Originally published In Press as doi:10.1074/jbc.M001392200 on March 23, 2000
J. Biol. Chem., Vol. 275, Issue 26, 20104-20109, June 30, 2000
Clitocypin, a New Type of Cysteine Proteinase Inhibitor from
Fruit Bodies of Mushroom Clitocybe nebularis*
Jo
e
Brzin
§¶,
Boris
Rogelj§,
Tatjana
Popovi
§,
Borut
trukelj§
, and
Anka
Ritonja§
From the § Department of Biochemistry and Molecular
Biology, Joef Stefan Institute, Jamova 39, 1000 Ljubljana,
Slovenia and Department of Pharmaceutical Biology, Faculty of
Pharmacy, University of Ljubljana, A
kereva 7, 1000 Ljubljana, Slovenia
Received for publication, February 17, 2000, and in revised form, March 17, 2000
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ABSTRACT |
A novel inhibitor of cysteine proteinases has
been isolated from fruit bodies of a mushroom Clitocybe
nebularis. The inhibitor was purified to homogeneity by affinity
chromatography and gel filtration, followed by reverse-phase high
pressure liquid chromatography. The active inhibitor has an apparent
molecular mass of about 34 kDa by gel filtration and by
SDS-polyacrylamide gel electrophoresis without prior boiling of the
sample. Boiling in 2.5% SDS or incubation in 6 M guanidine
hydrochloride resulted in a single band of 17 kDa, indicating homodimer
composition with no intersubunit disulfide bonds. The inhibitor in
nondenaturing buffer is resistant to boiling in water, retaining its
activity and dimer composition. The mushroom protein is a tight binding
inhibitor of papain (Ki = 0.59 nM),
cathepsin L (Ki = 0.41 nM), cathepsin B
(Ki = 0.48 µM), and bromelain
(Ki = 0.16 µM) but is inactive toward
cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and
sugar analysis indicates the absence of carbohydrate. A single protein
sequence of 150 amino acids, containing no cysteine or methionine
residues, was obtained by amino acid sequencing. The calculated
molecular mass of 16854 Da corresponds well with the value obtained by
mass spectrometry. A major part of this sequence was verified by
molecular cloning. The monomer sequence is clearly devoid of typical
cystatin structure elements and has no similarity to any other known
cysteine proteinase inhibitors but bears some similarity to a
lectin-like family of proteins from mushrooms. The inhibitor, which is
present in at least two other members of the Clitocybe
genus, has been named clitocypin (Clitocybe cysteine
proteinase inhibitor).
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INTRODUCTION |
Cysteine proteinases are involved in a diverse array of functions
involving specific processing or more general degradation of proteins
in a wide variety of organisms, including viruses, fungi, plants, and
animals. Their activity is regulated by limited proteolysis of inactive
precursors (1, 2), by pH and redox potential of the surroundings, and
tight binding with proteinaceous inhibitors (3).
Five structurally different groups of protein cysteine proteinase
inhibitors have been reported: cystatins (4), thyroglobulin type-1
domain inhibitors or thyropins (5), soybean trypsin inhibitor-like
inhibitors of cysteine proteinases from potato (6), pineapple
inhibitors of cysteine proteinases (7, 8), and very recently,
inhibitors of cysteine proteinases homologous to propeptide regions of
cysteine proteinases (9). So far, only the mechanism of interaction of
the cystatin superfamily of inhibitors has been elucidated (10, 11),
followed recently by that of thyropins (12), but overall there is very
little information available on all other cysteine proteinase
inhibitors concerning specificity, kinetics, and mode of binding.
Since cysteine proteinases in mammals have been implicated also in many
pathological events, such as tumor invasion and metastasizing cancer
(13), bone resorption (14), periodontitis (15), and rheumatoid
arthritis (16), there is a need for new specific, efficient, and
accessible inhibitors of the enzymes responsible for diagnosis and
treatment of these conditions. Fungi (Mycophyta) have been used for
religious, medical, and other purposes since ancient times. To our
knowledge, no protein cysteine proteinase inhibitors have been
characterized in higher fungi (Basydiomyceta), popularly called
mushrooms. We report here the identification, some properties, and
cloning of a new proteinaceous cysteine proteinase inhibitor from
Clitocybe nebularis fruit bodies, which we have called
clitocypin, a member of what is very likely a new structural superfamily of cysteine proteinase inhibitors.
