethyl ketone; TLCK, tosyl+lysine chloromethyl ketone.

117

albicans

(Isco, TRIS). The column was calibrated with standards (Sigma Chemical Co.) and the elution volume was determine lowing the absorbance at 280 nm. The voi volume was determined using tran 2000. Ion-exchange chromatography formed using a 1.0 x 18-c taining DEAE-Sephadex Aameter, 40-120 pm) beads with 0.1 M imidazole-HCl ( protein was eluted with a line NaCl (0.05-0.5 m. RESULTS

Carboxypeptidase specific activity (5 unitslmg protein) was 2.5-fold higher in tracts of stationary cells than in lo rithmic-phase cells (200 units/mg protein). The hydrolysis of N-Cbz-Phe-Leu linear for approximately 20 min, using 20 pg of protein in the reaction mixture. The pH for optimal N-C drolysis in crude extracts

sliced

and peptidase

Methods.

activity

was deter-

A single

When the salt-pr protein was fracti cry1 S-200 column, of N-Cbz-Phe-Leu was seen.

one symmetric hydro

analytical isoelectric polyac electrophoresis, a single ba activity was obtained with an

118

DAVID A LOGAN

plied to a DEAE-cellulose A-50 column previously equilibrated in the same buffer. Protein was eluted with an increase in NaCl concentration (O-O.5 M). Carboxypeptidase began to elute from DEAE-cellulose at about 0.25 M NaCl (Fig. 1). Fractions containing high activity were pooled, dialyzed against buffer to remove the NaCl, and stored in glycerol (20%, v/v) at - 20°C. To determine the rate of heat inactivation, the enzyme was incubated for various times at 50 or 60°C in 0.1 M Tris-HCl (pH 6.5), cooled on ice, and assayed at 37°C. Full carboxypeptidase activity was recovered at 50°C. At 6o”C, the half-time for inactivation was 18 min (Fig. 2). The slope of the In (natural logarithm) of the rate of activity vs time was linear and the apparent first-order rate constant for inactivation was 0.04 min-l (Fig. 2, inset). The effect of pH was determined using 0.05 M glycine-HCl (pH 2.5-4), 0.05 M potassium acetate-HAc (pH 4-6), and 0.05 M Tris-HCl (pH 6-8) buffers. The pH optimum for N-Cbz-Phe-Leu hydrolysis was 6.5 (Fig. 3). The initial rate of peptide hydrolysis was determined at various concentrations of peptide. Using Wilkinson’s (1961) method, the apparent K,,, and V, for N-Cbz-PheLeu hydrolysis was calculated to be 2 x

10h4 M and 20 pmol Leu released min-’ mg protein-‘, respectively. Several N-Cbz peptides were incubated with carboxypeptidase and the rate of amino acid release was compared with N-Cbz-Phe-Leu hydrolysis (Table 1). The best substrates for Candida carboxypeptidase were N-Cbz-Ala-X peptides, with N-Cbz-Ala-Leu giving the highest rate of peptide hydrolysis. The N-Cbz peptides that were poor substrates for the enzyme were Gly-Leu, Gly-Phe, Ile-Phe, IleMet, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. No amino acid release was detected from the N-Cbz peptides Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. The effect of various inhibitors on carboxypeptidase activity was determined (Table 2). Phenylmethylsulfonyl fluoride (PMSF), a potent inhibitor of serine proteinases, completely blocked enzyme activity. However, antipain, also a serine proteinase inhibitor, gave only 10% reduction in peptidase activity. The mercurial compound, p-chloromercuribenzoate (PCMB), reduced activity by 84% at 100 pM, while benzyloxy-carbonyl-L-phenylalanine chloromethyl ketone (ZPCK) and tosyl-L-phenylalanine chloromethyl ketone (TPCK) blocked enzyme activity by 68 and 43%, respectively. Proteolytic activity was not in-

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FIG. 1. DEAE-cellulose A-50 chromatography of carboxypeptidase. Fractions containing high peptidase activity from S-200 column chromatography were pooled, dialyzed, and loaded on the column. Protein was eluted with NaCl (O-O.5 kf) and peptide (N-Cbz-Phe-Leu) hydrolyzing activity was assayed as described under Materials and Methods.

