Identification and partial characterization of two proteinases from the cell envelope of Candida albicans blastospores

Zbl. Bakt. Hyg. A 260, 523-538 (1985)

Identification and Partial Characterization of Two Proteinases from the Cell Envelope of Candida albicans Blastospores 1 R.RUCHEL, BIRGITT BONING, and E. JAHN Abt. Medizinische Mikrobiologie, Hygiene-Institut der Universitat, D-3400 Cottingen

With 7 Figures· Received August 1, 1985 . Accepted September 13, 1985

Summary As we have communicated previously, the fungal opportunist Candida albicans produces an angiotensin-I liberating proteinase and a serine proteinase that acts as a converter of the coagulation factor X in vitro (25). Both enzymes are attached to delipidated cell fragments of fungal blastospores. They copurify upon ion exchange chromatography and gel filtration. The enzymes could be separated by hydrophobic chromatography on Phenylsepharose. According to their substrate and inhibition pattern.the enzymes have been classified as an aspartyl proteinase and a chymotrypsin-like proteinase. Their partial characterization includes estimates of the molecular weight and isoelectric points.

Zusammenfassung Der opportunistische hefeahnliche Pilz Candida albicans besitzt neben seiner sekretorischen Proteinase wenigstens zwei weitere proteolytische Enzyme, von denen eines Angiotensin-I freisetzt. Das andere Enzym ist eine Serinproteinase, die in vitro den Gerinnungsfaktor X aktiviert (25). Beide Enzyme sind assoziiert mit delipidierten Zellfragmenten von Blastosporen; sie konnten durch nicht-ionisches Detergenz in Losung gebracht werden. Die Enzyme waren weder durch DEAE-Chromatographie noch durch Gelfiltration an Sephacryl S200 zu trennen. Die Trennung gelang jedoch durch hydrophobe Chromatographie an

1

Dedicated to Prof. Dr. Dr. Friedrich Staib on the occasion of his 60th birthday.

Abbreviations used: AMC : 7-amino-4-methylcoumarin EDTA : Ethylenediaminetetraacetic acid DEAE : Diethylaminoethyl DMSO: Dimethyl sulfoxide MCA : 4-methylcoumaryl-7-amide TCA : Trichloroacetic acid Tris : Tris-Ihydroxymethyl) aminomethane For amino acids the common three letter notation was used.

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R. Biichel, B.Boning, and E.Jahn

Phenylsepharose. Anhand der Substrat- und Inhibitionsmuster konnte das Angiotensin-I freisetzende Enzym als Carboxyl protease und die Serinproteinase als chyrnotrypsin-ahnliches Enzym klassifiziert werden. Das Molgewicht beider Enzyme liegt im Bereich von ?: 80 000, wahrend die isoelektrischen Punkte bei pH 5 liegen.

Introduction Among the yeast-like fungi of the genus Candida, a few species are opportunists, causing superficial and deep mycosis in compromised patients; among these species, C. albicans is of foremost medical importance (1). With the rising incidence of serious Candida infections, the elucidation of pathomechanisms of mycosis, and the identification of fungal factors of virulence became increasingly important. Potential factors of virulence are secretory hydrolytic enzymes (6). They comprise acid proteinases, which have been discovered by Staib twenty years ago (31). Recently the secretory Candida proteinases have attracted much attention (for review see 28). All the Candida proteinases investigated so far are acid proteinases (E.C.3.4.23) which individually undergo denaturation at a pH above 6.5-8.4. Thus acid proteinases are potent tools for the fungus only as long as they stay in the acid microenvironment of the fungal cell. Such a restriction would not apply to structurally bound "membrane proteinases" as have been demonstrated in many' types of cells including fungi such as Aspergillus oryzae (34). In this communication we describe two proteinases which are associated with the cellular envelope of C. albicans CBS 2730.

Materials and Methods Azocoll was from Calbiochem, Frankfurt; Yeast Carbon Base was from Difco, Detroit; 4aminobenzamidine bound to succinyl-aminododecylcellulose was obtained from Merck, Darmstadt, as were most of the common laboratory chemicals, bovine serum albumin, butanedione, and Tween-20. Aminomethylcoumarin and the peptide Boc-Ile-Glu-Gly-Arg-MCA were from the Peptide Institute, Osaka. Phenylsepharose CL 4B, Sephacryl S 200, Sephadex G 25 and IEFSephadex were from Pharmacia, Freiburg. Azocasein, standard marker proteins, precast thin layer polyacrylamide gels for isoelectric focusing, DEAE-cellulose, and the following fluorogenic peptides were from Serva, Heidelberg: Benzoyl-Arg-MCA, Ala-Ala-Phe-M'CA, Glutaryl-Phe-MCA, Sue-Ala-Ala-ProPhe-MCA and Suc-Leu-Leu-Val-Tyr-MCA. Bovine coagulation factor X, bovine plasma devoid of factor X, Emulphogene BC 720, and crude bovine hemoglobin were from Sigma, Taufkirchen, as were the following inhibitors: chymostatin, diazoaceryl-Dl.-norleucine methylester (DAN), 1,2-epoxy-3-(p-nitrophenoxyl-propane (EPNP), pepstatin-A, phenylmethylsulfonylfluoride (PMSF), tosyl-lysine-chloromerhylketone (TLCK), and tosyl-phenylalanine-chloromethylketone (TPCK). Secretory Candida proteinases, that have been employed for comparison, were purified in this laboratory according to (23). Human monoclonal immunoglobulins were obtained from the same sources as indicated previously (27).