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EXPERIMENTAL PROCEDURES |
Fungal Material--
Edible mushrooms, C. nebularis,
were collected in their natural habitat in a forest in November and
frozen at 
20 or 70 °C until use. A specimen is deposited at the
Department of Pharmaceutical Biology, Faculty of Pharmacy, Ljubljana.
Chemicals and Enzymes--
Iodoacetate and
benzoyl-Arg-2-naphthylamide were from Sigma (Germany); CNBr-activated
Sepharose 4B and Sephacryl S-200 were from Amersham Pharmacia Biotech.
Z-Phe-Arg-MCA1 and
Z-Arg-Arg-MCA were from Bachem (Switzerland). Ep-475 was purchased from
Peptide Research Foundation (Japan).
Stem bromelain (EC 3.4.22.32), bovine trypsin (EC 3.4.21.4), and
porcine pepsin (3.4.23.1) were from Sigma (Germany). Papain (EC
3.4.22.2) 2× crystallized, also from Sigma, was additionally purified
by affinity chromatography (17). Glycyl endopeptidase (EC 3.4.22.25)
was a gift from Dr. Alan J. Barrett (The Babraham Institute, Cambridge,
United Kingdom) and was prepared as described (18). Endoproteinase
Lys-C (EC 3.4.21.50) was from Roche Molecular Biochemicals. 
-Trypsin
(EC 3.4.21.4) was prepared from type IX trypsin (Sigma) as described
(19). Cathepsin B (EC 3.4.22.1), cathepsin H (EC 3.4.22.16), and
cathepsin L (EC 3.4.22.15) were purified from human kidney by the
method already described (20).
Inhibitor Purification--
Frozen fruit bodies of C. nebularis (500 g, fresh weight) were homogenized in 1000 ml of
Tris/HCl buffer, pH 7.5, containing 0.5 M NaCl (Buffer A).
The homogenate was centrifuged at 8000 × g for 30 min.
The supernatant was applied in aliquots of 300 ml to a column of
carboxymethylpapain-Sepharose (2.5 × 15 cm) prepared according to
the manufacturer's instructions, carboxymethylated as described in
Ref. 21, and equilibrated with Buffer A. Bound inhibitory fractions
were eluted with 0.01 M NaOH, pooled, neutralized with
dilute HCl, concentrated on an Amicon UM-10 membrane, and chromatographed on a Sephacryl S-200 column (4 × 110 cm) washed with buffer A. For the purpose of amino acid composition and sequence analysis, the inhibitor was additionally purified on a reverse-phase HPLC (Milton Roy LCD, UK) on a Vydac C8 column (Alltech; 4.6 × 250 mm), using a gradient of 0-80% (v/v) acetonitrile in water containing 0.1% (v/v) trifluoroacetic acid over 25 min.
Isolation of RNA, Reverse Transcription-PCR, and Sequencing of
the cDNA Clone--
Total RNA was isolated from the fungal
material stored at 70 °C according to the method of Puissant and
Houdebine (22). The quality of the RNA was checked by electrophoresis
in a formaldehyde/formamide system (23) followed by the downward
alkaline transfer procedure of Chomczynski (24). The RNA was
transferred to Hybond-N nylon membranes and dyed with 0.04% methylene
blue. Degenerate primers were constructed with a linker restriction
site on the 5'-end. Forward primer CnF
(5'-GCGAATTCCCIGGIGTIGGIGGIGARTAYGC) was constructed from
the Pro18-Ala25 region of the protein sequence
with the EcoRI restriction site on the 5'-end, and the
reverse primer CnR (5'-CGGGATCCTGICKYTCRAAICKCCAIGCNGG) was constructed
from the Pro142-Arg148 region of the protein
sequence with a BamHI restriction site on its 5'-end. Both
primers contained inosine in the place of 4-nucleotide degeneration.
