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Purification and characterisation of a novel 34,000-Mr cell-associated proteinase from the dermatophyte Trichophyton rubrum
&MDy;f;OGY
AND
MICROBIOLOGY FEMS
Immunology
and Medical
Microbiology
I3 (1996)
I3 I - I40
Purification and characterisation of a novel 34,000-M, cell-associated proteinase from the dermatophyte Trichophyton rubrum Imelda Lambkin * , Andrew J. Hamilton, Rod J. Hay Drrnzntolo~~~
Unit.
Clinico1
Scierws
.!dm-orory,
Received
26 September
18th
Floor,
GU,Y.T Tower,
Gum
1995; accepted 27 October
Ho.spikd.
London
SE/
YRT, UK
1995
Abstract A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000-M, (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15.000-M, (reduced SDS-PAGE) and 37.000-M, (substrateSDS-PAGE).It had a pH optimumof 7.5 and a pi of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000-M, proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides irnmitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinaseto be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the rubrurn
proteinaseremainsspeculativealthoughit may play a role in the developmentandsubsequent proliferationof the fungusin vivo. Keww&:
Trichoph~or~
rrrhrron:
Cytoplasmic
antigen:
Cell-associated
1. Introduction
proteinase
* Corresponding author. Present address: Elan Corporation Research Institute. Trinity College, Dublin 2. Ireland. Tel.: + 353 (I) 67 I-090 I : Fax: + 353 (I ) 67 I-0920.
on the production of extracellular proteinases[5-81. However, apart from one early study which described the extraction of peptidases and enzymes with the specificities of trypsin and chymotrypsin from T. rubrum mycelia [9], there have been no definitive reports on the purification or characterisation of intracellular proteinasesfrom T. rubrurn or on the potential relationship of intracellular proteinaseswith the previously described extracellular proteinases.The potential significance of intracellular proteinasesin dermatophytes is illustrated by the two cell-bound keratinasespurified from liquid cultures of T. mentugrophytes rar. granulosum [lo],
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Societies.
Trichophyton rubrum is a cosmopolitan anthropophilic dermatophyte which causesinfection of the trunk, groin, foot and nail. It is the most common recorded cause of human dermatophytoses worldwide accounting for approximately 70-89% of all reported cases[l-4]. Studies on the factors influencing the virulence of this dermatophyte have focused
SSDl
5.00 8 1996 Federation
0928-8243(95)00095-X
of European
Microbiological
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which have been shown to induce delayed type hypersensitivity reactions in cutaneously infected guinea pigs [ 11,121. There have been a number of reports of intracellular proteinases in other pathogenic fungi. Proteinases have been observed in the vacuoles of Cundidu albicans, Saccharomyes cerecisiae, Neurosporu crassa, and the dermatophyte fungus Microsporum gypseum [ 131 and it has been suggested that intracellular proteinases are contained in membrane-bound compartments similar to the mammalian lysosome in eucaryotic organisms [ 141. Signal peptidases, which catalyse the removal of signal peptides from secretory proteins have also been observed in the rough and smooth microsomes of Aspergillus oryae [ 151. A number of studies have focused on the production and secretion of the 43,000-M, C. albicans aspartic proteinase (CAP) [ 16- 181 and these have provided the first information on the relationship between proteinases observed within the fungal cell and secreted proteinases. Intracellular forms of CAP have been detected by immunoblotting using anti-extracellular proteinase antiserum [ 1S] and it has been hypothesised that a 45,000-M, protein is a precursor form of the 43,000-M, secretory enzyme. In this paper the purification of a novel 34,000-M, proteinase from preparations of T. rubrum cytoplasmic antigen is described. The proteinase was purified to homogeneity by sequential Rotofor isoelectric focusing and gel filtration fast protein liquid chromatography (FPLC) and the purification process was monitored using SDS-PAGE and substrate SDSPAGE. The 34,000-M, proteinase was characterised by pH optima, isoelectric point, inhibition profile and N-terminal amino acid sequence analysis and the specificity of the proteinase for a range of substrates was determined. The relationship of the 34,000-M, intracellular proteinase with the previously isolated T. rubrum extracellular proteinases and other fungal proteinases is discussed.
2. Materials
and methods
2. I. Organisms and cultication
methods
A fresh clinical isolate of T. rubrum (strain F1478) was obtained from the Department of Medical My-
cology, St. John’s Institute of Dermatology, St Thomas’s Hospital, London, England. It was maintained on Sabouraud glucose agar slants (containing 2% w/v D( + )-glucose (Sigma, Poole: England), 1% w/v Bacto-peptone (Gibco, Uxbridge. England). 1.5% w/v Oxoid bacteriological agar (Unipath, Basingstoke, England), in I 1 of distilled water) stored at 4°C and was subcultured every 3 months. Spore suspensions were prepared by washing agar slants with distilled water after incubation for 14 days at room temperature. To examine proteinase production the spore suspensions were incubated in Sabouraud glucose broth (containing 2% w/v D( +)-glucose (Sigma), 1% w/v Bacto-peptone (Gibco). and 0.25% w/v sodium-P-glycerophosphate (Fisons, Loughborough, UK)) without agitation until cultures had reached late log/early stationary phase (approximately 21 days at room temperature). 2.2. Pur$ication
of the proteinase
Cultures were filtered using Whatman no. 1 paper and the harvested mycelia were humogenised in a bead beater (Biospec Products. Oklahoma, USA) containing 5 mm glass ball ballotini (Stratech Scientific, Bedfordshire, UK) and 50 ml of phosphatebuffered saline (PBS, 0.01 M, pH 7.4). The homogenate was centrifuged at 10,000 X g for 2 h and the supernatant cytoplasmic antigen preparation was recovered. After dialysis against distilled water for 2 h at room temperature the cytoplasmic antigen was loaded onto a Rotofor isoelectric focusing cell (BioRad, Hemel Hempsted. England). Broad range (pH 3-10) Biolyte ampholytes (Bio-Rad) were used and the system was focused for 4 h at 12 W constant power. After focusing the Rotofor fractions were collected and tested for pH, protein concentration and azocasein degrading activity (see below). The relevant fractions were concentrated on Microsep 1 kDa centrifugal concentrators (Flowgen Instruments Ltd., Kent, England), analysed by conventional and substrate SDS-PAGE (see below) and were subsequently pooled and refocused on the Rotofor system. Following the same procedure selected secondary Rotofor fractions were pooled, concentrated and loaded on to a Superose 12 HR lo/30 FPLC gel filtration column (separation range 1000-300,000M,) (Pharmacia, Milton Keynes, UK). The column
I. Lumbkirz
et ul./
FEMS
Immunolng~
and
was equilibrated using 0.05 M Tris hydrochloride (pH 8.4). Samples were eluted at a flow rate of 0.6 ml/min in the same buffer and 1 ml fractions were collected and tested for protein concentration and azocasein degrading activity (see below). Two to three gel filtration chromatography cycles were performed. Culture filtrates were concentrated to l/500 of the original volume by dialysis against neat polyethylene glycol 8000 (BP Chemicals, UK) in presoaked dialysis visking membrane (Medicell International, London, UK). Samples were then processed for substrate SDS-PAGE analysis as described below. 2.3. SDS-PAGE and substrate SDS-PAGE analysis Purification was monitored by both conventional SDS-PAGE and substrate SDS-PAGE. The modified method of Laemmli was used for conventional SDS-PAGE [19]. Samples in 0.5 M Tris hydrochloride (pH 6.8), (containing 20% (v/v) glycerol and 2% (w/v> SDS) were loaded on to 15% resolving gels with 4% stacking gels, with or without boiling and pretreatment with 5% (v/v) 2-mercaptoethanol (2-ME). Sample protein concentrations were determined by the method of Bradford [20]. Gels were electrophoresed at 25 mA/gel and protein bands were identified by staining with Coomassie brilliant blue R-250. Low range molecular mass markers (Bio-Rad) in the range 18,500-106,000-M,, were also electrophoresed on the gels. General proteinase activity was examined using SDS-PAGE gels copolymerized with 0.5% gelatin (Sigma). A modification of the method of Heussen and Dowdle was used [21]. Samples were not boiled or reduced, and the stacking gel did not contain substrate. After electrophoresis at 25 mA/gel, the gels were washed in 2.5% (v/v) Triton X-100 (Sigma) for 30 min prior to overnight incubation at 37°C in PBS. Bands of proteolytic activity were demonstrated by staining the gels with Coomassie brilliant blue R-250. In some instances samples of purified proteinase were incubated with the irreversible serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF, at a concentration of 1 mM or 10 mM) as described below, prior to loading on to the gels.