PEPTIDASE

OF Can&& albicans

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TIME (min)

FIG. 2. Heat inactivation of carboxypeptidase. Fractions containing high peptidase activity from DEAE-cellulose chromatography were pooled. Enzyme was incubated at 60°C for the times indicated, rapidly placed on ice, and assayed at 37°C as described under Materials and Methods. The inset is a plot of the In (natural logarithm) of the rate of activity vs time of incubation at 60°C.

hibited by either the carboxyl and metallo proteinase inhibitor, pepstatin, or the metal chelator, ethylenediaminetetraacetate (EDTA). The carboxypeptidase of C. albicans was insensitive to the thiol proteinase inhibitor, leupeptin, the trypsin and papain inhibitor, tosyl-L-lysine chloromethyl ketone (TLCK), as well as the aminopeptidase inhibitor, bestatin. DISCUSSION

A single class of intracellular carboxypeptidase was detected in C. albicans. Fractionation of the enzyme by native polyacrylamide gel electrophoresis, molecular sieve chromatography, and ion-exchange chromatography produced symmetrical peaks with no shoulders. In addition, inactivation at 60°C over time was linear. However, it is possible that enzymes with closely related properties were present but not detected under the conditions employed.

119

A number of N-Cbz peptides were teste against Candida carboxypeptidase to gai information about its substrate speciality (Table 1). Substrates that gave the highest rate of hydrolysis were N-Cbz-Ala tides while, except for Gly-Leu, a Phe, there was no detectable hydro~y any N-Cbz-Gly-X peptides test disadvantage of the assay system this study is that only peptides c residues that are substrates for amino acid oxidase can be tested. A different assay system could perhaps be employed to gain a better picture of the substrate s~e~ifi~~ty of the enzyme. Candida peptidas tested against several inhibitors to mine its classification. Peptidase was inhibited by PMSF, P and TPCK, (Table 2); ther zyme is probably similar to carbo dase Y (EC 3.4.12.-), a we serine sulfhydryl exopeptidase of S. cerevi-

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FIG. 3. The effect of pH on the activity of carboxypeptidase. Fractions containing high peptidase activity from DEAE-cellulose chromatography were pooled. The initial rate of N-Cbz-Phe-Leu hydrolysis was determined after incubation at 37°C for 10 min in the various buffers as indicated in the figure.

120

DAVID

A. LOGAN

TABLE 2 siae (Wolf, 1980; Achstetter and Wolf, Effect of Various Inhibitors on the Activity of 1985). Candida albicans Carboxypeptidase” The enzyme of the present study appears Compound nmol min-’ % to be quite different from the well-charactested mg protein- lb Inhibition terized secretory proteinases of C. albicans that are induced by growth on albumin as a PMSF 200 (14,000) 98 1,400 (8,800) 84 nitrogen source or the extracellular kera- PCMB 4,900 (15,100) 68 tinolytic enzyme (Hattori et al., 1984; Ru- ZPCK 43 TPCK 9,800 (17,200) chel, 1981) produced in medium containing TLCK 13,500 (15,600) 13 human stratum corneum as the source of Leupeptin 13,800 (15,600) 11 nitrogen. The latter proteinase is characterAntipain 14,000 (15,600) 10 15,100 (15,600) 3 ized by a molecular weight of 42,000, an EDTA Bestatin 16,900 (15,600) 0 isoelectric point of 4.5, and a pH optimum 17,200 (16,100) 0 of 4.0, and it is inhibited by pepstatin and Pepstatin A chymostatin (Negi et al., 1984). The physia N-Cbz-Phe-Leu was the substrate. All inhibitors ological function of the intracellular car- were at 100 uLM and were incubated with the enzyme boxypeptidase of C. albicans remains to be (0.5 ug) for 10 min prior to addition of substrate. The number in parentheses represents the activity determined. Preliminary results show an of bthe solvent control (no inhibitor). almost threefold increase in specific activity levels when cells are transferred into nitrogen-free medium and activity levels vary in response to addition of different carbon sources to stationary cells (unpublished observations). However, previous TABLE 1 studies in S. cerevisiae (Saheki and Holzer, Hydrolysis of Various Peptides by Carboxypeptidase of Candida albicansa 1975) and C. albicans (Farley et al., 1986) have revealed the presence of endogenous Ratec inhibitors in these organisms. Whether an N-Cbz peptideb (nmol min-’ mg protein-‘) inhibitor of the intracellular carboxypeptiPhe-Leu 20,267 f 170 (100) dase is present in Candida remains to be Ala-Leu 24,633 -t 665 (122) determined. Therefore, interpretation of Ala-Phe 12,167 2 94 (60) any data concerning specific activity levels Ala-Ile 8,000 k 356 (40) Ala-Met 7,433 2 170 (37) must be tempered with this consideration. Ala-Val 7,533 + 377 (37) Studies in this laboratory are presently Phe-Met 9,233 i 125 (46) concerned with understanding the role of Val-Phe 1,397 ? 29 (7) proteinases and peptidases in the cellular Ile-Phe 1,097 i 45 (5) metabolism of this fungus. Ile-Met 903 f 19 (4) Glu-Phe Glu-Tyr Pro-Phe Gly-Leu Gly-Phe

507 400 103 207 207

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a There was no detectable hydrolysis of Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. b Peptide concentration was 1.0 mM and protein concentration was 0.5 kg. c All rates represent the means and standard deviations of triplicate determinations. The number in parentheses represents the percentage of N-Cbz-PheLeu (control) hydrolysis.