Reverse transcription was performed in a reaction mix containing 1×
PCR buffer II, 5 mM MgCl2, 1 mM each dNTP, 1 unit/µl RNasin, 0.5 µg of CnR primer, and 2.5 units/µl of murine leukemia virus reverse transcriptase. The reaction
mixture was incubated for 10 min at 23 °C, 15 min at 42 °C, and 5 min at 99 °C. 30 cycles of PCR were performed by adding 1× PCR
buffer II, 5 mM MgCl2, 0.83 µg CnF primer,
0.33 µg CnR primer, and 2.5 units of AmpliTaq DNA polymerase
(Perkin-Elmer) to the final volume of 100 µl. After electrophoresis
on 1.7% agarose gel, a band at about 400 base pairs was excised,
inserted into EcoRI/BamHI-digested pUC19, and
sequenced using the Pharmacia T7 sequencing kit following the
appropriate manufacturer's protocol.
Protein and Sugar Determination--
Protein concentration of
the purified inhibitor was determined by absorbance at 280 nm using a
Perkin-Elmer UV-visible spectrometer 
18. A molar absorbance
coefficient of 22,900 M
1 cm1
was calculated from the amino acid sequence (25).
Clitocypin was assayed for potential glycosylation with incubation with
N-glycosidase F (Roche Molecular Biochemicals), according to
the manufacturer's instructions. The reaction was incubated overnight
at 37 °C, and the products were followed by SDS-PAGE. The inhibitor
was also tested with a phenol-sulfuric acid assay for hexoses and
pentoses as described (26).
SDS-PAGE--
A Pharmacia Phast System unit and 8-25% gradient
gels were used, following the instructions of the manufacturer. Samples
were prepared by mixing with equal volumes of 80 mM
Tris/HCl buffer, pH 8.0, containing 5% SDS, and were applied to the
gel with or without previous boiling at 100 °C for 5 min. For sample
reduction, 2-mercaptoethanol in a final concentration of 5% (v/v) was
included in the mixture before boiling. Molecular masses were
determined using LMW markers of 14.4-94 kDa (Amersham Pharmacia
Biotech). Gels were stained with 0.1% (w/v) Coomassie Brilliant Blue
R-250.
Isoelectric Focusing--
Samples were run on a Pharmacia Phast
System using commercial precast pH 3-9 gradient gels following the
instructions provided. pI values were determined using the Pharmacia
broad-pI calibration kit (pI range 3.65-9.30)
Electrospray-Ionization Mass Spectrometry--
HPLC-purified
protein was dissolved in water/methanol (1:1, v/v) solution containing
1% acetic acid and analyzed on a high resolution magnetic sector
Autopsied Q mass spectrometer (Micromass, Manchester, UK). The protein
sample was introduced at a flow rate of 10 µmol/min using a syringe
pump. Spectra were obtained by scanning from a mass/charge ratio of
2000 to 400 at 10 s/scan. Sodium iodide ions were used for calibration.
Each molecular species produced a series of multiply charged protonated
ions from which the molecular mass was determined by simple calculation.
Protein Sequence Analysis--
Chemical cleavage of 100 pmol of
the inhibitor was performed by boiling it in 0.1% (v/v)
trifluoroacetic acid for 20 min. For enzymatic cleavages, 350 pmol of
native inhibitor was dissolved in 0.1 M phosphate buffer,
pH 6.5, and 6 M guanidine HCl and incubated for 48 h
at 37 °C. After removal of guanidine HCl by HPLC, 100 pmol of sample
was first fragmented using 2% (w/w) glycyl endopeptidase as described
(6). Hydrolysis of 100 pmol of sample by -trypsin was carried out at
37 °C in 0.5 M N-methylmorpholine, pH 8.2, for 30 min at an enzyme/substrate ratio of 1:100 (w/w). Endoproteinase Lys-C at an enzyme/substrate molar ratio of 1:30 was used for proteolytic digestion of 100 pmol of the inhibitor for 10 h at room temperature in 0.3 M Tris buffer, pH 8.6, containing
0.1 mM CaCl2 and 5 M urea. All
three enzyme hydrolyses were performed in a final volume of 200 µl.
