Purification and properties of three intracellular proteinases from Candida albicans

Biochimica et Biophysica Acta 881 (1986) 229-235

229

Elsevier BBA 22251

Purification and properties of three intracellular proteinases from Candida albicans Francisco Portillo * and Carlos Gancedo Departamento de Bioqulmica, Facultad de Medicina UAM and lnstituto de lnvestigaciones Biombdicas CS1C, 28029 Madrid (Spain)

(ReceivedNovember12th, 1985)

Key words: Proteinase;Dipeptidase;Aminopeptidase;Asparticproteinase; (C. albicans)

Three intracellular proteinases termed A, B and C were purified to homogeneity from the unicellular form of the yeast Candida albicans. Enzyme A is an aspartic proteinase that acts on a variety of proteins. Its optimal pH is around 5 and it is displaced to 6.5 by KSCN. It is not significantly inhibited by PMSF, TLCK (Tos-Lys-CHCI2) or soybean trypsin inhibitor but it is inhibited by pepstatin. Its molecular weight is 60000. Enzyme B is a dipeptidase that acts on esters or on dipeptides without blocks in either the carboxyl or amino ends. Its pH optimum is around 7.5 and the molecular weight is 57 000. It is inhibited by PMSF, TLCK and DANME (N2Ac-Nle-OMe). Proteinase C is an aminopeptidase with an optimum pH around 8. Its molecular weight was 67000 when determined by SDS gel electrophoresis and 243000 when determined by gel filtration. It is active towards dipeptides in which at least one amino acid is apolar and is not active when the N-terminal amino acid is blocked. It is inhibited by EDTA or o-phenanthroline and activated by several divalent cations.

Introduction Candida albicans is an opportunistic pathogen, the clinical importance of which is increasingly appreciated. Several basic problems related to the physiology of Candida, e.g., the control of its yeast-mycelium dimorphism or the mechanisms of invasion of the host, remain unsolved until now. Hydrolytic enzymes have received attention in some cases either as possible agents of the pathogenicity [1] or as possible tools in the diagnosis of

* To whom correspondence should be addressed at: Departamento de Bioqulmica, Facultad de Medicina UAM, Arzobispo Morcillo4, 28029 Madrid, Spain. Abbreviations: PMSF, phenylmethylsulfonylfluoride; TLCK, tosyl-L-lysinechloromethylketone; DANME, diazoaeetyl-DLnorleucine methyl ester; TPCK, tosyl-L-phenylalaninechloromethyl ketone; Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine.

candidosis [2] but to our knowledge only the purification of an external proteinase has been reported [2-4]. However, little attention has been given to intracdlular proteinases that in Saccharomyces cerevisiae play an important role in the life of the organism (for a review see Ref. 5). During a study of certain aspects of the physiology of Candida we have identified three intracellular proteinases: an aspartic proteinase, a dipeptidase and an aminopeptidase. In this work we describe the purification and characterization of these proteinases.

Material and Methods Chemicals. Most of the substrates used to measure proteolytic activity and auxiliary enzymes were from Sigma, St. Louis, MO, U.S.A. DEAE-cellulose (DE-52) was from Whatman (U.K.). Con-

0304-4165/86/$03.50 © 1986 ElsevierSciencePublishers B.V. (BiomedicalDivision)

230 canavalin A-agarose was from Pharmacia (Uppsala, Sweden), hydroxyapatite and Bio-Gel A-1.5 from Bio-Rad (Richmond, CA, U.S.A.). Diaflo membranes were from Amicon (Lexington, MA, U.S.A.). Yeast strains and cultivation conditions. A clinical isolate from C. albicans was used (kindly provided by Dr. M. Sousa, Centro Ram6n y Cajal, Madrid). The organism was grown on 1% glucose/ 1% yeast extract until the stationary phase, harvested by centrifugation, washed with 50 mM Tris-HCl (pH 8) and stored at - 2 5 ° C until use. Preparation of cell extracts. 40 g of cells (wet wt.) were thawed and brought up to 90 ml with 50 mM Tris-HCl (pH 8). 170 ml of glass beads (0.5 mm diameter) were added and the mixture was shaken for 5 rain in a Vibrogen cell mill (E. Biihler, Ttibingen, F.R.G.). The temperature of the vessel was kept low by continuous circulation of cold water. The glass beads were separated by filtration, washed with 30 ml of the same buffer, and then the wash was added to the filtrate. The cell extract was adjusted to pH 8 with 1 M Tris and was centrifuged at 1500 × g for 10 min. The supernatant was used for purification of proteinase C. For purification of proteinases A and B the supernatant was centrifuged for 90 min at 110 000 × g. The supernatant of this step was then used for further fractionation. Enzymatic assays. The proteolytic activities present in extracts were measured with different substrates as indicated below. Reactions were carried out at 30°C in a final volume of 0.5 ml. Values given are corrected for spontaneous hydrolysis of the substrates determined in parallel under identical conditions. Activity on azocasein. The method of Fritz and Hochstrasser [6] was used. The reaction mixture contained 1% azocasein, 50 mM citrate-phosphate (pH 5) and an adequate amount of enzyme. After 10 min incubation, 0.5 ml 10% trichloroacetic acid were added and after centrifugation the absorbance of the supernatant was read at 360 nm. One unit is defined as the amount of the enzyme that in these conditions causes an increase in absorbance of 0.1. Activity on L-leucine p-nitroanilide. The reaction mixture contained 50 mM Tricine (pH 8), 0.5 mM L-leucine p-nitroanilide (stock solution 5 mM in