Medical
Microbiology
13 (IYY6)
13/-140
133
2.4. isoelectric focusing Purified enzyme was electrophoresed on an isoelectric focusing gel (1% (w/v) agarose (Sigma), 11% (w/v) sorbitol (Sigma)) containing 2% Biolyte 3/ 10 ampholytes. Standards (Sigma) in the pH range 4.5-10.5 were used. 2.5. Proteolytic assays 2.5.1. Azocasein degradation 200 ~1 of 2% (w/v) azocasein (Sigma) in 0.1 M phosphate buffer (pH 8.5) supplemented with 0.01 M MgC12 was incubated with 20 ~1 of the sample to be tested (or 1 pg of purified proteinase) for 24 h at 37°C. The reaction was terminated by the addition of 400 ~1 of 5% (w/v) trichloroacetic acid. After centrifugation at 13,000 X g for 5 min 400 ~1 of the supematant was added to 800 ~1 of 0.4 M sodium hydroxide and the optical density COD) was determined at 440 nm. One unit of azocasein degrading activity was defined by an increase in OD of 0.01 under the conditions described. 2.5.2. Azoalbumin degradation Methodology was as described for azocasein. A solution of 59.4 mg ml-’ azoalbumin (Sigma) in 0.5 M Tris hydrochloride (pH 8.5) was used.One unit of azoalbumin degrading activity was defined by an increase in OD of 0.01 under the conditions described. The azocasein degradation assay and all other assays described below were performed in duplicate. 2.6. pH optimum The pH optimum of the purified proteinase was determined using azocasein and azoalbumin substrates as described above, with the following overlapping buffer systems replacing the 0.1 M phosphate buffer; for the pH range 5.0-8.0, 0.05 M citric acid, 0.1 M disodium phosphatebuffer: for pH 7.09.5, 0.05 M Tris hydrochloride and for pH 8.5-10.5, 0.05 M sodium carbonate. Samples without added proteinasewere used as controls over the various pH ranges.
134
I. Lcrmhkin
et al. / FEMS
Imnwdo,~~
trnd Medical
2.7. Inhibition assr~ys Stock solutions (100 ~1) of proteinase inhibitors (EDTA, N-ethylmaleimide (NEM), tosyl Iysyl chloromethyl ketone (TLCK), iodacetamide (IAA), leupeptin and L-trclns-epoxysuccinyl leucylamido(4guanidino)butane (E64) in water; pepstatin, I,1 Ophenanthroline, phenylmethylsulfonyl fluoride (PMSF), tosyl phenylalanyl chloromethyl ketone (TPCK) in methanol; and chymostatin in dimethylsulfoxide (DMSO) (all reagents from Sigma)) were incubated with 1 pg of purified proteinase solution in azocasein assays. Final concentrations of all reagents are shown in Table I. Residual activity was compared to that of a reaction mixture without inhibitors. For inhibitors made up in methanol and DMSO an equivalent amount of solvent was run as a control. 2.8. Substrate specijkity 2.8. I. A,-ocoll degrudution A 3-5 mg sample of azocoll (Sigma) in 500 ~1 of 0.05 M Tris hydrochloride (pH 8.5), supplemented with 0.005 M CaCI, was incubated with 1 pg of purified proteinase for 24 h at 37°C. The reaction was terminated by the addition of 0.5 ml of 0.01 M EDTA. After centrifugation at 13,000 X g for 5 min the released dye in the supernatant was measured Table I Effect of proteinase inhibitors and metal ions seinolytic activity of the 34,000-M, proteinase
on the azoca-
Inhibitor
Final concentration
Inhibition
Chymostatin PMSF EDTA I. IO-Phenanthroline TPCK TLCK Pepstatin A Leupeptin Iodacetamide E64 N-ethylmaleimide Zn’+ ca? +
0.1 mM I mM I mM I mM 0.1 mM 0.2 mM I mM 0.1 /.LM 0.1 /JM I mM IO PM 5mM 5mM 5mM
66 64 30 20 0 0 0 0 0 0 0 46 23 23
Mg” ‘I All assays were reported.
performed
in duplicate
(‘7&J a
and mean values
Microbiology
13/L
I-IO
spectrophotometrically at 530 nm. One unit of azoco11 degrading activity was defined as the amount of enzyme which produced an increase in OD of 0.1 under the conditions described. 2.8.2. Kerutin Five mg of keratin azure (Sigma) in 300 ~1 of 0.1 M glycine-sodium hydroxide (pH 8.5) was incubated with I pg of purified proteinase for 24-72 h at 37°C prior to centrifugation at 13,000 X g for 5 min. The OD of the supernatant was measured at 595 nm. One unit of keratinase activity was defined as the amount of enzyme which produced an increase in OD of 0.01 under the conditions described. 2.8.3. Laminin and ,fibronectin Ten ~1 of each substrate (at a concentration of 1 mg ml-’ - both from Sigma) was incubated with 1 pg of purified proteinase solution for 24-72 h at 37°C prior to analysis on a 10% SDS-PAGE gel. Samples were pretreated with 5% (v/v> 2-ME and boiled for 3 min. 2.84. Elastin Five pg of elastin orcein (Sigma) in 300 ~1 of 0.2 M Tris hydrochloride (pH 8.5) was incubated with 1 pg of purified proteinase for 24 h at 37°C prior to centrifugation at 13,000 X g for 5 min. The released dye in the supematant was measured spectrophotometrically at 530 nm. One unit of elastin orcein degrading activity was defined as the amount of enzyme which produced an increase in OD of 0. I under the conditions described. 2.9. N-terminal
are
13 ClYY6)
urnino acid sepencing
Purified proteinase samples were boiled, treated with 2-ME and electrophoresed on 15% SDS-PAGE gels (stored overnight at 4°C with 0.002 M thioglycollie acid (Sigma) in the upper buffer chamber prior to use). Electrophoresed proteinase was transferred onto a polyvinylidene fluoride membrane using standard Western blotting methodology [22] and stained with Coomassie brilliant blue R-250. The proteinase band was excised and analysed on a 477A Protein Sequencer (Applied Biosystems, England) at The Protein Sequencing Unit, Biochemistry Department, University of Cambridge. Cambridge, England.
3. Results 3.1. Proteinase pur$cation Two hundred ml volumes of cytoplasmic antigen (70 mg total protein) from 5 1 of T. rubrum F1478 Sabouraud glucose broth cultures in late log to early stationary phase were utilised for proteinase purification. Preliminary studies had indicated that the 34,000-M, proteinase was not detectable prior to late log phase and that after this time it represented the dominant proteolytic species in cytoplasmic antigen preparations (data not shown). Fig. 1 illustrates the typical elution profiles obtained during Rotofor isoelectric focusing (Fig. la). refractionation on the Rotofor system (Fig. lb) and Superose 12 gel filtration chromatography (Fig. 1~). Two peaks of azocasein degrading activity (Peak I, fractions 7-10 and Peak 2, fractions l3- 16) were obtained by isoelectric focusing (Fig. la). Substrate SDS-PAGE analysis indicated that two major bands of gelatin degrading activity with relative molecular masses of 37.000-M, and 23,000-M, correlated with the azocasein degrading activity in Peak 1 (Fig. 2, lane A). Refractionation of the Peak 1 fraction on the Rotofor system resulted in further separation of the two proteolytic species (Fig. lb and Fig. 2, lanes B and C) and after 1-3 cycles of gel filtration chromatography (Fig. lc and Fig . 2, lanes D-G) the 37,000-M, species was purified to homogeneity by removal of the contaminating 23.000-M, species. The purification of the 37.000-M, proteinase as viewed on reduced SDS-PAGE gels is summarised in Fig. 3. The relative molecular masses of the purified proteinase were estimated as 37,000-M, on substrate SDS-PAGE gels (Fig. 2). 15.000-M, on reduced conventional SDS-PAGE gels (Fig. 3) and 34,000-M, on non-reduced SDS-PAGE gels (data not shown). It was concluded that the proteinase existed as a 34,000-M, molecule in its native state and that it was composed of subunits of 15.000-M, which could be visualised after disruption with 5% 2-ME. The discrepancy in the relative molecular masses of the 34,000-M, nonreduced protein on SDS-PAGE gels and the 37,000M, proteolytic band on substrate SDS-PAGE gels almost certainly reflected the interaction of the enzyme with the gelatin substrate during electrophoresis on substrate SDS-PAGE gels. The relative molec-
ular mass of 34,000-M, was deemed more accurate and will be used to define the proteinase. Attempts to quantify the purification, yield and specific activity
a
Fig. I. Purification of the 34,000-M, proteinase by (a) Rotofor isoelectric focusing. (b) refractionation on the Rotofor system and (c) gel filtration FPLC (example of final gel filtration cycle). The histogram represents azocasein degrading activity (U m- ’ ), closed squares gradient
the protein (the latter
concentration (mg ml-’ ) and is shown in a and b only).