Production ofdelipidated cell fragments A variant of C. albicans CBS2730 (serotype A) was grown in Yeast Carbon Base (Difco) with 1% (w/v) of hemoglobin as described previously (23). After two days at 3rc under aerobic conditions and constant agitation, yeasts were centrifuged at 10000 x g (20 min,

Proteinases from the Cell Envelope of C. albicans Blastospores

525

4°C). Under these conditions the variant strain employed grew in yeast phase only.The type strain under comparable conditions grew preferentially in mycelial phase. The blastospores were washed in saline by centrifugation as above. Subsequently they were submitted to disintegration in the MSK-mill (Braun, Melsungen, Germany), which was operated at 4000 rpm for 5 min under carbon dioxide refrigeration as suggested by the manufacturer. The cell fragments were washed subsequently in 50 mM sodium acetate buffer with 5 mM MgCl z, 1 mM EDTA and 0.25 M sucrose; the final pH was 5.8. In the course of this procedure, removal of soluble acid proteolytic activity was monitored by the hemoglobin assay. The pellet was suspended finally in saline and was transferred to stainless steel buckets. Twenty volumes of cold ethanol ether (3+1) were added and the suspension was shaken vigorously for 5 min at -18°C. Subsequently the suspension at -18 °C was centrifuged as above. Delipidation was repeated three times. Finally the sediment was dried under vacuum and was stored at -18°C. Extraction of membrane proteinase Delipidated cell fragments at a ratio of 1 mg/l00 ~l were suspended in 0.2 M sodium citrate buffer pH 6.3 and 0.4% of Emulphogene BC 720. A small amount of sterile quartz sand was added as a grinder. After 1 h on a rotary mixer at 35°C, a clear supernatant was produced by centrifugation (1 h, 30000 X g, 4°C). Excess of detergent was removed from the supernatant by extraction with cold acetone (3). A sediment was collected by centrifugation at -18°C; it was freely soluble in 0.2 M citrate buffer pH 6.2. The solution was stable at -18°C at least for a year, it is referred to as crude extract.

Anion exchange chromatography Anion exchange chromatography of the crude extract was performed essentially as described for the secretory Candida proteinase (23). In order to prevent hydrophobic adsorption of protein, a minimum (0.05% v/v) of Emulphogene BC 720 was added to the crude extract and to the starting buffer (20 mM citrate buffer, pH 6.5). Desorption of protein was performed without detergent; 0.2 M citrate buffer pH 6.2 was used. Fractions with acid proteolytic activity were monitored by the hemoglobin assay. They were pooled and stored at-18°C. Gel filtration Partially purified acid proteinase was submitted to gel filtration at Sephacryl S 200. The common buffer was 30 mM sodium phosphate with 0.05% Tween-20, pH 6.6. As in the previous purification step, detergent was added to prevent adsorption. Prior to and after gel filtration, the pooled proteolytic fractions were concentrated by nitrogen pressure dialysis; they are referred to as mCBS-fraction.

Hydrophobic chromatography Hydrophobic chromatography of the mCBS-fraction was performed essentially as described by Meussdoerffer et al. (18). A column of 9 rnrn ID and 15 ern length was filled with Phenylsepharose CL-4B and was equilibrated with 0.1 M phosphate buffer pH 6. A sample of the mCBS-fraction (0.3 ml) was adsorbed to the column. Subsequently the gel was rinsed with 3 volumes of starting buffer plus 1.4 M NaCl, followed by starting buffer plus 35% v/v ethylene glycol. Finally the column was developed in opposite direction by a continuous gradient of ethylene glycol (up to 85%) in starting buffer.