N, N-dimethylformamide) and an adequate amount of enzyme. Increase in absorbance at 405 nm was followed in a spectrophotometer. The molar extinction coefficient for p-nitroanilide is 9.62.103 M - 1. c m - 1. One unit is defined as the amount of enzyme that liberates 1 /~mol p-nitroanilide per min [7]. Activity on benzyloxycarbonyl-L-amino acid pnitrophenyl ester (Z-L-amino acid ONp). The procedure of Martin et al. [8] was used following hydrolysis of the ester at 400 nm. The assay mixture contained 50 mM Tricine buffer (pH 8), 50 #1 methanol, 0.1 mM ester (stock solution 5-10 mM in dimethyl sulfoxide) and the appropriate amount of enzyme. One unit is defined as the amount liberating 1/Lmol p-nitrophenol per minute under the conditions used. An E400 of 0.1-10 4 M -1. c m - 1was used for p-nitrophenol. Ammonium ions, imidazole or mercaptoethanol interfered with the assay. Activity on peptides. The assay was performed according to Wolf and Weiser [9]. The assay mixture contained 0.1 M potassium phosphate buffer (pH 7), 40/lg o-dianisidine, 10 ktg peroxidase, 0.1 mg L-amino-acid oxidase (Sigma type IV), 10 mM substrate and the purified enzyme. The reaction was followed at 405 nm. One unit is defined as the amount of enzyme liberating 1 /~mol of L-amino acid per min in the conditions used. The change in absorption corresponding to the oxidation of 1 /~mol L-leucine was used as the standard, and all the results referred to it. Other techniques. Polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate was carried out as described by Laemmli [10]. For molecular weight determination the following markers were employed: phosphorylase b ( M r 94000), albumin ( M r 67000) ovalbumin ( M r 43 000) and carbonic anhydrase ( M r 30 000). Gels were stained with 0.1% Coomassie brilliant blue and destained with 50% methanol/10% acetic acid. Protein was assayed as described by Lowry et al. [11]. Results and Discussion

Purification of the enzymes All the operations described were carried out at 4°C. Activities were followed during the purifica-