wedges
the pH
0' 5
Fig. 2. Analysis of the purification of the 37,000-M, proteinase by substrate SDS-PAGE. Lane A, Rotofor isoelectric focusing cycle I fraction 7 (tracks 7-11 contained the 37.000-M, and 23,000-M, proteinases); lanes B and C. refractionation of Peak 1 fractions on the Rotofor system, fractions 7 and 9 respectively; lanes D-G. Superose 12 HR IO/30 gel filtration fractions IS-21 containing the pure 37,000-M, proteinaae after 2 gel filtration cycles.
of the proteinase were hindered by the presence of multiple proteolytic species with varying specificities for the azocasein substrate. Estimates of the total protein concentration and total enzyme activity prior to purification (70 mg and 2400 Units respectively) and after purification to a homogeneous band ( < 0.04 mg and 6 Units respectively) provide a partial indication of the extent of purification achieved.
5.5
6
6.5
7
8
7.5
6.5
9
9.5
I 10 10.:
PH Fig. 4. pH profile of the 34,000-M, proteinase using azocasein and azoalbumin substrates. The wedges and closed squares represent the azocasein and azoalbumin substrates respectively.
was active over a broad pH range with high activity against azocasein in the pH range 6.5-10.5. Using azoalbumin the enzyme was highly active over the pH range 6.0-8.5 and maintained a constant level of activity from pH 8.5-10.5. The proteinase migrated during Rotofor isoelectric focusing with a pl of between 3.4-4.4. The pl was more accurately determined as 4.5 on an isoelectric focusing gel (data not shown). 3.3. Effect of proteinuse inhibitors und metal ions
3.2. Optimal pH and isoelectric point (pli Using azocasein and azoalbumin substrates in spectrophotometric assays the optimal pH of the 34.000-M, proteinase was 7.5 (Fig. 4). The enzyme A
-
6
CD
E
FGH zf
1’8”o” -49
Table 1 illustrates the inhibition profile of the 34.000-M, proteinase. The serine proteinase inhibitor PMSF inhibited the enzyme, together with chymostatin. which inhibits chymotrypsin-type serine proteinases. The metalloproteinase inhibitors EDTA and 1.10 phenanthroline inhibited the proteinase to a A
B
C
D
-32.5 w-
-27.5
Fig. 3. Analysis of the purification of the proteinase by conventional SDS-PAGE. Lane A, crude cytoplasmic antigen; lanes B and C. refractionated Rotofor fractions 7 and 9 respectively: lane D. Superose 12 HR IO/30 gel filtration fraction 19; lanes E-G. gel filtration fractions 18-20 after 2 gel filtration cycles; lane H. Bio-Rad low range standard molecular mass marker proteins.
Fig. 5. Substrate SDS-PAGE analysis of the inhibition of proteinase activity by the irreversible serine proteinase inhibitor PMSF. Lane A, 37,000-M, uninhibited proteolytic band; lane B, 37,000-M, proteolytic band after exposure to I mM PMSF; lane C, 37,000-M, proteolytic band after exposure to IO mM PMSF; lane D. Bio-Rad low range standard molecular mass marker proteins.
Table 2 Substrate
specificity
of the 33.000-M,
Suhstrate
Activity (Units
Arocasein
mg-
’ protein)
“
14.600 12.600
Aroalbumin Azocoll h Keratin El&in ’ All assays reported.
proteinase
I52 140 122 were
performed
m duplicate
and
mean
values
are
h One unit of azocoll degrading activity was defined by an increase of 0.1 in optical density under the conditions described. For all other substrates an increase of 0.01 was used.
lesser extent in these assays. Of the metal ions Zn’+ showed marked inhibitory activity and there was some inhibition by Ca” and Mg”. No other inhibitors, including cysteine and aspartic proteinase inhibitors, had any effect. Fig. 5 illustrates the effect of the irreversible serine proteinase inhibitor PMSF on the 37.000-M, proteolytic band on substrate SDS-PAGE gels. Following incubation with 1 mM PMSF the intensity of the 37.000-M,. proteolytic band was reduced by approximately 50% (Fig. 5. lane B). No proteolytic band could be detected after incubation with 10 mM PMSF indicating that complete inhibition had occurred (Fig. 5. lane C).
was less affected after exposure to the proteinase for 24 h (Fig. 6b, Lanes C. D) but progressive degradation occurred with incubation time (Fig. 6b, lanes E, F and G. H, respectively). After exposure to the proteinase for 72 h multiple weak degradation products in the range > 106,000-M, to approximately 32,500-M, were observed. The complete degradation of laminin to products of less than 49.000-M, observed with the positive control proteinase (Src~~l~~lococc~rs uureeus XVII, Fig. 6b. lane I) did not occur. 3.5. N-trrminal
amino acid sequence
Table 3 illustrates the N-terminal amino acid sequence of the 34,000-M, proteinase (in its reduced 15.000-hil, form) and the homology shared by this sequence with the previously published deduced
(a)
A
B
C
D
E
F
G
H
I
-106 -80
3.4. Substrute specijki~ The substrate specificity of the 34,000-M,. proteinase was assayed at pH 7.5 using azocoll, azocasein. azoalbumin, keratin and elastin substrates as well as laminin and fibronectin. The proteinase actively degraded azocasein. azocoll and azoalbumin while weaker activity (ten fold less) was detected against keratin and elastin (Table 2). Fibronectin (Fig. 6) was actively degraded within 24 h of exposure to the proteinase producing multiple degradation products of less than 106.000-M, (Fig. 6a, lanes C, D). Similar degradation products were observed after incubation for 48 h and 72 h (Fig. 6a, lanes E, F and G, H. respectively). The complete degradation of fibronectin to products of less than 49,000-M, observed with a positive control proteinase (Sraph$ococcus c7~1re14s XVII (Sigma P8400). Fig. 6a. lane I) did not occur with the 34,000-M, proteinase. Laminin
(blA \ ,
B
C
D
E
F
G
H
I
-106 -80
Fig. 6. Degradation 34,000-M, proteinase.
of
(a) All
fibronectin samples
and (b) were boiled
laminin and
by the reduced.
Lanes A. B, fibronectin or laminin untreated, 24 h; lanes CD, EF. GH. fihronectin or laminin proteinase treated, 24 h, 4X h and 72 h respectively: lane 1. fibronectin or laminin treated with Stophdoco~xs
UY~II.S
XVII
control
proteinase.
24 h.
Table 3 N-terminal amino acid sequence of the 34,000-M, proteinase and its homology with the C’. imnlirit proteinase (from GENEMBL sequence data bank)
Rotein
Amino acid sequence
T rubrum pmteinase
WS~PK~NAG~l,T~ T
C immiris
proteinax
’ Percentage
sequence
LSYD
YD
Identity
LL
v
(%%)
50% 120 amino acids)
TADhSQTHYDDPSIJ’UFY
identity
in amino acid overlap.
amino acid sequence of a Coccidioides immitis chymotrypsin-type serine proteinase. The 15,000-M, proteinase shared 50% homology with the C. immitis 34,000-M, proteinase.
4. Discussion A novel 34.000-M, intracellular proteinase has been purified to homogeneity from cytoplasmic antigen preparations of T. rubrum. To our knowledge this is the first report of the purification and characterisation of an intracellular proteinase from T. rubrum and it confirms the observation of Chattaway et al. that T. rubrum mycelia contained enzymes with the specificities of trypsin and chymotrypsin [9]. The purification of the 34,000-M, proteinase was relatively straightforward due to the high protein content of the late log phase mycelial mat and the high levels of the proteinase present. However the presence of other intracellular proteinases during the purification of the 34,000-M, enzyme makes it inappropriate to quantitate values for specific activity and total yield for the 34,000-M, molecule alone as it is likely that the other proteolytic species may contribute to these parameters prior to purification. A 34,000-M, proteinase was also detectable in culture filtrate in late log/early stationary phase cultures. This may be related to the intracellular proteinase of the same molecular mass and this possibility is currently being investigated. The age of the cultures from which this proteinase was identified makes it likely that this represents a lysis product rather than a genuine extracellular product.