Reactions were stopped by the addition of trifluoroacetic acid. The
peptide mixtures obtained were separated by HPLC (Milton Roy Co.) using
a reverse phase Vydac C18 column equilibrated with 0.1% (v/v)
trifluoroacetic acid and eluted with a linear gradient of acetonitrile
from 0 to 80% in 0.1% aqueous trifluoroacetic acid over 60 min. The
absorbance of the eluant was monitored at 215 nm. Amino acid
composition was determined by hydrolysis of samples in 6.0 M HCl at 110 °C for 24 h and analysis of the
obtained hydrolysates on an Applied Biosystems 421 amino acid analyzer
with precolumn phenylthiohydantoin derivatization. Automated Edman
degradation and sequence analysis was carried out on an Applied
Biosystems liquid pulse sequencer 475 A connected on-line to a model
120 A phenylthiohydantoin analyzer from the same manufacturer. For
sequence comparison of clitocypin with other known protein sequences,
data bases were searched with ExPASy protein sequence similarity search
using the BLAST algorithm (27) and SAMBA (28).
Inhibitor Assay--
The inhibitory activities of samples and
fractions during the isolation procedure were measured against papain.
A sample of 100 µl was added to 0.05 µM papain in 0.85 ml of buffer solution (0.1 M sodium phosphate, pH 6.5, containing 5 mM cysteine and 1.5 mM EDTA).
After 10 min of preincubation at room temperature, the reaction was
initiated by the addition of 25 µl of 0.1 M substrate benzoyl-Arg-2-naphthylamide in Me2SO, and the mixture was
incubated for 10 min at 37 °C. The reaction was stopped, and
A520 was read, following the procedure of
Barrett (29).
Active Site Titrations--
Active concentrations of cathepsins
B and L and papain were determined by titration with Ep-475 (30).
Residual activities were determined with benzoyl-Arg-2-naphthylamide as
substrate for cathepsin B and papain (29) or with Z-Phe-Arg-MCA as
substrate for cathepsin L (30). The active concentration of clitocypin was determined by the same method using previously active site-titrated papain. All concentration values given below refer to active concentrations.
Determination of Inhibition Constants--
Inhibition kinetics
of papain and cathepsin L were studied under pseudo-first-order
conditions with at least a 10-fold molar excess of inhibitor over
enzyme and in the presence of substrate (31) in continuous kinetic
assays followed by a Perkin-Elmer LS50B fluorimeter, connected to an
IBM personal computer, running Flusys software (32). Various amounts of
the inhibitor in the final concentration range of 2.5-50
nM were mixed with 10 µM Z-Phe-Arg-MCA as
substrate in the assay buffer in a final volume of 2 ml in a
fluorescence cuvette thermostated at 25 °C. The assay buffer for
papain was 0.1 M sodium phosphate buffer, pH 6.5, containing 2 mM dithioerythritol and 1.5 mM
EDTA, while cathepsin L was assayed in 0.4 M sodium acetate
buffer, pH 5.5, containing 2 mM dithioerythritol and 1.5 mM EDTA. To initiate the reaction, papain (final
concentration 0.2 nM) or cathepsin L (final concentration
0.1 nM) was added in a negligible volume. Product formation
was monitored continuously at excitation and emission wavelengths of
370 and 460 nm, respectively. The progress curves were fitted by
nonlinear regression analysis to the equation of Morrison for the model
of slow binding kinetics (33), and kd and
ka values were obtained using Km
of 80 µM for papain (34) and 2 µM for
cathepsin L (35). The equilibrium constants (Ki)
were calculated from ka and kd
(Ki = kd/ka).