231 tion with azocasein (proteinase A), benzyloxycarbonyl-L-phenylalanine p-nitrophenyl ester (proteinase B) and t-leucine p-nitroanilide (proteinase C). Initial specific activities ( m U / m g protein) were: 20 for A, 40 for B and 40 for C. Proteinase A. Step 1: chromatography on DAE-cellulose. 330 ml of extract (see Material and Methods) were applied to a DEAE-cellulose column (30 x 2 cm) previously equilibrated with 20 mM phosphate buffer (pH 7). The column was washed with the same buffer containing 0.1 M KC1 until the absorbance at 280 nm was less than 0.1. Then the column was eluted with a gradient of KC1 from 0.1-0.5 M in the equilibration buffer. Activity appeared at 0.2 M KC1. Step 2: chromatography on concanavalin A-agarose. The active fractions of the previous step (160 ml) were applied to a column of concanavalin A-agarose (5 × 1 cm) equilibrated with a mixture containing 20 mM phosphate/0.5 M NaC1/1 mM MgCI2/5 mM CaC12 (pH 7). The column was washed with the same mixture until an absorbance of 0.07 was reached and then it was eluted with 0.5 M a-methyl mannoside in the same buffer. Active fractions (11.5 ml) were pooled and concentrated with a Diaflo UM10 membrane to a final volume of 2 ml. The enzyme was purified about 1000-times with a yield of 20% and appeared homogeneous in SDS-polyacrylamide electrophoresis (Fig. 1A). Proteinase B. Step 1: chromatography on DEAE-cellulose. 155 ml of extract (see Material and Methods) were applied to a DEAE-cellulose column (30 × 2 cm) equilibrated with 20 mM TrisHCI (pH 8). The column was washed with the same buffer containing 0.1 M KC1 until the absorbance was inferior to 0.1. A linear gradient of KCI from 0.1-0.3 M in the same buffer was applied to the column. The peak of activity appeared at 0.16 M KC1. Step 2: ammonium sulfate fractionation. Active fractions of previous step were pooled and brought to 60% saturation with solid ammonium sulfate. After stirring for 60 min the suspension was centrifuged at 15 000 × g for 30 min and the precipitate was discarded. Ammonium sulfate was added to 90% saturation, stirred for 60 rain, and the suspension was centrifuged at 15 000 x g for 30 min. The precipitate was suspended in 20 mM Tris-HC1 (pH 8).

Step 3: gel filtration on Sephadex G-150. The suspension from the previous step was applied to a Sephadex G-150 column (160 × 1.8 cm) previously equilibrated with 20 mM Tris-HC1 (pH 8); the same buffer was used to elute the column. Active fractions were concentrated 5-times with a Diaflo UM10 membrane to a final volume of 3 ml. Step 4: chromatography on hydroxyapatite. The concentrated fractions were applied to hydroxyapatite column (10 × 2 cm) equilibrated with 0.2 M Tris-HC1 (pH 8). The column was washed with 0.1 M KHEPO 4 until the effluent presented an absorbance of 0.02. A continuous gradient from 0.1-0.4 M K H 2 P O 4 was applied to the column. Activity was eluted in a single peak ~around 0.14 M phosphate. Active fractions were pooled and concentrated as in step 3 to a final volume of 2 ml (about five-times concentration). The purification was 1200-times and the yield about 40%. Fig. 1B shows a scan of an SDS gel electrophoresis of the preparation. Proteinase C. Step 1: chromatography on DEAE-cellulose. 150 ml of extract prepared as described in Material and Methods were applied to a DEAE-cellulose column (30 × 2 cm) equilibrated with 20 mM Tris-HCl (pH 8). The column was washed with the same buffer until the absorbance was equal t o that of the original buffer. Then a linear KC1 gradient from 0-0.35 M in the same buffer was applied. Activity eluted at 70 mM KC1. Step 2: chromatography on hydroxyapatite. The active fractions of the previous step were pooled and applied to a 12 × 1.5 cm column filled with hydroxyapatite equilibrated with 0.2 M Tris-HC1 (pH 8). The column was washed with the same buffer and elution was carried out with a linear gradient of phosphate from 0-0.4 M. Activity appeared at 0.1 M phosphate. The active fractions were concentrated to one-tenth of their original volume (final volume about 2 ml) with a Diaflo XM100 membrane. Step 3: gel filtration on Bio-Gel A-1.5. The concentrated fractions were filtered through a column of Bio-Gel A-1.5 (80 × 1.2 cm) equilibrated with 20 mM Tris-HCl (pH 8). Elution was carried out with the same buffer. The active fractions were concentrated 20-times as in the previous step and ZnSO 4 was added to a final concentration of 0.1

232

B

C

20

o E

30.000

43D00 68000 94D~0 30.000

43000 68.000 94000 30.000

mM. The enzyme was purified about 1000-times with a yield of 20%. Fig. 1C shows an electrophoretogram of the purified preparation.

Properties of the enzymes Molecular weight. The molecular weight of the proteinases was determined by gel filtration on Sephadex [12] and by gel electrophoresis in the presence of SDS [13] (Fig. 1). In the case of proteinases A and B the results obtained with both methods were equivalent (60000-55 000 for A and 57 000-54 000 for B). In the case of proteinase C, however, the molecular weight found by gel filtration was 243 000 and that found by electrophoresis was 67 000. It seems therefore that while A and B are monomeric proteins, C either forms aggregates or is a multimeric protein.