The single 37,000-M,. proteolytic species on substrate SDS-PAGE gels represented the gelatin degrading activity of the single protein band of 34,000M, and 15,000-M, on non-reduced and reduced SDS-PAGE gels respectively. Retardation of fungal proteinase migration in substrate SDS-PAGE gels due to the interaction of the enzyme with the substrate has been reported with a difference in apparent molecular mass of 6000-M, observed between the proteolytic band on substrate SDS-PAGE gels and the non-reduced proteinase on conventional SDSPAGE gels [23]. The 34,000-M, non-reduced molecular mass of the T. rubrum intracellular proteinase is accordingly the more accurate representation of the protein in its native state. Disruption of the protein produced non-proteolytic bands of 15,000-M, suggesting that the protein normally exists as an active disulfide-linked dimer in a similar manner to the extracellular T. rubrum 7 1,000-M, and 90,000-M, dimeric proteinases which consist of subunits of 36,000-M, and 44,000-M,. respectively [5]. The relationship of the 34,000-M, enzyme to previously described proteinases from both T. rubrum and other fungal pathogens is of considerable interest. The 34,000-M, proteinase shares some general characteristics with the previously described T. rubrum extracellular proteinases. A T. rubrutn proteinase of similar molecular mass (34.700-M,) [6] was previously described as an extracellular product. In contrast to the proteinase described here however the molecular mass reported was the same for this enzyme in both the reduced and non-reduced states [6]. The optimum activity against azocasein and azoalbumin substrates at pH 7.5 correlated relatively closely with the alkaline pH optima of 8.0 against azocoll exhibited by the previously reported 27,000M, [7]. 71.000-M, and 90,000-M, proteinases [5] together with the 235,000-M, proteinase recently described by our group [24] which exhibited an optimum pH of 8.5 against azocasein and azoalbumin substrates. All of the above mentioned proteinases exhibited substantial activity within the pH range 7.0 to 10.0. In common with the 27,000-M, and 235,000-M, extracellular proteinases and the majority of T. rubrum proteins (as demonstrated by Rotofor isoelectric focusing) the 34,000-M, proteinase had an isoelectric point of less than 5.0. This contrasts with the previously described 7 1,000-M,
and 90,000-M, extracellular proteinases which exhibit isoelectric points of 6.5 and 7.8 respectively. The 34,000-M,. intracellular proteinase demonstrated a similar pattern of inhibition to the previously described T. rubrum 23.5,000-M, extracellular proteinase when incubated with a range of proteinase inhibitors and metal ions [24]. Thus the inhibitory action of the serine proteinase inhibitor phenylmethylsulfonyl fluoride together with the chymotrypsintype serine proteinase inhibitor chymostatin suggested that the 34,000-M, enzyme was a chymotrypsin-type serine proteinase. However there are two observations which may elucidate the relationship of the 34,000-M, proteinase with other fungal proteinases. Firstly, the 34,000-M, proteinase is weakly inhibited by the metalloproteinase inhibitors EDTA and 1. IO-phenanthroline as well as the metal ions Ca’+. Mg’+ and Zn’+. With the exception of the 235,000-M, proteinase this pattern of inhibition has not been identified in T. rubrum proteinases. although similar inhibition profiles have been observed for an A. niger semi-alkaline aspergillopeptidase [25] and an A. ,fi4n~igarus elastase 1261. It is of significance that the A. niger aspergillopeptidase has an isoelectric point of 4.1 (compared to 4.5 for the T. rubrum enzyme) and a pH optimum of 7.8 (compared to 7.5) suggesting a relationship between the two enzymes. Secondly. based on N-terminal amino acid sequence data, the 34,000-M, T. rubrum proteinase appears to be related to a C. immitis wall-associated chymotrypsin-type serine proteinase. The C. immitis proteinase exhibits collagenolytic and elastinolytic activity, has an isoelectric point of 4.5 and is inhibited by chymostatin. While this enzyme is larger than the T. rubrum proteinase it shares the low pl and the active disulphide-linked dimeric native state of the T. rubrum enzyme. The C. immitis proteinase appeared to be concentrated in the walls of the parasitic cells at stages of active growth and it has been suggested that it plays a role in cell wall turnover or in wall plasticization. The C. imrnitis proteinase shares a conserved region of 75% homology with human ru-chymotrypsin and chymotrypsinogen from various sources and the gene for the proteinase is the only fungal chymotrypsin-type serine proteinase gene isolated to date. Indeed. of the fungal serine proteinases so far
characterised most. with the limited exception of enzymes such as the C. imnzitis 34.000-M, wall-associated proteinase and the A. niger 21,000-M, semi-alkaline aspergillopeptidase described above, have been classed as subtilisin-type serine proteinases [25]. Our data is strongly suggestive of a relationship between the 34,000-M, T. rubrum enzyme described here and both the C. immitis and A. rziger enzymes. It has been postulated that the latter play a role in the pathogenesis of their respective diseases and. based on biochemical similarities, the T. rubrum enzyme may fulfill a similar function. Preliminary investigations. via the production of antisera for immunochemical studies, are under way to explore this possibility.
Acknowledgements We wish to thank Dr. Mary Moore and Dr. Gillian Midgely of the Department of Medical Mycology. St. John’s Institute of Dermatology, St. Thomas’s Hospital. London for supplying the fungal isolate. This research was funded by a grant from Sandoz Pharma Ltd., whose help is gratefully acknowledged.
References [I]
Philpot. phyteh:
[2]
Sinski. phyte\
M. (1978) a review.
Geogmphical diatributmn J. Hyg. 80. 30-3 13.
J.T. and Fluoras. isolated from 1979
of worldwide inciidence from the United States. [x] [3]
K. (1984) A survey of to 1981 with chronological
dermatolistings
of five dermatophytes often Mycopathologia 85. 97-120.
isolated
De Vroey. C. (1985) Epidemiology of ringworm (dermatophytosis). Semin. Dermatol. 3. I X5- I9 I Elewski. B.E. (1992) Superficial mycoses. dermatophytoses and selected ics in Clinical pp. 12-6X.
[s]
of the dermato-
Asahi, stein.
M.. W.L.
dermatomycoaes. Dermatology:
In: Elewslil. Cutaneous
Lindquist, R.. Fukuyama, and McKerrow. J.H.
characteriwtion of major extracellular c/w~~hw~n ruhnrrn. Biochem. J. 332.
K.. (1985)
B.E. Fungal
(Ed.). TopInfections.
Apodaca. Purification
proteinases 139-I-W.
G.. Epand from
Tri-
161 Sanyal. A.K.. Da\. S.K. and Banerjee. A.B. (1985) Purification and partial characterisation of an extracellular protcinase from Tri&yhvtofz n~hnrr~~. J. Med. Vet, Mycol. 23, 165- 178. [7] Apodaca. G. and McKerrow. J.H. (1989) Purification and characterisation Tric+wph~ton
of a 27.000-M, whnrr~ Infect.
extracellular proteinase Immun. 57. 3072-3080.
from
140
1.
[8] Apodaca, TrichophWm
Lmnhkir7
G. and McKerrow. r&rum proteolytic
~1 al. / FEMS
Immunobg~
J.H. (1989) Regulation activity. Infect. Immun.
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308 I-3090. [9] Chattaway, F.W., Ellis, D.A. and Barlow, J.E. (1963) Peptidases of dermatophytes. J. Inv. Derm. 41, 3 l-37. [IO] Yu. R.J., Harmon, S.R.. Grappel, SF. and Blank, F. (1971) Two cell-bound keratinases of Trichophyton nzenrn~roph+a. J. Inv. Dermatol. 56, 27-32. [I I] Grappel, S.F. and Blank, F. (1972) Role of keratinasea in dermatophytosis. 1. Immune responses of guinea pigs infected with Trichophwm menttrgroph~tes and guinea pigs immunised with keratinases. Dermatologica. 145. 245-255. [I21 Eleuterio, M.K.. Grappel, S.F.. Caustic. C.A. and Balkk, F. (1973) Role of keratinases in dermatophytosis. III. Demonstration of delayed hypersensitivity to keratinases by the capillary tube migration test. Dermatologica 147. 255-260. [13] Kalisz. H.M. (1988) Microbial proteinases. Adv. Biochem. Eng/Biotechnol. 36, 3-65. [14] Holzer, H., Betz, H. and Ebner, E. (1975) Intracellular proteinases in microorganisms. Curr. Top. Cell. Regul. 9. 103-156. [I51 Tsujita, Y. and Endo, A. (1980) Intracellular localization of two molecular forms of membrane acid protease in Aspergillus orwze. J. B&hem. 88. 113- 120. [ 161 Ross. I.K., De Bernardis. F.. Emerson. G.W., Cassone. A. and Sullivan. P.A. (1990) The secreted aspartate proteinase of Candida alhicrrrzs: physiology of secretion and virulence of a proteinase-deficient mutant. J. Gen. Microbial. 136. 687-694.