526

R. Biichel, B.Boning, and E.] ahn

Benzamidine affinity chromatography

Affinity chromatography of the trypsin-like proteinase was performed with benzamidine as a solid phase ligand essentially as described by Scbleuning et aI. (30), with 0.1 M triethanolamine-HCI containing 0.15 M NaCl , pH 7.3 as starting buffer, and 0.1 M glycineHCI containing 0.15 M NaCl as desorption buffer. Assay of acid membrane proteinase

Proteolytic activity at pH 3 was determined by the Anson test with hemoglobin as a substrate (23). The use of enzyme samples containing more than 0.05 % detergent occasionally caused turbidities in the blanks when sample was added directly prior to trichloroacetic acid precipitation. Use of pepstatin-inhibited or heat-denatured enzyme in the blanks over the full length of incubation solved the problem. Assay of neutral proteolytic activity

Proteolytic activity around neutrality was tested by various procedures: a) chromogenic substrates like azocoll and azocasein were employed according to Moore (19) and Hasche et al. (9), respectively. Sodium pyrophosphate - HCI buffers (pH 6.5-7.3) served as assay buffers in these and the following tests. b) liberation of the fluorophore aminomethylcoumarin (AMC) from synthetic peptides such as Benzoyl-Arg-MCA, Ala-Ala-Phe-MCA, Succinyl-Ala-Ala-Pro-Phe-Mt.A, Butyloxycarbonyl-I1e-Glu-Gly-Arg-MCA, Succinyl-Leu-Leu-Val-Tyr-MCA and Glutaryl-Phe-MCA. Stock solutions(10 mM ) of these subst ances were made up in dimethyl sulfoxide (Uvasol, Merck). Prior to use they were diluted 100 x. After incubation at 37 °C, the reaction was sto pped by addition of 1.5 volumes of 17% acetic acid. The concentration of liberated AMC was determined by fluorometry at 380 nm excitation and 460 nm emission (37). c) Proteolytic activation of bovine coagulation factor X at pH 6.8 was tested as described previously (25). Essentially this involves the preincubation of inactive factor X with the sample at pH 6.8 and Aliquots of this mix were then added to a solution of the prothrombin analogue peptide Boc-I1e-Glu-Gly-Arg-MCA at pH 8. After another incuba tion at the reaction was stopped and fluorescence of AMC was measured as outlined above.

3rc.

3rc

Electrophoresis

Electrophoresis was performed in continuous polyacr ylamide grad ient gel slabs essentially as described previously (22). The gradient ranged from 2.5 to 20% total acrylamide at a constant cross linkage of 2.5%. Anionic electrophoresis at pH 6.6 was performed in a continuous buffer (20 mM NaKphosphate) with 0.1 % Emulphogene BC 720 to prevent adsorption. This buffer was introduced into the gel by electrophoretic premn (overnight, 10 Wcm). Electrophoresis was performed under constant cycling of the buffer at the electrodes. Isoelectric focusing

Analytical electrofocusing was performed in precast thin layer polyacrylamide gels of 5% total acrylamide and 3% cross linkage as suggested by the manufacturer (Serva, Heidelberg). Fast green and methyl green were used as anionic and cationic marker, respectively. Phosphoric acid (1 M) and ethylenediamine (2.5% ) were used as terminal electrolytes. Inhibitors

For inhibition of acid proteolytic acnvity pepstatin, epoxy-p-nitrophenoxy propane (EPNP) and diazoacetyl norleuc ine methylester (DAN) were employed as previousl y de-

Proteinases from the Cell Envelope of C. albicans Blastospores

527

scribed (23). Butanedione was used as suggested by Gripon and Hofmann (8) at pH 6.2 and 4 hours exposure to fluorescent light. Serine proteinases were inhibited by phenylmethylsulfonyl-fIuoride (PMSF) at 1 mM (13). Tosyl-phenylalanine-chloromethylketone (TPCK) and tosyl-Iysine-chloromethylketone (TLCK) were employed according to Carpenter (5). Chyrnostatin was used out of a stock solution (1 mM in DMSO), which was diluted 100 X in the assay. For use in the two stage factor X convertase assay (see above) free inhibitor after preincubation was removed by extensive dialysis.

Results Delipidated cell fragm ent s

Blastospores of C. albicans CBS 2730 that had been grown in proteinaceous medium as described above, were submitted to various treatments for the production of cell fragments. These methods (in the sequence of increasing efficacy) included sonication, blendi ng, mortar, freeze thawi ng, aggravated osmotic shock, Hughes press, and the MSK-mill. In our hands the last three techniques only produced cell fragments with less th an 2% of the yeasts remaining viable. For osmotic shock treatment the cells were equilibrated with absolute ethylene glycol fod h prior to lysis in distilled water. The Hugh es press (12) was employed th ree times as suggested by the manufacturer (AB Biox, j arfalla.Sweden), The MSK-homogenizer (Braun, Melsungen , Germany) was ope rated at 4000 rpm for 5 min with carbon dioxide refrigeration. For further experimentation only cell fragments derived from the MSK mill were employed; 0.3% only of the yeasts survived this treatment. The cell fragments were washed repeatedly in acetate buffer of pH 5.8 , and the removal of soluble proteolytic activity was monitored by the hemo globin test. After the fourth centrifugation virtually all acid proteolytic activity had been removed from the supernatant. Subsequentl y the cell fragment s were delipidated as outlined above and were dried for storage at -18 °C. Extraction of proteinase