TABLE I SUBSTRATE SPECIFICITY OF P U R I F I E D PROTEINASE A F R O M CANDIDA A L B I C A N S Activity on azocasein, azocoll or hide powder azure was measured as described in Material and Methods for azocasein. Activity on hemoglobin and bovine serum albumin were assayed as previously described [22]. 15 /,tg of purified enzyme were used in the assays. Substrate

Activity ( u n i t s / m g protein)

Azocasein

7 880

AzocoU Hide powder azure Hemoglobin Bovine serum albumin

7 470 6 530 6 330 < 150

4~000

68000 9~,000

Fig. 1. Electrophoretic patterns of purified proteinases A, B and C. Electrophoresis was carried out as indicated in Material and Methods. Positions of the markers and their molecular weights are given. 50 /~g protein were applied for proteinase A and C and 100 /*g in the case of proteinase B.

Substrate specificity. Proteinase A was active on denatured haemoglobin, azocasein, azocoll and hide powder azure but it was without significant activity towards bovine serum albumin (Table I). The activity on azo-substrates cannot be directly compared with that on hemoglobin or albumin TABLE II SUBSTRATE SPECIFICITY OF P U R I F I E D PEPTIDASES F R O M C. A L B I C A N S Activities are expressed relative to the rate of L-Leu-Leu hydrolysis. Substrate concentrations were 10 mM. Assays as described in Material and Methods, with the enzymes purified as described in the text. Substrate

Relative activities proteinase B

L-Leu-Leu L-Phe-Phe L-Leu-Phe L-Val-Phe L-Ala-Phe L-Phe-Leu L-Gly-Phe L-Phe-Val L-Gly-Leu L-Phe-Gly r-Lys-Leu L-Phe-Phe-Phe L-Phe-Leu-amide L-Gly-Phe-Phe L-Leu-Gly-fl-naphthylamide N-Ac-L-Phe-Phe Z-L-Val-Phe Z-L-Gly-Phe

< < < < < < < <

1 1 1 0.8 0.6 0.6 0.3 0.2 0.08 0.05 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01

proteinase C 1 0.5 0.7 0.5 0.5 0.5 0.4 0.5 0.4 0.3 0.05 0.5 0.3 0.2 0.2 < 0.01 < 0.01 < 0.01

233

due to the different assay methods. The low activity on bovine serum albumin has also been observed in the case of a neutral endoproteinase from S. cerevisiae (proteinase B) [14]. No activity was found towards synthetic anilides. This behaviour is characteristic of the majority of fungal acid proteinases [15]. Proteinase B was assayed against a variety of different substrates. The enzyme was active against dipeptides that had both their carboxyl and amino groups free (Table II). No activity was observed against tripeptides or proteins. Table III shows the results obtained with different esters. Although no definite conclusions can be reached due to the limited number of substrates tested, it appears that good esterolytic activity is observed when the amino acid involved is nonpolar and unbranched. The difference between Z-Leu-ONp and Z-IleONp is illustrative of this point. Proteinase C was active towards dipeptides in which at least one of the components was apolar (Table II). The enzyme was not active when the N-terminal amino acid was blocked. It presented esterolytic activity towards L-leucine p-nitroanilide, although not towards the nitroanilides of L-phenylaniline, L-alanine, L-lysine and L-valine. Influence of p H and inhibitors on the activity of the proteinases A, B and C. Optimal pH for activities was 4-5.5 for proteinase A, 7.5-8 for proteinase B and 8-8.5 for proteinase C. Chaotropic agents such as KSCN displaced the pH optimum of proteinase A to 6.5 (Fig. 2). This displacement was observed with the purified enzyme as well as with crude extracts. Table IV shows the effects of a series of inhibi-

tors on the activity of the different proteinases under study. Proteinase A was not significantly inhibited by PMSF or soybean trypsin inhibitor, thus indicating that serine is not implicated in its active center. Histidine also seems not to be part of the active center, as shown by the lack of effect of TLCK. Antipain partially inhibited proteinase A and pepstatin was strongly inhibitory. Inhibition by this pentapeptide indicates the presence of a carboxyl group of an aspartic or glutamic acid at the active site of the enzyme [16]. Proteinase B possesses a serine residue in its active center, as shown by its inhibition by PMSF; however, it is not a trypsin-like proteinase, as shown by the lack of effect of soy-bean trypsin inhibitors. The obsrved inhibition by TLCK and TPCK indicates the existence of a histidine residue necessary for activity. Inhibition by DANME suggests that a carbonyl residue may be implicated in the catalytic action. Inhibition by miconazole and econazole, two imidazole derivatives used in the treatment of candidosis [17] may be explained by the similarities of part of their structures with that of the substrate. Ketoconazole, another antibiotic of the same family but with a different structure, was without effect. 1200

I 0 O0

800

600

J

TABLE III SPECIFICITY OF THE ESTERASE ACTIVITY OF PROTEINASE B

400

The purified enzymatic preparation was used and the assay was carried out as described in Material and Methods. The activity towards Z-L-Phe-ONp was taken as 10070.