The Ki values for the inhibition of cathepsin B and
stem bromelain by the inhibitor were determined from the linear equation of Henderson (36) derived for kinetics of tight binding competitive inhibitors. Each enzyme was incubated at 25 °C with different amounts of the inhibitor (0.05-1.5 µM) for 15 min in 0.3 ml of 0.1 M phosphate buffer, containing 10 mM cysteine and 1.5 mM EDTA. Cathepsin B was
assayed at pH 6.0 and bromelain at pH 6.8. The reaction was initiated
by the addition of the substrate in the final concentration of 10 µM in a negligible volume. Z-Phe-Arg-MCA was used for
cathepsin B and Z-Arg-Arg-MCA for bromelain. After 10 min of
incubation, the reaction was stopped with 5 mM iodoacetic acid. The released MCA was measured on a Perkin-Elmer LS 30 fluorimeter. The apparent Ki values were obtained
graphically (36), and true inhibition constants (Ki)
were obtained after correction for substrate competition using the
equation Ki = Ki(app)/(1 + [So]/Km), where [So] is
the initial substrate concentration and Km values
are 150 µM for cathepsin B (37) and 15 µM
for bromelain (38).
Testing the Inhibitory Activity against Other Classes of
Enzymes--
Trypsin activity was measured using the synthetic
substrate benzoyl-arginyl-p-nitroanilide as described by
Erlanger (39). Pepsin was assayed using fluorogenic biopodyl-labeled
casein as described in Ref. 40. Clitocypin inhibitory activity was
determined by titrating 2 µg of trypsin or 0.1 µg of pepsin with
increasing amounts of clitocypin.
Assay of Hemagglutinating Activity--
Human (phenotypes A, B,
and O) and rabbit red blood cells were extensively washed and suspended
in buffered saline. 50-µl samples of crude C. nebularis
extract and purified clitocypin (1 mg/ml) and their serial dilutions
were mixed with 1 ml of 3% (v/v) erythrocyte suspension in test tubes
and incubated for 3 h at room temperature. Agglutinated red blood
cells formed a pellet, which was not resuspended upon shaking.
Temperature Stability--
Purified clitocypin was boiled in
buffer A for 5 min in a sealed microcentrifuge tube. After cooling on
ice for 5 min, it was used for titration of papain or run on calibrated
gel filtration, as described above.
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RESULTS |
Purification of 34-kDa Inhibitory Protein--
The purification
scheme required for purification of clitocypin was rather
straightforward. Affinity chromatography proved to be an
efficient first step, since it specifically removed other noninhibitory
proteins, so that after gel filtration the inhibitor was practically
homogeneous (Figs. 1 and 3). In a typical
preparation, we obtained 2 mg of purified inhibitor from 100 g of
fresh mushrooms.
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Fig. 1.
Analysis of the purification of clitocypin
from C. nebularis by SDS-PAGE. Samples were
run as follows: C. nebularis extract (lane
1), purified clitocypin after gel filtration
(lanes 2-5), the first (lane
6) and the second (lane 7) peak after
HPLC, clitocypin boiled in nondenaturing buffer and eluted from gel
filtration (lane 9), and guanidine HCl-denatured
clitocypin (lane 10). MW indicates
molecular markers (lane 8), where sizes in kDa
are indicated. Samples were treated in 2.5% SDS at room temperature
(rt) before application to wells or boiled in 2.5% SDS for
5 min (bt). Samples analyzed in the presence of 5%
2-mercaptoethanol are indicated as ME. The gel was
Coomassie-stained.
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The molecular mass of clitocypin under native conditions, as estimated
by calibrated gel filtration, as well as that obtained in nonreducing
SDS-PAGE without boiling of the sample was 34 kDa. The same mass of 34 kDa was obtained in the presence of reducing agent. Reduced and
nonreduced samples, boiled in the presence of SDS, gave only a single
band of 17 kDa (Fig. 1).
Subsequent purification by RP HPLC resulted in two peaks Cn1 and Cn2,
both showing inhibitory activity. Cn1 gave a single band of 34 kDa, and
Cn2 gave two bands of 34 and 17 kDa on SDS-PAGE without boiling (Fig.
2). Fraction Cn1 is thus composed solely of dimers (about 
of the total), and Cn2 is composed of a
mixture of dimers and monomers (about 
of the total).
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Fig. 2.
HPLC chromatography of clitocypin. The
elution profile of clitocypin obtained from gel filtration
(solid line). Linear gradient from 0-80% of
acetonitrile in 0.1% trifluoroacetic acid (dashed
line).