[17] Banerjee. A.. Ganesan. K. and Datta A. (1991) Induction of secretory acid proteinase in Ccrndidu ~lhicnns. J. Gen. Microbiol. 137. 2455-2461.
Medical
Mrcroholog~
13 (IW6J
131-140
[ 181 Homma. M.. Kanbe, T., Chibana, H. and Tanaka. K. (1992) Detection of intracellular forms of secretory aspartic proteinase in Crrrzdidr~ nlhicans. J. Gen. Microbial. 138, 627633.
[ 191 Laemmli. U.K. (I 970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227. 680-685. [20]
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. 248254.
[2 I] Heussen, C. and Dowdle. E.B. ( 1980) Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulphate and copolymerised substrate. Anal. Biochem. 102, 196-202. [22] Towbin, H., Staehelin. T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Nat]. Acad. Sci. USA 76, 4350-4354. [23] Cole, G.T., Zhu. S.. Pan, S., Yuan, L.. Kruse, D. and Sun. S.H. (1989) Isolation of antigens with proteolytic activity from Coccidioidus hnitis. Infect. Immun. 57, 1524- 1534. [24] Lambkin. 1.. Hamilton. A.J. and Hay, R.J. (1994) Partial purification and characterisation of a 235,000-M, extracellular proteinase from Tric.ho@,vron rzthrum. Mycoses 37, 8592.
Barthomeuf, C.. Pourmt, H. and Pourrat. A. (1989) Properties of a new alkaline proteinase from Aspergillus niger. Chem. Pharm. Bull. 37. 1333-1336. [26] Fresco. M., Chase, T. and MacMillan, J.D. (1992) Purification and properties of the elastase from Asprrgillus $migurus. Infect. Immun. 60. 728-734.
[25]
AND
MICROBIOLOGY FEMS
Immunology
and Medical
Microbiology
I3 (1996)
I3 I - I40
Purification and characterisation of a novel 34,000-M, cell-associated proteinase from the dermatophyte Trichophyton rubrum Imelda Lambkin * , Andrew J. Hamilton, Rod J. Hay Drrnzntolo~~~
Unit.
Clinico1
Scierws
.!dm-orory,
Received
26 September
18th
Floor,
GU,Y.T Tower,
Gum
1995; accepted 27 October
Ho.spikd.
London
SE/
YRT, UK
1995
Abstract A novel cell-associated proteinase was purified to homogeneity from cytoplasmic antigen preparations of Trichophyton by sequential isoelectric focusing and gel filtration chromatography. The enzyme exhibited relative molecular masses of 34,000-M, (non-reduced sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)), 15.000-M, (reduced SDS-PAGE) and 37.000-M, (substrateSDS-PAGE).It had a pH optimumof 7.5 and a pi of 4.5. The proteinase exhibited broad substrate specificity and it was strongly inhibited by the serine proteinase inhibitors phenylmethylsulfonyl fluoride and chymostatin. The N-terminal amino acid sequence of the 34,000-M, proteinase shared 50% homology with the deduced amino acid sequence of a Coccidioides irnmitis wall-associated chymotrypsin-type serine proteinase. This is the first cell-associated proteinaseto be purified and characterised from T. rubrum and it would appear to be related to the chymotrypsin-type serine proteinases, a class of enzymes that have rarely been isolated from fungi. The function of the rubrurn
proteinaseremainsspeculativealthoughit may play a role in the developmentandsubsequent proliferationof the fungusin vivo. Keww&:
Trichoph~or~
rrrhrron:
Cytoplasmic
antigen:
Cell-associated
1. Introduction
proteinase
* Corresponding author. Present address: Elan Corporation Research Institute. Trinity College, Dublin 2. Ireland. Tel.: + 353 (I) 67 I-090 I : Fax: + 353 (I ) 67 I-0920.
on the production of extracellular proteinases[5-81. However, apart from one early study which described the extraction of peptidases and enzymes with the specificities of trypsin and chymotrypsin from T. rubrum mycelia [9], there have been no definitive reports on the purification or characterisation of intracellular proteinasesfrom T. rubrurn or on the potential relationship of intracellular proteinaseswith the previously described extracellular proteinases.The potential significance of intracellular proteinasesin dermatophytes is illustrated by the two cell-bound keratinasespurified from liquid cultures of T. mentugrophytes rar. granulosum [lo],
0928-X241/96/$1
Societies.
Trichophyton rubrum is a cosmopolitan anthropophilic dermatophyte which causesinfection of the trunk, groin, foot and nail. It is the most common recorded cause of human dermatophytoses worldwide accounting for approximately 70-89% of all reported cases[l-4]. Studies on the factors influencing the virulence of this dermatophyte have focused
SSDl
5.00 8 1996 Federation
0928-8243(95)00095-X
of European
Microbiological
All rights reserved
which have been shown to induce delayed type hypersensitivity reactions in cutaneously infected guinea pigs [ 11,121. There have been a number of reports of intracellular proteinases in other pathogenic fungi. Proteinases have been observed in the vacuoles of Cundidu albicans, Saccharomyes cerecisiae, Neurosporu crassa, and the dermatophyte fungus Microsporum gypseum [ 131 and it has been suggested that intracellular proteinases are contained in membrane-bound compartments similar to the mammalian lysosome in eucaryotic organisms [ 141. Signal peptidases, which catalyse the removal of signal peptides from secretory proteins have also been observed in the rough and smooth microsomes of Aspergillus oryae [ 151. A number of studies have focused on the production and secretion of the 43,000-M, C. albicans aspartic proteinase (CAP) [ 16- 181 and these have provided the first information on the relationship between proteinases observed within the fungal cell and secreted proteinases. Intracellular forms of CAP have been detected by immunoblotting using anti-extracellular proteinase antiserum [ 1S] and it has been hypothesised that a 45,000-M, protein is a precursor form of the 43,000-M, secretory enzyme. In this paper the purification of a novel 34,000-M, proteinase from preparations of T. rubrum cytoplasmic antigen is described. The proteinase was purified to homogeneity by sequential Rotofor isoelectric focusing and gel filtration fast protein liquid chromatography (FPLC) and the purification process was monitored using SDS-PAGE and substrate SDSPAGE. The 34,000-M, proteinase was characterised by pH optima, isoelectric point, inhibition profile and N-terminal amino acid sequence analysis and the specificity of the proteinase for a range of substrates was determined. The relationship of the 34,000-M, intracellular proteinase with the previously isolated T. rubrum extracellular proteinases and other fungal proteinases is discussed.