200

Ester

Relative activity (%)

Z-L-Phe-ONp N-Ac-DL-Phe-ONp Z-L-Leu-ONp Z-L-Gly-ONp Z-L-Ile-ONp

100 70 63 3 1

i

iI 0~1/ I I % %2

3

4

,5

6 7 pH

8

9

Fig. 2. pH-dependence of the activity of proteinase A from Candida albicans. Proteinase A was assayed as described in Material and Methods with azocasein as substrate, in the absence (O) or the presence (e) of 0.5 M KSCN.

234 Proteinase C is i n h i b i t e d b y E D T A a n d o - p h e n a n t h r o l i n e a n d is activated by divalent cations, the more powerful b e i n g Z n 2÷ or Co 2+, that stimulated activity 6-times at 1 mM.

Final remarks The results o b t a i n e d allow proteinase A to be classified as a n aspartic proteinase (EC 3.4.23.6). Its affinity for c o n c a n a v a l i n suggests that it is a glycoprotein, a feature shared b y m a n y fungal acid proteinases [15]. Proteinase A from Candida is similar in molecular weight, p H o p t i m u m a n d the absence of a serine residue in its active center to the proteinase A from S. cerevisiae [18]. However, we did not find a high molecular weight i n h i b i t o r in extracts as is f o u n d in S. cerevisiae [19]. Proteinase A from Candida is different from the external acid proteinase purified from this yeast [3,4], b o t h in molecular weight a n d in p H o p t i m u m . The behavior of proteinase B indicates that it m a y be classified as a dipeptidase (EC 3.4.13). This enzyme differs from the dipeptidase described in S. cerevisiae. T h e baker's yeast enzyme prefer-

entially cleaves the dipeptide G l y - L e u which is a poor substrate for the Candida enzyme (Table II). The specificity of proteinase C allows it to be considered as an a m i n o p e p t i d a s e (EC 3.4.11). One a m i n o p e p t i d a s e described in S. cerevisiae, the a m i n o p e p t i d a s e I of Frey a n d R S h m [20] (app a r e n t l y equivalent to a m i n o p e p t i d a s e II of M a s u d a et al. [21]), is also activated by Z n 2 ÷ but presents a much higher molecular weight (640 000) than that reported here for the Candida enzyme. The results presented show that the proteolytic e q u i p m e n t of C. albicans is different from the one described for S. cerevisiae although it is still too early to allow a definitive comparison. The use of an increased range of substrates is likely to increase the n u m b e r of proteolytic activities detected in Candida, as occurred in S. cerevisiae [5].

Acknowledgements This work was partly supported by a grant of the C o m i s i o n Asesora Cientifica y T6cnica.

References TABLE IV INHIBITION OF THE ACTIVITY OF PROTEINASES A, B AND C BY DIFFERENT COMPOUNDS Proteinases A, B and C were assayed with azocasein, Z-L-PheONp and L-leucinep-nitroanilide, respectively, as substrates as described in Material and Methods, with the addition of the different inhibitors as indicated. Values of inhibition were calculated with reference to a test without added inhibitor. PHMB, p-hydroxymercuribenzoate; STI, soybean trypsin inhibitor; n.t., not tested. Inhibitor

Antipain DANME Econazole EDTA Ketoconazole Miconazole o-Phenanthroline Pepstatin PHMB PMSF STI TLCK TPCK

Concentration 1 #g/ml 0.01 mM 0.1 mM 0.1 mM 1 mM 0.1 mM 0.1 mM 0.2 mM 1 mM 1 mM 10 ~ g/ml 1 mM 0.1 mM

Inhibition(%) with proteinase A

B

C

36 <1 <1 <1 <1 <1 <1 90 12 11 <1 <1 <1

n.t. 97 40 <1 <1 80 <1 <1 90 97 <1 84 70

<1 <1 <1 96 n.t. <1 94 <1 <1 <1 <1 <1 <1

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