ACKNOWLEDGMENT This work was supported by a Biomedical Research Support Grant from Drexel University to D.A.L. REFERENCES ACHSTETTER, T., AND WOLF, D. H. 1985. Proteinases, proteolysis and biological control in the yeast Sacchavomyces cevevisiae. Yeast 1: 139-157. 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.

PEPTIDASE

OF

BUDTZ-JORGENSEN, E. 1971. Proteolytic activity of Gandidu spp. as related to the pathogenesis of denture stomatitis. Subouruudiu 12: 266-271. CHATTAWAY, F. W., ODDS, F. C., AND BARLOW, A. J. E. 1971. An examination of the production of hydrolytic enzymes and toxins by pathogenic strains of Candida albicans. J. Gen. Microbial. 67: 255-263. DAVIS, B. J. 1964. Disc electrophoresis II. Method and application to human serum proteins. Ann. N. Y. Acad. Sci. 121: 404-427. FARLEY, P. C. SHEPHERD, M. G., AND SULLIVAN, P. A. 1986. The purification and properties of yeast proteinase B from Candida albicans. Biochem. J. 236: 177-184. Fu~rr~, M., PARSONS, D. S., AND WOJNAROWSKA, F. 1972. Qligopeptidases of brush border membranes of rat small intestinal mucosal cells. J. Physiol. 227: 337-394. GERMAINE, 6. R., AND TELLEFSON, L. M. 1981. Effect of pH and human saliva on protease production by Candida albicans. Infect. Immun. 31: 323-326. HATTORI, M., YOSHIURA, K., NEGI. M., AND OGAWA, H. 1984. Keratinolytic proteinase produced by Candida albicans. Sabouraudia 22: 175- 183. HERMSDORF,~. L., ANDSIMMONDS,~. 1980.Roleof peptidases in utilization and transport of peptides by bacteria. In Microorganisms and Nitrogen Sources (J. W. Payne, Ed.), pp. 301-334. Wiley, New York. LOGAN, D. A. 1986. Neutral proteinase activity of Cundida albicans. Exp. Mycol. 10: 157-160. LOGAN, D. A,, NAIDER, E, AND BECKER, J. M. 1983. Peptidases of yeast and filamentous forms of Candida albicans. Exp. Mycol. 7: 116-126.

Candida albicans

121

LOWRY, 0. H., ROSEBROUGH, N.J., FARR, A. L, AND RANDALL, R.9. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. NEGI, M., TSUBO~, R., MATSUI, T., AND OGAWA, H. 1984. Isolation and characterization of proteinases from Cundida albicans: Substrate specificity. J. Invest. Dermatol. 83: 32-36. REMOLD, H., FASOLD, H., AND STAIB, F. 1968. Pm% cation and characterization of a proteolytic enzyme from Candidu albicans. Biochim. Biophys. Acta 167: 399-406. RWCHEL, R. 1981. Properties of a purified proteinase from the yeast Candidu albicans. Biochim. Biophys. Acta 659: 99-113. RUCHEL, R., TEGELER, R., AND TRQST, M. 1982. A comparison of secretory proteinases from different strains of Cundida ulbicans. Sabourattdia 20: 233-244. SAHEIU, T., AND HOLZER, H. 1975. Proteolytic activities in yeast. Biochim. Biophys. Acta 384: 203--214. STAIB, l? 1965. Serum-proteins as nitrogen source for yeast-like fungi. Sabouraudia 4: 187- 193. STRONGIN, A. Y., AZARENKOVA, N. M., VAOANO~A, T. I., LEVIN, E. ID., AND STEPANQV, V. Visualization of leucineaminopeptidase activity after acrylamide gel electrophoresis. Anal. Biothem. 74: 597-599. WILKINSON, G. N. 1961. Statistical estimations in enzyme kinetics. Biochem. J. 80: 324. WOLF, D. H. 1980. Control of metabolism in yeast and other lower eukaryotes through action of proteinases. Adv. Micro. Physiol. 21: 267-338. WOLF, D. H., AND HOLZER, H. 1980. PrOteOIysis ifi yeast. In Microorganisms and Nitrogen Sources (J. W. Payne, Ed.), pp. 431-458. Wiley, New York.