2. Materials
and methods
2. I. Organisms and cultication
methods
A fresh clinical isolate of T. rubrum (strain F1478) was obtained from the Department of Medical My-
cology, St. John’s Institute of Dermatology, St Thomas’s Hospital, London, England. It was maintained on Sabouraud glucose agar slants (containing 2% w/v D( + )-glucose (Sigma, Poole: England), 1% w/v Bacto-peptone (Gibco, Uxbridge. England). 1.5% w/v Oxoid bacteriological agar (Unipath, Basingstoke, England), in I 1 of distilled water) stored at 4°C and was subcultured every 3 months. Spore suspensions were prepared by washing agar slants with distilled water after incubation for 14 days at room temperature. To examine proteinase production the spore suspensions were incubated in Sabouraud glucose broth (containing 2% w/v D( +)-glucose (Sigma), 1% w/v Bacto-peptone (Gibco). and 0.25% w/v sodium-P-glycerophosphate (Fisons, Loughborough, UK)) without agitation until cultures had reached late log/early stationary phase (approximately 21 days at room temperature). 2.2. Pur$ication
of the proteinase
Cultures were filtered using Whatman no. 1 paper and the harvested mycelia were humogenised in a bead beater (Biospec Products. Oklahoma, USA) containing 5 mm glass ball ballotini (Stratech Scientific, Bedfordshire, UK) and 50 ml of phosphatebuffered saline (PBS, 0.01 M, pH 7.4). The homogenate was centrifuged at 10,000 X g for 2 h and the supernatant cytoplasmic antigen preparation was recovered. After dialysis against distilled water for 2 h at room temperature the cytoplasmic antigen was loaded onto a Rotofor isoelectric focusing cell (BioRad, Hemel Hempsted. England). Broad range (pH 3-10) Biolyte ampholytes (Bio-Rad) were used and the system was focused for 4 h at 12 W constant power. After focusing the Rotofor fractions were collected and tested for pH, protein concentration and azocasein degrading activity (see below). The relevant fractions were concentrated on Microsep 1 kDa centrifugal concentrators (Flowgen Instruments Ltd., Kent, England), analysed by conventional and substrate SDS-PAGE (see below) and were subsequently pooled and refocused on the Rotofor system. Following the same procedure selected secondary Rotofor fractions were pooled, concentrated and loaded on to a Superose 12 HR lo/30 FPLC gel filtration column (separation range 1000-300,000M,) (Pharmacia, Milton Keynes, UK). The column
I. Lumbkirz
et ul./
FEMS
Immunolng~
and
was equilibrated using 0.05 M Tris hydrochloride (pH 8.4). Samples were eluted at a flow rate of 0.6 ml/min in the same buffer and 1 ml fractions were collected and tested for protein concentration and azocasein degrading activity (see below). Two to three gel filtration chromatography cycles were performed. Culture filtrates were concentrated to l/500 of the original volume by dialysis against neat polyethylene glycol 8000 (BP Chemicals, UK) in presoaked dialysis visking membrane (Medicell International, London, UK). Samples were then processed for substrate SDS-PAGE analysis as described below. 2.3. SDS-PAGE and substrate SDS-PAGE analysis Purification was monitored by both conventional SDS-PAGE and substrate SDS-PAGE. The modified method of Laemmli was used for conventional SDS-PAGE [19]. Samples in 0.5 M Tris hydrochloride (pH 6.8), (containing 20% (v/v) glycerol and 2% (w/v> SDS) were loaded on to 15% resolving gels with 4% stacking gels, with or without boiling and pretreatment with 5% (v/v) 2-mercaptoethanol (2-ME). Sample protein concentrations were determined by the method of Bradford [20]. Gels were electrophoresed at 25 mA/gel and protein bands were identified by staining with Coomassie brilliant blue R-250. Low range molecular mass markers (Bio-Rad) in the range 18,500-106,000-M,, were also electrophoresed on the gels. General proteinase activity was examined using SDS-PAGE gels copolymerized with 0.5% gelatin (Sigma). A modification of the method of Heussen and Dowdle was used [21]. Samples were not boiled or reduced, and the stacking gel did not contain substrate. After electrophoresis at 25 mA/gel, the gels were washed in 2.5% (v/v) Triton X-100 (Sigma) for 30 min prior to overnight incubation at 37°C in PBS. Bands of proteolytic activity were demonstrated by staining the gels with Coomassie brilliant blue R-250. In some instances samples of purified proteinase were incubated with the irreversible serine proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF, at a concentration of 1 mM or 10 mM) as described below, prior to loading on to the gels.
Medical
Microbiology
13 (IYY6)
13/-140
133
2.4. isoelectric focusing Purified enzyme was electrophoresed on an isoelectric focusing gel (1% (w/v) agarose (Sigma), 11% (w/v) sorbitol (Sigma)) containing 2% Biolyte 3/ 10 ampholytes. Standards (Sigma) in the pH range 4.5-10.5 were used. 2.5. Proteolytic assays 2.5.1. Azocasein degradation 200 ~1 of 2% (w/v) azocasein (Sigma) in 0.1 M phosphate buffer (pH 8.5) supplemented with 0.01 M MgC12 was incubated with 20 ~1 of the sample to be tested (or 1 pg of purified proteinase) for 24 h at 37°C. The reaction was terminated by the addition of 400 ~1 of 5% (w/v) trichloroacetic acid. After centrifugation at 13,000 X g for 5 min 400 ~1 of the supematant was added to 800 ~1 of 0.4 M sodium hydroxide and the optical density COD) was determined at 440 nm. One unit of azocasein degrading activity was defined by an increase in OD of 0.01 under the conditions described. 2.5.2. Azoalbumin degradation Methodology was as described for azocasein. A solution of 59.4 mg ml-’ azoalbumin (Sigma) in 0.5 M Tris hydrochloride (pH 8.5) was used.One unit of azoalbumin degrading activity was defined by an increase in OD of 0.01 under the conditions described. The azocasein degradation assay and all other assays described below were performed in duplicate. 2.6. pH optimum The pH optimum of the purified proteinase was determined using azocasein and azoalbumin substrates as described above, with the following overlapping buffer systems replacing the 0.1 M phosphate buffer; for the pH range 5.0-8.0, 0.05 M citric acid, 0.1 M disodium phosphatebuffer: for pH 7.09.5, 0.05 M Tris hydrochloride and for pH 8.5-10.5, 0.05 M sodium carbonate. Samples without added proteinasewere used as controls over the various pH ranges.
134
I. Lcrmhkin
et al. / FEMS
Imnwdo,~~
trnd Medical
2.7. Inhibition assr~ys Stock solutions (100 ~1) of proteinase inhibitors (EDTA, N-ethylmaleimide (NEM), tosyl Iysyl chloromethyl ketone (TLCK), iodacetamide (IAA), leupeptin and L-trclns-epoxysuccinyl leucylamido(4guanidino)butane (E64) in water; pepstatin, I,1 Ophenanthroline, phenylmethylsulfonyl fluoride (PMSF), tosyl phenylalanyl chloromethyl ketone (TPCK) in methanol; and chymostatin in dimethylsulfoxide (DMSO) (all reagents from Sigma)) were incubated with 1 pg of purified proteinase solution in azocasein assays. Final concentrations of all reagents are shown in Table I. Residual activity was compared to that of a reaction mixture without inhibitors. For inhibitors made up in methanol and DMSO an equivalent amount of solvent was run as a control. 2.8. Substrate specijkity 2.8. I. A,-ocoll degrudution A 3-5 mg sample of azocoll (Sigma) in 500 ~1 of 0.05 M Tris hydrochloride (pH 8.5), supplemented with 0.005 M CaCI, was incubated with 1 pg of purified proteinase for 24 h at 37°C. The reaction was terminated by the addition of 0.5 ml of 0.01 M EDTA. After centrifugation at 13,000 X g for 5 min the released dye in the supernatant was measured Table I Effect of proteinase inhibitors and metal ions seinolytic activity of the 34,000-M, proteinase
on the azoca-
Inhibitor
Final concentration
Inhibition
Chymostatin PMSF EDTA I. IO-Phenanthroline TPCK TLCK Pepstatin A Leupeptin Iodacetamide E64 N-ethylmaleimide Zn’+ ca? +
0.1 mM I mM I mM I mM 0.1 mM 0.2 mM I mM 0.1 /.LM 0.1 /JM I mM IO PM 5mM 5mM 5mM
66 64 30 20 0 0 0 0 0 0 0 46 23 23
Mg” ‘I All assays were reported.
performed
in duplicate
(‘7&J a
and mean values
Microbiology
13/L
I-IO
spectrophotometrically at 530 nm. One unit of azoco11 degrading activity was defined as the amount of enzyme which produced an increase in OD of 0.1 under the conditions described. 2.8.2. Kerutin Five mg of keratin azure (Sigma) in 300 ~1 of 0.1 M glycine-sodium hydroxide (pH 8.5) was incubated with I pg of purified proteinase for 24-72 h at 37°C prior to centrifugation at 13,000 X g for 5 min. The OD of the supernatant was measured at 595 nm. One unit of keratinase activity was defined as the amount of enzyme which produced an increase in OD of 0.01 under the conditions described. 2.8.3. Laminin and ,fibronectin Ten ~1 of each substrate (at a concentration of 1 mg ml-’ - both from Sigma) was incubated with 1 pg of purified proteinase solution for 24-72 h at 37°C prior to analysis on a 10% SDS-PAGE gel. Samples were pretreated with 5% (v/v> 2-ME and boiled for 3 min. 2.84. Elastin Five pg of elastin orcein (Sigma) in 300 ~1 of 0.2 M Tris hydrochloride (pH 8.5) was incubated with 1 pg of purified proteinase for 24 h at 37°C prior to centrifugation at 13,000 X g for 5 min. The released dye in the supematant was measured spectrophotometrically at 530 nm. One unit of elastin orcein degrading activity was defined as the amount of enzyme which produced an increase in OD of 0. I under the conditions described. 2.9. N-terminal
are
13 ClYY6)
urnino acid sepencing
Purified proteinase samples were boiled, treated with 2-ME and electrophoresed on 15% SDS-PAGE gels (stored overnight at 4°C with 0.002 M thioglycollie acid (Sigma) in the upper buffer chamber prior to use). Electrophoresed proteinase was transferred onto a polyvinylidene fluoride membrane using standard Western blotting methodology [22] and stained with Coomassie brilliant blue R-250. The proteinase band was excised and analysed on a 477A Protein Sequencer (Applied Biosystems, England) at The Protein Sequencing Unit, Biochemistry Department, University of Cambridge. Cambridge, England.