For the extraction of bound proteolytic activity the detergent Emulph ogene BC 720 was chosen. It is a relat ive of Triton X-I 00 and Nonidet P-40, but it lacks the phenol ic ring of these two detergents, which accounts for the high UV-absorption (7, 11). The optimum concentration of the detergent (in 20 mM sodium citrate buffer, pH 6.5 ) was determined at 0.4 % (v/v). When delipidated cell fragments at a ratio of 1 mg per ml were shaken vigorously in this medium in the presence of quartz sand for 2 h at 22 °C, subsequent centrifugation (8000 x g, 2 min) yielded a supernatant that contained two thirds of the acid proteolytic activity. Elevated temperatures are raising the yield, but autodigestion of the different enzymes has to be considered . Solubl e proteolytic activity as detected by the hemoglobin test was subsequently freed of unbound detergent by acetone precipitation. The precip itated protein was dissolved in 0.2 M citrate buffer at pH 6.2 and was stored at -18 °C. Ion exc hange chromatography

The crude enzyme was submitted to DEAE-chromatograph y as described above. The yield of acid proteolytic activity was 85%, pro ving that the cont inued presence of free

528

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Fig. 1. Ion exchange chromatography of detergent extracted proteins. The Emulphogene extract of cell fragments of C. albicans CBS 2730 was submitted to DEAE-chromatography after acetone precipitation. The starting buffer was 20 mM sodium citrate, pH 6.5 with 0.05 % Emulphogene Be-nO. After adsorption of the sample and rinsing of the column with starting buffer desorption of proteins was performed in a stepwise fashion in inverted direction with 0.2 M sodium citrate buffer pH 6.2. Fr: Fractions of the eluate; -,.-: profile of conductivity (mSi)j - 0- : profile of the absorption (A) of protein-like substances at 280 nm; proteolytic activity at pH 3.5: - e-. detergent was no essential requirement . By this purification step the bulk of contaminating hemoglobin fragments was remo ved. Th e concentration of the acid proteinase was raised three times while its specific activity went up ten times. It is noteworthy that the major peak of protein-like sub stances desorbed prior to the enzyme, thu s offering an opportunity for sequential desorption by a buffer grad ient (Fig. 1).

Gel filtration The fractions with acid proteolytic activity derived from the DEAE gel were pooled and submitted to gel filtration on Sephacryl 5-200 at pH 6.6 in the pr esence of 0.05% Tween-20 . This purification step removed another fraction of proteinaceous contaminants (Fig. 2). Once again fractions with acid proteolytic activity were pooled. This pool was stored as before. It was used for characterization experiments if not stated otherwise; it is subsequently referred to as "mCB5 fraction".

Acid m embrane proteinase The acid proteolytic enzyme of the mCBS fraction was characterized by the course of its alkaline denaturation. As comp ared with its secretory counterpart, it proved less stable (Fig. 3a ).

Proteinases from the Cell Envelope of C. albicans Blastospores 10

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Fig. 2. Gel filtration of partially purified acid proteinase. Acid proteolytic activity after ion exchange chromatography was pooled and submitted to gel filtration on Sephacryl S-200. The common buffer was 30 mM sodium phosphate pH 6.6; 0.05% Tween-20 was added to prevent hydrophobic adsorption. Fr: fractions of the eluate; - : profile of desorbed protein expressed as absorption (A) at 280 nm wavelength; - - -: profile of conductivity (mSi); -e-: profile of acid proteolytic activity, expressed by absorption (A) at 280 nm of TCA-soluble fragments of hemoglobin. Pooled proteolytic fractions are referred to as mCBS fraction. The peak of conductivity is due to salt of the sample.

Differences among the two enzymes were also revealed upon comparison of the pHdependent activity profiles (Fig. 3b), and by comparison of their electrophoretic mobilities under non-denaturing conditions. After electrophoresis, the acrylamide gel was sliced and assayed for acid proteolytic activity. Thus, the two enzymes could clearly be assigned to different positions in the gel (Fig. 4). Apparently the acid membrane proteinase has a higher molecular weight than the secretory enzyme. For reasons given in the next chapter the approximate molecular weight of the acid membrane proteinase is in the range of 86000. The pattern of inhibition of the two acid proteinases were fairly identical (Table 1). They were in agreement with both the pattern of the secretory proteinase and the acid membrane proteinase of C. albicans ATCC 48867. The latter enzyme had been purified by the procedure described here.

Chymotrypsin-like proteinase Our previous investigation of the influence of the mCBS fraction on blood coagulation in vitro led to the discovery of a potent activator of the Stuart factor (25). This factor X converting activity had its optimum at pH 6.8; it was found to be sensitive to inhibition by PMSF and TPCK but not by TLCK. The enzyme thus is a serine proteinase of the chymotrypsin type as was confirmed by inhibition with chymostatin, Separation of this enzyme from the acid membrane proteinase of the mCBS fraction was accomplished by hydrophobic chromatography (Fig.