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By ES mass spectrometry, a single mass of 16,863 Da was obtained for
clitocypin from gel filtration and from both HPLC peaks.
Isoelectric Focusing--
On isoelectric focusing, the inhibitory
protein ran as a single band at a pH value of 4.4 (Fig.
3), which agrees well with the value of
4.42 calculated from the amino acid composition.
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Fig. 3.
Isoelectric focusing. Samples were
loaded onto Phast Gel IEF with a gradient ranging from pH 3 to 9. Isoelectric point markers (lane 1), purified
clitocypin (lane 2), and C. nebularis
extract (lane 3).
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Subunit Composition Analysis and Stability of
Clitocypin--
Clitocypin proved to be an extremely stable protein.
When boiled in nondenaturing buffer (see details under "Experimental Procedures"), it remained, as judged by inhibitory activity, elution volume in gel filtration, and behavior on SDS-PAGE, indistinguishable from the initial inhibitor (Fig. 1). Incubation in denaturing buffer
with guanidine HCl, however, resulted in only the 17-kDa band on
SDS-PAGE with and without boiling (Fig. 1) and complete loss of
inhibitory activity.
Sequence Analysis of Clitocypin--
The position of peptides and
overall strategy used for the complete amino acid sequence
determination of clitocypin is shown in Fig.
4. The N-terminal sequence analysis of
clitocypin established the initial 11 N-terminal residues with a
sequence yield in the range of 10%. Five peptides were obtained by
acid hydrolysis (A1 to A5) and sequenced through to their C termini.
Glycyl endopeptidase yielded the second set of peptides. G1 filled the
gap between A2 and A3 and together with G2 established the order and
overlaps of A2, A3, and A4. Tryptic peptide T1 contributed to the
alignment and overlapping of the A1 and A2 peptides on the N-terminal
part of the molecule. Based on the low content of Lys, the final set of
peptides was obtained by endoproteinase Lys-C digestion. Peptide L1
filled the remaining gap between A3 and A4 and together with L2
provided the order of the peptides A3, A4, and A5 on the C terminus of
the protein. From the sequence data, we conclude that 17-kDa monomeric
clitocypin is composed of 150 amino acids with a calculated molecular
mass of 16,854 Da. The molecule contains no cysteinyl residues, only
one histidine and two tryptophanes, and is rich in proline and glycine.
Amino acid composition analysis of undegraded clitocypin and its
fragments provided the same 150 amino acid residues (results not
shown). No sequence polymorphism was observed. Clitocypin contained no
inhibitor consensus sequences characteristic of the cystatin
superfamily members. No attachment site for oligosacharide chains was
present, which is compatible with the observed absence of sugars by
both methods used.
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Fig. 4.
Summary of the determination of the complete
amino acid sequence of clitocypin. The sequenced regions are
marked by a solid line. N represents
the N-terminal sequence of the inhibitor, and the origins of the
peptides are designated by A for acid, T for
tryptic, G for glycyl endopeptidase, and L for
endopeptidase Lys-C hydrolysis.
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A primer pair designed to span from the previously determined N
terminus to the apparent C terminus was used for the reverse transcription-PCR amplification of mRNA isolated from C. nebularis fruit bodies. A product of approximately 400 base pairs
termed Cn c1 was isolated, subcloned, and sequenced (Fig.
5B). Analysis of the deduced
amino acid sequence and comparison with the directly determined protein
sequence showed 95% identity (Fig. 5A). All seven amino
acid residues that differ between the cDNA derived and protein
sequences are located in the C-terminal region of the protein.
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Fig. 5.
A, comparison of clitocypin protein
sequence aligned with the cDNA derived clitocypin sequence Cn c1
and with the lectin-related 16.5-kDa protein sequence PC-LRP reported
by Oguri and Nagata (41). Dashed lines represent
a sequence identical to the clitocypin protein sequence.
Periods represent the frameshifting of proteins for maximal
alignment between sequences. Numbering is according to the clitocypin
sequence. B, the cDNA and deduced amino acid sequences
of the clitocypin amino acid residues 18-146.