3. Results 3.1. Proteinase pur$cation Two hundred ml volumes of cytoplasmic antigen (70 mg total protein) from 5 1 of T. rubrum F1478 Sabouraud glucose broth cultures in late log to early stationary phase were utilised for proteinase purification. Preliminary studies had indicated that the 34,000-M, proteinase was not detectable prior to late log phase and that after this time it represented the dominant proteolytic species in cytoplasmic antigen preparations (data not shown). Fig. 1 illustrates the typical elution profiles obtained during Rotofor isoelectric focusing (Fig. la). refractionation on the Rotofor system (Fig. lb) and Superose 12 gel filtration chromatography (Fig. 1~). Two peaks of azocasein degrading activity (Peak I, fractions 7-10 and Peak 2, fractions l3- 16) were obtained by isoelectric focusing (Fig. la). Substrate SDS-PAGE analysis indicated that two major bands of gelatin degrading activity with relative molecular masses of 37.000-M, and 23,000-M, correlated with the azocasein degrading activity in Peak 1 (Fig. 2, lane A). Refractionation of the Peak 1 fraction on the Rotofor system resulted in further separation of the two proteolytic species (Fig. lb and Fig. 2, lanes B and C) and after 1-3 cycles of gel filtration chromatography (Fig. lc and Fig . 2, lanes D-G) the 37,000-M, species was purified to homogeneity by removal of the contaminating 23.000-M, species. The purification of the 37.000-M, proteinase as viewed on reduced SDS-PAGE gels is summarised in Fig. 3. The relative molecular masses of the purified proteinase were estimated as 37,000-M, on substrate SDS-PAGE gels (Fig. 2). 15.000-M, on reduced conventional SDS-PAGE gels (Fig. 3) and 34,000-M, on non-reduced SDS-PAGE gels (data not shown). It was concluded that the proteinase existed as a 34,000-M, molecule in its native state and that it was composed of subunits of 15.000-M, which could be visualised after disruption with 5% 2-ME. The discrepancy in the relative molecular masses of the 34,000-M, nonreduced protein on SDS-PAGE gels and the 37,000M, proteolytic band on substrate SDS-PAGE gels almost certainly reflected the interaction of the enzyme with the gelatin substrate during electrophoresis on substrate SDS-PAGE gels. The relative molec-
ular mass of 34,000-M, was deemed more accurate and will be used to define the proteinase. Attempts to quantify the purification, yield and specific activity
a
Fig. I. Purification of the 34,000-M, proteinase by (a) Rotofor isoelectric focusing. (b) refractionation on the Rotofor system and (c) gel filtration FPLC (example of final gel filtration cycle). The histogram represents azocasein degrading activity (U m- ’ ), closed squares gradient
the protein (the latter
concentration (mg ml-’ ) and is shown in a and b only).
wedges
the pH
0' 5
Fig. 2. Analysis of the purification of the 37,000-M, proteinase by substrate SDS-PAGE. Lane A, Rotofor isoelectric focusing cycle I fraction 7 (tracks 7-11 contained the 37.000-M, and 23,000-M, proteinases); lanes B and C. refractionation of Peak 1 fractions on the Rotofor system, fractions 7 and 9 respectively; lanes D-G. Superose 12 HR IO/30 gel filtration fractions IS-21 containing the pure 37,000-M, proteinaae after 2 gel filtration cycles.
of the proteinase were hindered by the presence of multiple proteolytic species with varying specificities for the azocasein substrate. Estimates of the total protein concentration and total enzyme activity prior to purification (70 mg and 2400 Units respectively) and after purification to a homogeneous band ( < 0.04 mg and 6 Units respectively) provide a partial indication of the extent of purification achieved.
5.5
6
6.5
7
8
7.5
6.5
9
9.5
I 10 10.:
PH Fig. 4. pH profile of the 34,000-M, proteinase using azocasein and azoalbumin substrates. The wedges and closed squares represent the azocasein and azoalbumin substrates respectively.
was active over a broad pH range with high activity against azocasein in the pH range 6.5-10.5. Using azoalbumin the enzyme was highly active over the pH range 6.0-8.5 and maintained a constant level of activity from pH 8.5-10.5. The proteinase migrated during Rotofor isoelectric focusing with a pl of between 3.4-4.4. The pl was more accurately determined as 4.5 on an isoelectric focusing gel (data not shown). 3.3. Effect of proteinuse inhibitors und metal ions
3.2. Optimal pH and isoelectric point (pli Using azocasein and azoalbumin substrates in spectrophotometric assays the optimal pH of the 34.000-M, proteinase was 7.5 (Fig. 4). The enzyme A
-
6
CD
E
FGH zf
1’8”o” -49
Table 1 illustrates the inhibition profile of the 34.000-M, proteinase. The serine proteinase inhibitor PMSF inhibited the enzyme, together with chymostatin. which inhibits chymotrypsin-type serine proteinases. The metalloproteinase inhibitors EDTA and 1.10 phenanthroline inhibited the proteinase to a A
B
C
D
-32.5 w-
-27.5
Fig. 3. Analysis of the purification of the proteinase by conventional SDS-PAGE. Lane A, crude cytoplasmic antigen; lanes B and C. refractionated Rotofor fractions 7 and 9 respectively: lane D. Superose 12 HR IO/30 gel filtration fraction 19; lanes E-G. gel filtration fractions 18-20 after 2 gel filtration cycles; lane H. Bio-Rad low range standard molecular mass marker proteins.
Fig. 5. Substrate SDS-PAGE analysis of the inhibition of proteinase activity by the irreversible serine proteinase inhibitor PMSF. Lane A, 37,000-M, uninhibited proteolytic band; lane B, 37,000-M, proteolytic band after exposure to I mM PMSF; lane C, 37,000-M, proteolytic band after exposure to IO mM PMSF; lane D. Bio-Rad low range standard molecular mass marker proteins.
Table 2 Substrate
specificity
of the 33.000-M,
Suhstrate
Activity (Units
Arocasein
mg-
’ protein)
“
14.600 12.600
Aroalbumin Azocoll h Keratin El&in ’ All assays reported.
proteinase
I52 140 122 were
performed
m duplicate
and
mean
values
are
h One unit of azocoll degrading activity was defined by an increase of 0.1 in optical density under the conditions described. For all other substrates an increase of 0.01 was used.
lesser extent in these assays. Of the metal ions Zn’+ showed marked inhibitory activity and there was some inhibition by Ca” and Mg”. No other inhibitors, including cysteine and aspartic proteinase inhibitors, had any effect. Fig. 5 illustrates the effect of the irreversible serine proteinase inhibitor PMSF on the 37.000-M, proteolytic band on substrate SDS-PAGE gels. Following incubation with 1 mM PMSF the intensity of the 37.000-M,. proteolytic band was reduced by approximately 50% (Fig. 5. lane B). No proteolytic band could be detected after incubation with 10 mM PMSF indicating that complete inhibition had occurred (Fig. 5. lane C).
was less affected after exposure to the proteinase for 24 h (Fig. 6b, Lanes C. D) but progressive degradation occurred with incubation time (Fig. 6b, lanes E, F and G. H, respectively). After exposure to the proteinase for 72 h multiple weak degradation products in the range > 106,000-M, to approximately 32,500-M, were observed. The complete degradation of laminin to products of less than 49.000-M, observed with the positive control proteinase (Src~~l~~lococc~rs uureeus XVII, Fig. 6b. lane I) did not occur. 3.5. N-trrminal
amino acid sequence
Table 3 illustrates the N-terminal amino acid sequence of the 34,000-M, proteinase (in its reduced 15.000-hil, form) and the homology shared by this sequence with the previously published deduced
(a)
A
B
C
D
E
F
G
H
I
-106 -80
3.4. Substrute specijki~ The substrate specificity of the 34,000-M,. proteinase was assayed at pH 7.5 using azocoll, azocasein. azoalbumin, keratin and elastin substrates as well as laminin and fibronectin. The proteinase actively degraded azocasein. azocoll and azoalbumin while weaker activity (ten fold less) was detected against keratin and elastin (Table 2). Fibronectin (Fig. 6) was actively degraded within 24 h of exposure to the proteinase producing multiple degradation products of less than 106.000-M, (Fig. 6a, lanes C, D). Similar degradation products were observed after incubation for 48 h and 72 h (Fig. 6a, lanes E, F and G, H. respectively). The complete degradation of fibronectin to products of less than 49,000-M, observed with a positive control proteinase (Sraph$ococcus c7~1re14s XVII (Sigma P8400). Fig. 6a. lane I) did not occur with the 34,000-M, proteinase. Laminin
(blA \ ,
B
C
D
E
F
G
H
I
-106 -80
Fig. 6. Degradation 34,000-M, proteinase.
of
(a) All
fibronectin samples
and (b) were boiled
laminin and
by the reduced.