530

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Fig. 3a. Alkaline denaturation of the acid membrane proteinase. Samples of the mCBS fraction were preincubated for 20 min at 22 °C at various pH ; residual acid pr oteolytic activity (A, - e -) was determined at pH 3.5 by the hemoglob in assay (for details see methods). The denatu ratio n profile of the secretory acid proteinase of the same fungal strain (- X - ) was entered for compar ison.

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Fig. 3b. pH-dependen t activity profile of the acid membrane proteina se. Aliquots of the mCBS fraction were submitted to the hemoglobin assay at varying pH under standard conditions.Proteolytic activity (A) is expressed as absorption of TCA-soluble peptides at 280 nm wavelength (- e- ). The activity profile of the homologous secretory acid proteinase was added for compari son (- X - ) .

Proteinases from the Cell Envelope of C. albicans Blastospores

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Fig. 4. Anionic polyacrylamide gradient gel electrophoresis of both acid proteinases of C. albicans CBS 2730. Samplesof the mCBS fraction containing the acid membrane proteinase (right lane), and of highly purified acid secretory proteinase (left lane) were submitted to gel electrophoresis under non-denaturing conditions at pH 6.6 in a continuous buffer system; image after Coomassie blue staining (for details see methods). The peak of acid membrane proteinase (~) was identified by slicing and subsequent hemoglobin assay.

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5). The sequence of elution of the two enzymes suggests that the acid proteinase is more hydrophobic than the chymotrypsin-like proteinase. The molecular weight of the latter was determined by gel gradient electrophoresis under non-denaturing conditions, followed by slicing and the factor X convertase test (Fig. 6). The comparison of the activity profile of the gel slices with positions of a panel of marker proteins suggests a molecular weight in the range of 86000. Since both the chymotrypsin-like proteinase and the acid mCBS proteinase were not separated clearly by gel filtration as described above, the latter enzyme possibly has a molecular weight in the same range. Electrofocusing followed by slicing of the gel and factor X convertase test allowed the isoelectric point of the chymotrypsin-like enzyme to be located at pH 4.9 (Fig. 7). While this is rather uncommon for a serine proteinase, it would have been expected for the acid mCBS proteinase. A proximity of the isoelectric points may explain why both proteinases could not be separated by ion exchange chromatography. The chymotrypsin-like enzyme was tried with various fluorogenic synthetic peptides. As shown in Table 2, four chymotrypsin substrates were cleaved with different efficacy at pH 6.8 while the trypsin substrate arginine-MCA remained untouched. This pattern of specificity confirms the classification of the enzyme. Both Candida membrane proteinases are able to cleave human immunoglobulins within the range of their optimum activity (pH 3.5 and 6.8, respectively), as could be demonstrated by subsequent analytical gel electrophoresis. While cleavage of the IgG1 heavy chain was obvious, cleavage of IgA2 , and more so IgM, was limited to proteoly-

532

R. Biichel, B. Boning, and E.Jahn

Table 1. Inhibition of acid Candida proteinases (++ : total, + : ;;, 50% , (+ ): < 50%, - : no inhibition) Pepstatin

DAN

EPNP

Butanedione

membrane proteina se

C. albicans CBS 2730

++

+

membrane proteinase

ATCC 4886 7

++

+

secretory proteinase

C. albicans CBS 2730

++

++

secretory prote inase

ATCC 48867

++

++

(The data on the secretory proteinases have been taken from 27).

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Fig. 5. Hydrophobic chromatography of acid and chymotrypsin-like proteinase . A sample of the mCBS fraction was adsorbed to Phenylsepharose at pH 6. Bound proteins were desorbed by a continuous gradient of ethyleneglycol (35-85% v/v) (for details see methods). The gradient was monitored by measurement of the conductivity (mSi) in the fractions (Fr) of the eluate. The chymotrypsin-like proteinase (-e-) was detected by its factor X converting activity which was expressed as relative fluorescence (RI). The acid proteolytic activity (-0-) was detected by hemoglobin assay; it was expressed by absorption (A) at 280 nm wavelength of TCA-soluble peptides.