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Amino Acid Sequence Comparison--
In a search for homologous
proteins in data bases, no protein showing high similarity with
clitocypin was found. However, three protein sequences with some degree
of similarity to clitocypin were disclosed. Relatively high similarity,
restricted to just two regions of clitocypin (34 and 35% identity) for
residues 47-89 and 95-136, respectively, was found for two parts of
the minor tail 43.0-kDa protein from Mycobacterium phage L5 (Swiss-Prot accession no. O05278). Clitocypin was also found to share significant
similarity (26% identity, 41% conservative residues throughout the
aligned sequences) with a 16.5-kDa lectin-related protein (PC-LRP) from
a lectin-deficient strain of mushroom Pleurotus cornucopiae
(PIR accession no. JC 2102) (41). Higher levels of similarity were
found in the N-terminal regions of the molecules as shown in Fig.
5A. Finally, when applying SAMBA computation of alignments,
clitocypin was found to share considerable sequence similarity (35%
amino acid identity) with 37 amino acids in the C-terminal region of
Rhesus macaque cystatin C precursor protein (Swiss-Prot accession no.
O19092) (42). In other parts of the sequences, the relatedness is not
apparent. No cystatin C consensus sequences are observed in clitocypin.
Kinetics of Inhibition--
Titration, together with the
determination of the concentration of the inhibitor, led to a value of
0.89 mol of clitocypin dimer needed to abolish enzymatic activity of 1 mol of papain (active concentration).
The pseudo-first-order rate constant, k, for binding of
clitocypin to papain and cathepsin L increased linearly with inhibitor concentration. The association (ka), and
dissociation (kd) rate constants and equilibrium
constants (Ki) are presented in Table
I. Both ka and
kd are 2- and 3-fold lower, respectively, for
cathepsin L than for papain, thus resulting in similar equilibrium
constants (Ki) for these two enzymes.
Ki for the inhibition of cathepsin B and bromelain
are substantially higher, in the micromolar range. Under the same
conditions, no inhibition of cathepsin H was observed, even at a
100-fold excess of the inhibitor.
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Table I
Kinetic data for the interaction of clitocypin with a different
cycteine proteinases
Kinetic and equilibrium constants for the inhibition of papain and
cathespin L were determined under pseudo-first-order conditions in
continuous kinetic assays and calculated as described under
"Experimental Procedures." Equilibrium constants for the inhibition
of cathepsin B and bromelain were determined in stopped assays as
described under "Experimental Procedures." Interactions were
performed at 25 °C. ND, not
determined.
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The ability of clitocypin to inhibit serine and aspartic proteinases
was tested by titration with trypsin and pepsin. No inhibition was
observed in either case.
Titration assays showed that among the red blood cells tested, only
human erythrocytes of type B were weakly and rabbit erythrocytes strongly agglutinated by C. nebularis raw extract. Purified
clitocypin in final concentrations up to 50 µg/ml had no effect under
the same conditions.
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DISCUSSION |
Basidiomycete mushroom C. nebularis contains a specific
inhibitor of cysteine proteinases, clitocypin. It was obtained in high
amounts from fruit bodies and shows marked stability at high temperatures. To our knowledge, no protein proteinase inhibitors of any
class have been reported from this or similar sources.
The molecular mass and sequence data show that purified clitocypin is a
dimer stabilized by noncovalent interactions. The absence of effect of
reducing agent confirms the lack of cysteine residues in the sequence.
The 34-kDa clitocypin band was also obtained following a modified
purification procedure, not including affinity chromatography, which
indicates that the dimer is present in mushroom juice and is not
introduced during the affinity chromatography step (results not shown).
The molecular mass of clitocypin calculated from the amino acid
sequence agrees well with that obtained by electrospray mass
spectrometry, indicating that no post-translational modification
occurs. Taken together, these data show that this inhibitor consists of
only one kind of polypeptide chain of 16,854 Da, having no sugar
moieties. One unanswered question is the nature of the second peak from
HPLC, a proportion of which runs on SDS-PAGE (without boiling) as a
monomer. It may reflect subtle differences in the hydrophobicity of a
partially denatured form following slow denaturation on hydrophobic
surfaces in the presence of organic phase in HPLC, with decreased
tendency to form dimers.