Lanes A. B, fibronectin or laminin untreated, 24 h; lanes CD, EF. GH. fihronectin or laminin proteinase treated, 24 h, 4X h and 72 h respectively: lane 1. fibronectin or laminin treated with Stophdoco~xs
UY~II.S
XVII
control
proteinase.
24 h.
Table 3 N-terminal amino acid sequence of the 34,000-M, proteinase and its homology with the C’. imnlirit proteinase (from GENEMBL sequence data bank)
Rotein
Amino acid sequence
T rubrum pmteinase
WS~PK~NAG~l,T~ T
C immiris
proteinax
’ Percentage
sequence
LSYD
YD
Identity
LL
v
(%%)
50% 120 amino acids)
TADhSQTHYDDPSIJ’UFY
identity
in amino acid overlap.
amino acid sequence of a Coccidioides immitis chymotrypsin-type serine proteinase. The 15,000-M, proteinase shared 50% homology with the C. immitis 34,000-M, proteinase.
4. Discussion A novel 34.000-M, intracellular proteinase has been purified to homogeneity from cytoplasmic antigen preparations of T. rubrum. To our knowledge this is the first report of the purification and characterisation of an intracellular proteinase from T. rubrum and it confirms the observation of Chattaway et al. that T. rubrum mycelia contained enzymes with the specificities of trypsin and chymotrypsin [9]. The purification of the 34,000-M, proteinase was relatively straightforward due to the high protein content of the late log phase mycelial mat and the high levels of the proteinase present. However the presence of other intracellular proteinases during the purification of the 34,000-M, enzyme makes it inappropriate to quantitate values for specific activity and total yield for the 34,000-M, molecule alone as it is likely that the other proteolytic species may contribute to these parameters prior to purification. A 34,000-M, proteinase was also detectable in culture filtrate in late log/early stationary phase cultures. This may be related to the intracellular proteinase of the same molecular mass and this possibility is currently being investigated. The age of the cultures from which this proteinase was identified makes it likely that this represents a lysis product rather than a genuine extracellular product.
The single 37,000-M,. proteolytic species on substrate SDS-PAGE gels represented the gelatin degrading activity of the single protein band of 34,000M, and 15,000-M, on non-reduced and reduced SDS-PAGE gels respectively. Retardation of fungal proteinase migration in substrate SDS-PAGE gels due to the interaction of the enzyme with the substrate has been reported with a difference in apparent molecular mass of 6000-M, observed between the proteolytic band on substrate SDS-PAGE gels and the non-reduced proteinase on conventional SDSPAGE gels [23]. The 34,000-M, non-reduced molecular mass of the T. rubrum intracellular proteinase is accordingly the more accurate representation of the protein in its native state. Disruption of the protein produced non-proteolytic bands of 15,000-M, suggesting that the protein normally exists as an active disulfide-linked dimer in a similar manner to the extracellular T. rubrum 7 1,000-M, and 90,000-M, dimeric proteinases which consist of subunits of 36,000-M, and 44,000-M,. respectively [5]. The relationship of the 34,000-M, enzyme to previously described proteinases from both T. rubrum and other fungal pathogens is of considerable interest. The 34,000-M, proteinase shares some general characteristics with the previously described T. rubrum extracellular proteinases. A T. rubrutn proteinase of similar molecular mass (34.700-M,) [6] was previously described as an extracellular product. In contrast to the proteinase described here however the molecular mass reported was the same for this enzyme in both the reduced and non-reduced states [6]. The optimum activity against azocasein and azoalbumin substrates at pH 7.5 correlated relatively closely with the alkaline pH optima of 8.0 against azocoll exhibited by the previously reported 27,000M, [7]. 71.000-M, and 90,000-M, proteinases [5] together with the 235,000-M, proteinase recently described by our group [24] which exhibited an optimum pH of 8.5 against azocasein and azoalbumin substrates. All of the above mentioned proteinases exhibited substantial activity within the pH range 7.0 to 10.0. In common with the 27,000-M, and 235,000-M, extracellular proteinases and the majority of T. rubrum proteins (as demonstrated by Rotofor isoelectric focusing) the 34,000-M, proteinase had an isoelectric point of less than 5.0. This contrasts with the previously described 7 1,000-M,
and 90,000-M, extracellular proteinases which exhibit isoelectric points of 6.5 and 7.8 respectively. The 34,000-M,. intracellular proteinase demonstrated a similar pattern of inhibition to the previously described T. rubrum 23.5,000-M, extracellular proteinase when incubated with a range of proteinase inhibitors and metal ions [24]. Thus the inhibitory action of the serine proteinase inhibitor phenylmethylsulfonyl fluoride together with the chymotrypsintype serine proteinase inhibitor chymostatin suggested that the 34,000-M, enzyme was a chymotrypsin-type serine proteinase. However there are two observations which may elucidate the relationship of the 34,000-M, proteinase with other fungal proteinases. Firstly, the 34,000-M, proteinase is weakly inhibited by the metalloproteinase inhibitors EDTA and 1. IO-phenanthroline as well as the metal ions Ca’+. Mg’+ and Zn’+. With the exception of the 235,000-M, proteinase this pattern of inhibition has not been identified in T. rubrum proteinases. although similar inhibition profiles have been observed for an A. niger semi-alkaline aspergillopeptidase [25] and an A. ,fi4n~igarus elastase 1261. It is of significance that the A. niger aspergillopeptidase has an isoelectric point of 4.1 (compared to 4.5 for the T. rubrum enzyme) and a pH optimum of 7.8 (compared to 7.5) suggesting a relationship between the two enzymes. Secondly. based on N-terminal amino acid sequence data, the 34,000-M, T. rubrum proteinase appears to be related to a C. immitis wall-associated chymotrypsin-type serine proteinase. The C. immitis proteinase exhibits collagenolytic and elastinolytic activity, has an isoelectric point of 4.5 and is inhibited by chymostatin. While this enzyme is larger than the T. rubrum proteinase it shares the low pl and the active disulphide-linked dimeric native state of the T. rubrum enzyme. The C. immitis proteinase appeared to be concentrated in the walls of the parasitic cells at stages of active growth and it has been suggested that it plays a role in cell wall turnover or in wall plasticization. The C. imrnitis proteinase shares a conserved region of 75% homology with human ru-chymotrypsin and chymotrypsinogen from various sources and the gene for the proteinase is the only fungal chymotrypsin-type serine proteinase gene isolated to date. Indeed. of the fungal serine proteinases so far
characterised most. with the limited exception of enzymes such as the C. imnzitis 34.000-M, wall-associated proteinase and the A. niger 21,000-M, semi-alkaline aspergillopeptidase described above, have been classed as subtilisin-type serine proteinases [25]. Our data is strongly suggestive of a relationship between the 34,000-M, T. rubrum enzyme described here and both the C. immitis and A. rziger enzymes. It has been postulated that the latter play a role in the pathogenesis of their respective diseases and. based on biochemical similarities, the T. rubrum enzyme may fulfill a similar function. Preliminary investigations. via the production of antisera for immunochemical studies, are under way to explore this possibility.
Acknowledgements We wish to thank Dr. Mary Moore and Dr. Gillian Midgely of the Department of Medical Mycology. St. John’s Institute of Dermatology, St. Thomas’s Hospital. London for supplying the fungal isolate. This research was funded by a grant from Sandoz Pharma Ltd., whose help is gratefully acknowledged.
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