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Fig. 6. Molecular weight of the chymotrypsin-like proteinase. Partiall y pur ified C. albicans membrane proteinases after DEAE-chromatography were submitted to polyacrylamide gel gradient electrophoresis at pH 8,4 under non-denaturing conditions. Three lanes of the slab gel were stained with Cooma ssie blue: The right lane shows the pattern of anionic membrane prote ins, the middle and left lane show marker proteins (L: ~-lactoglobulin, 17500 d; T: soy bean trypsin inhibitor, 21000 d; 0 : ovalbumin, 45000 d; R1,2: bovine serum albumin monomer (68000 d) and dimer ; Fl , 2: horse ferritin, monomer (440000 d) and dimer). Another lane of the gel slab wa s sliced and analyzed for factor X converting activity among the Candida membrane proteins (for detail see methods). RI: relative intensity of the fluorescence of liberat ed AMC. P: peptides within the buffer front. The peak of fluorescence represents convert ase activity; its projection on the profile of the marker proteins indicates a molecular mass of approximately 86000 d for the chymotr ypsin-like enzyme.

Table 2. Degradation of fluorogenic pept ides by the mCBS fraction and chymotrypsin at pH 6.8 (the figures represent the relative velocity of degradation under comparable conditions of the assay)

Phe-MCA Ala-Ala-Phe-MCA Leu-Leu-Val-Tyr-M CA Ala-Ala-Pro-Phe-MC A

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Fig. 7. Isoelectric point of the chymotrypsin-like membrane proteinase. Isoelectric focusing was performed in a precast thin layer polyacrylamide gel (Serva,Heidelberg) of 0.1 mm thickness, containing 1.8% carrier ampholytes pH 3-6. 40 mM glutamic acid and 0.2 M L-histidine were used as terminating electrolytes. At a length of 11.5 cm eleetrofocusing was performed for 5h at 15 "C, The maximum voltage was 1.2 kY. The chymotrypsin-like proteinase was detected by its factor X converting activity at pH 6.8 (for detail see methods). Fr: Gel fractions of 5 mm width; -e- : pH profile after elution of the gel slices in water; -0-: profile of factor X converting activity expressed by fluorescence (RI) of liberated AMC.

tic nicking; it became apparent onl y upon reduction of intramolecular disulfide bonds (results not shown). Both Candida membrane proteinases are largely stable against freezing at pH 6.2 in 0.2 M citrate buffer.

Exclusion of trypsin-like proteinase The crude detergent extract of cell fragments of C. albicans CBS 2730 was checked for trypsin-like proteinase employing the specific inhibitor benzamidine as a solid phase ligand'. Affinity chromatography was performed essentiall y as described previously (30). No evidence of a fungal tryp sin-like enzyme was gathered. However, attention had to be payed to an unidentified trypsin-like enzyme, that was already attached to the commercially obtained affinity gel prior to use. Thi s activity can be monitored conveniently at pH 7.8 by azocoll assay ; it can be removed from the gel matrix by repeated desorption at pH 2 (see above ).

Proteinases from the Cell Envelope of C. albicans Blastospores

535

Discussion Twenty years ago Staib (31) discovered the secretory proteolytic activity of C. albicans, and subsequently he suggested that this activity might represent an important factor of fungal virulence in animals and humans (32). Staib initiated also the first attempts to purify and characterize a Candida proteinase (21). Ever since, these enzymes have attracted the interest of mycologists (4,10,15-17,28,33), and by now trials to identify and clone the proteinase gene are underway in several laboratories. Studies concerning the biochemical stability of the Candida proteinase (24), and immunohistochemical findings (16,26) suggested that the secretory Candida proteinases are predominantly active in the acid microenvironment around the yeast cell that is maintained by secretion of organic acids. These acids are products of incomplete degradation of glucose, and consequently it has been shown that the proteolytic activity of C. albicans depends on the level of glucose in the medium (29). Secreted enzymes are likely to diffuse quickly out of the narrow microenvironment and are then prone to alkaline denaturation. Teleologically, the situation calls for structurally bound enzymes that stay within the range of the fungal cell. Such "membrane proteinases" have already been identified in the industrially used mould Aspergillus oryzae (34). Following the scheme of these authors, we were able to prove the existence of an acid membrane proteinase in C. albicans CBS 2730 (serotype A). A comparable enzyme was discovered in C. albicans ATCC 48867 (25). The acid membrane proteinase has approximately twice the molecular weight of its secretory counterpart, but both pattern of inhibition are almost identical. It is intriguing to speculate that the acid membrane proteinase is. the active precursor of the secretory enzyme. Functionally both enzymes differ in their abiliy to cleave an angiotensinogen-analogous peptide (25). Upon ion exchange chromatography and gel filtration, a chymotrypsin-like proteinase copurified with the acid membrane proteinase. Separation of both enzymes was finally achieved by hydrophobic chromatography. Its molecular weight is in the range of 86000, and its behaviour upon ion exchange chromatography was explained by its pI (pH 4.9). The respective data of the acid proteinase are possibly in the same range as suggested by copurification. Acid isoelectric points are not uncommon among serine proteinases, as exemplified by various proteinases of the blood coagulation cascade (2). The chymotrypsin-like enzyme turned out to be a potent activator of coagulation factor X at pH 6.8 (25). The enzyme possibly represents the "procoagulant substance" of C. albicans, that was predicted on the basis of pathohistological findings (20). The properties of the chymotrypsin-like enzyme are reminiscent of the proteinase B of Saccharomyces cerevisiae,which is located in the vacuole of the fungal cell (14). Proteinase B is sensitive to sulfhydril reagents (36), which was also observed with the enzyme of C. albicans (Ruchel, unpublished). Beyond the establishment of membrane proteinases of C. albicans the available data give no indication of the amounts of the enzymes with respect to dry weight of the yeast. No statement can be made concerning the relationship between proteinases and fungal dimorphism, and generalizations beyond the single strain tested have to be avoided. Indeed there are indications that other strains of C. albicans such as ATCC 48867 (serotype B) hardly produce the chymotrypsin like proteinase (Ruchel, unpublished). The yield of solubilized enzyme from delipidated cell fragments needs technical improvement. However, any consideration of the yield is hampered by the fact that

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structurally bound enzymes are prone to loss of activity upon solubilization. Thus, the activity of the related acid proteinase cathepsin-D was found to depend stro ngly on the presence of specific lipids (35 ). The true localization of the enzymes within the cellular envelope ha s to be investigated. They may be a genuine part of th e cell wall or they may be components of the plasma membrane that attach to the cell wall in the course of delipidation. Antibodies, that had been elicited in guinea pig against the mCBS-fraction, reacted equally strong with int act blastospores or cell fragments. This indicates that most of the antigens of the mCBS fraction are exposed on the outer surface of the yeast. Finally, no specific function of the two membrane proteinases in fungal biology ha s yet been proposed. It is suggestive to ass ume that C. a/bicans has an equally elaborate system of cellular proteolytic enz ymes and endogenous inh ibitors as ha s been discovered in Saccharomyces cerevisiae (fo r review see 14, 36 ). The acid membrane proteinase of C. a/bicans may match proteinase A of Saccharomyces, and the chymotrypsin-like Candida proteinase appears to be related to proteinase B. However, concerning fungal virulence, it is still the secretion of acid proteinase as discovered by Staib in 1965 (31), that makes the difference between the truly apathogenic yeasts and the opportunistic pathogen C. a/bicans.

Acknowledgement. We are indebted to Mrs . Anneli Siegmann for expert secretarial work. The investigation was supported by a grant from the Deutsche Forschungsgemeinschaft .

References

1. Ah earn, D. G.: Medically importa nt yeasts. Ann. Rev. Microb iol. 32 (197 8) 59-68 2. Andersson, L.a.: Haem ophili a Bvfactor (Factor IX), in: Plasma Protein s; pp. 281-285, ed. by B. Blombdck and L. A. Hanson. Wiley, Chichester (1979) 3. Barritault, D., A. Expert-B ezanson, H. Guerin, and D. Hayes: The use of acetone precip itat ion in the isolation of ribosomal proteins. Europ . J. Biochem. 63 (197 6) 131- 135 4. Budtz-jorg ensen, E.: Proteol ytic activity of Candida spp . as related to the pathogenesis of denture stomatitis. Sabouraudi a 12 (197 1) 266-271 5. Carpenter, F. H.: Treatment of trypsin with TPCK. Meth. Enzymol. 11 (1967) 23 7 6. Chattaway, F. W., F. C. Odds , and A. J. E. Barlow: An examinati on of the production of hydrol ytic enzymes and tox ins by pathogenic strains of Candida albicans. ]. gen. Microbiol. 67 (1971) 255-263 7. Furth, A. J., H. Balton, J. Potter, and J. D. Priddle: Separating detergent from proteins. Meth. Enzymol. 104 (1984) 318-328 8. Gripon,J.-c. and T. Hofmann: Inactivation of aspartyl proteinases by butane-2,3-dione. Biochem. ] . 193 (1981) 55-65 9. Hasche, K. D., W.Schaeg, H. Blobel, and J. Briickler: Reinigung einer Protease von Staphylococcus aureus. Zbl. Bakt. Hyg., I. Abt. Orig. A 238 (1977) 300- 309 10. Hatt ori, M., K. Yoshiura, M. Negi, and H. Ogawa: Keratinolytic proteinase produced by Candida albicans. Sabouraudia 22 (1984) 175-183 11. Helenius, A. and K.Simons: Solub ilization of membranes by detergents. Biochim. Bioph ys. Acta 415 (1975) 29-79 12. Hughes, D. E.: A press for disrupting bacteria and other microorganisms. Brit. J. expo Path . 32 (1951) 97-109

Proteinases from the Cell Envelope of C. albicans Blastospores

537

13. James, G. T.: Inactivation of the protease inhibitor phenylmethylsulfonylfluoride in buffers. Analyt. Biochem. 86(1978) 574-579 14. Jones, E. W.: Genetic approaches to the study of protease function and proteolysis in Saccharomyces cerevisiae, in: Yeast Genetics; pp. 167-203, ed. by]. F. T. Spencer, D. M. Spencer, and A. R. W. Smith. Springer, New York (1983) 15. Ku/on-Chung, K.]., C. Good, D. Lehman, and P. T. Magee: Genetic evidence for the role of extracellular proteinase in the virulence of Candida albicans. Infect. Immun. 49 (1985) 571-575 16. Macdonald, F. and F. C. Odds: Inducible proteinase of Candida albicans in diagnostic serology and in the pathogenesis of systemic candidosis. J. med. Microbiol. 13 (1980) 423-435 17. Macdonald, F.: Secretion of inducible proteinase by pathogenic Candida species. Sabouraudia 22 (1984) 79-82 18. Meussdoerfer, F., P. Tortora, and H. Holzer: Purification and properties of proteinase A from yeast. J. BioI. Chern. 255 (1980) 12087-12093 19. Moore, G. L.: Use of azo-dye-bound collagen to measure reaction velocities of proteolytic enzymes. Analyt. Biochem. 32(1969) 122-127 20. Myerowitz, R.L.: Ultrastructural observations in disseminated candidasis. Arch. Path. Lab. Med. 102 (1978) 512-514 21. Remold, H., H. Fasold, and F. Staib: Purification and characterization of a proteolytic enzyme from Candida albicans. Biochim. Biophys. Acta 167 (1968) 399-406 22. Riichel, R. and J. Gross: Preparations of continuous gradient gel slabs: A simple technique. Analyt. Biochem. 92 (1979) 91-98 23. Riichel, R.: Properties of a purified proteinase from the yeast Candida albicans. Biochim. Biophys. Acta 659 (1981) 99-113 24. Ruchel. R. and M. Trost: A study of the structural conversions of two carboxyl proteinases employing electrophoresis across a pH-gradient, in: Electrophoresis '81; pp. 667-676, ed. by R. Allen and P. Arnaud. de Gruyter, Berlin (1981) 25. Ruchel, R.: On the renin like activity of Candida proteinases and activation of blood coagulation in vitro. Zbl. Bakt. Hyg., I.Abt. Orig. A 255 (1983) 368-379 26. Ruchel, R.: On the role of proteinases from Candida albicans in the pathogenesis of acronecrosis. Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 524-536 27. Ruebel, R., K. Ublemann, and B. Boning: Secretion of acid proteinases by different species of the genus Candida. Zbl. Bakt. Hyg., I. Abt. Orig. A 255 (1983) 537-548 28. Riichel, R.: A variety of Candida proteinases and their possible targets of proteolytic attack in the host. Zbl. Bakt. Hyg., A 257 (1984) 266-274 29. Samaranayake, L. P., D. A. M. Geddes, D. A. Weetman, and T. W. MacFarlane: Growth and acid production of Candida albicans in carbohydrate supplemented media. Microbios 37 (1983) 105-115 30. Schleuning, W. D., H. Schiessler, and H. Fritz: Highly purified acrosomal proteinase (Boar acrosin): Isolation by affinity chromatography using benzarnidine-cellulose and stabilization. Hoppe Seyler's Z. Physiol. Chern. 354 (1973) 550-554 31. Staib, F.: Serum-Protein-Agar-pl-I 4.2 und 5.0 fur Sprofspilze. Zbl. Bakt.,I. Abt. Orig. 195 (1965) 265-267 32. Staib, F.: Proteolysis and pathogenicity of Candida albicans strains. Mycopathologia 37 (1969) 345-348 33. Staib, F., S. K.Mishra, and T. Abel: Serodiagnostic value of extracellular antigens of an actively proteolysing culture of Candida albicans. Zbl. Bakt. Hyg., I. Abt. Orig. A 238 (1977) 284-287 34. Tsujita, Y. and A. Endo: Intracellular localization of two molecular forms of membrane acid protease in Aspergillus oryzae. Europ. J. Biochem. 88 (1980) 113-120 35. Watabe, S. and N. Yago: Phospholipids activate cathepsin D. Biochem. Biophys. Res. Commun. 110 (1983) 934-939 36. Wolf, D. H. and H. Holzer: Proteolysis in yeast. In: Microorganisms and nitrogen

538

R. Buche], B. Boning, and E. Jahn

sources:Transport and utilization of amino acids, peptides, proteins and related substrates; pp. 431-458, ed. by]. W. Payne. Wiley, Chichester (1980) 37. Zimmerman, M., B. Ashe, E. C. Yureioicz, and G. Patel: Sensitive assays for trypsin, elastase, and chymotrypsin using new fluorogenic substrates. Analyt. Biochem. 78 (1977) 47-51 Dr. R.Ruchel, Abt. Med. Mikrobiologie, Hygiene-Institut der Universitat, Kreuzbergring 57, D-3400 Gottingen