The inhibition spectrum of clitocypin is similar but not identical to
that of other classes of cysteine proteinase inhibitors. Among the
cysteine endopeptidases tested, clitocypin inhibits most strongly
papain and cathepsin L, as do cystatins (4), thyropins (5), and potato
cysteine proteinase inhibitors (43). Clitocypin differs significantly
from them, however, in that it is a relatively poor inhibitor of
cathepsin B and completely ineffective against cathepsin H. Interestingly, it inhibits bromelain reasonably well, as has been found
for potato cysteine proteinase inhibitor (PCPI 8.3), but not for
cystatins and thyropins. Whether this apparent specificity for
endopeptidases is general will be shown in future experiments. Since
clitocypin is a dimer, with the potential of binding two molecules of
proteinase simultaneously, the inhibitor dimer/enzyme binding
stoichiometry of 1:1.1 could be explained in two different ways. Either
the inhibitor domains bind independently to protease but only 55% of
them are active monomers, or only one monomer binds to the enzyme
active site while binding of the other domain is sterically hindered.
Further studies will be needed to clarify the inhibitory mechanism and
to elucidate the structure of the complex of clitocypin with a cysteine proteinase.
Our data base search disclosed no highly related proteins, just a few
limited sequence similarities. A relatively high value of identity was
found (using only one search engine) between short regions of residues
in the C-terminal region of a cystatin C precursor protein from monkey
(but not, apparently, other species) and the N-terminal region of
clitocypin. Together with the absence of any structurally or
functionally significant sequence similarities, this similarity cannot
be considered as significant. The local similarity observed for the
mycobacterial tail protein is, on the same grounds, not significant.
None of the three critical elements involved in the inhibitory
mechanism characteristic for the cystatin superfamily of CPIs was
identified in clitocypin sequence. These are the N-terminally conserved
Gly-9 residue, the central Gln-Xaa-Val-Xaa-Gly motif (first hairpin
loop), and the C-terminally located Pro-Trp element (second hairpin
loop), all forming the wedge-shaped hydrophobic edge that inserts into
the active site cleft of the proteinase (10, 11). It is possible that
the inhibitory edge of cystatins could be reproduced by other
structural elements as a result of convergent evolution similar to that
recently shown for thyropins (12).
In the search for related proteins, the significance threshold of about
26% identity was, however, reached with a lectin-like protein from a
lectin-deficient strain of P. cornucopiae mushroom. The
physiological function of this protein in mushroom is not known.
Structurally, it appears to be related to a lectin family of proteins
with agglutination activity that have been extensively characterized in
several basidiomycete and parasitic deuteromycete fungi such as
Ganoderma lucidium (44, 45), Agaricus bisporus (46), and Arthrobotrys oligospora (47), where they
presumably play a role in fungal growth, morphogenesis, and
mycorrhization (48). In contrast to the alignment with cystatin C
precursor, the level of identity applies over the entire primary
structures. In addition, there are some additional common structural
traits between this lectin-like group of proteins and clitocypin that appear to be meaningful: the lack of cysteine and methionine residues, closely similar acidic isoelectric points, similar molecular masses, and almost exclusively homodimeric structure under nondenaturing conditions. An early report (49) describes C. nebularis
lectin as an oligomeric protein, with subunit molecular mass in the
range of 15-20 kDa, isoelectric point around pH 4.4, no cysteines, a lack or only trace amounts of methionine and histidine, and similar percentage composition of another 9 amino acids to that reported here
for clitocypin. As expected, therefore, our experiments showed that
C. nebularis juice contains lectin activity agglutinating rabbit and human type B, but not A and O, erythrocytes. In contrast, purified clitocypin showed no activity against these red blood cells.
At this stage, the conclusion that the inhibitor is not a lectin is
premature, since the existence of highly specific and also
nonagglutinating lectins in fungi has been demonstrated (48).
Evidently, a group of structurally related proteins is present in
fungi, some lectin-related but with yet unknown functions, others as
inhibitors of cysteine proteinases described in this paper, and most of
them as lectins with differing specificities. Although tentative until
homology has been confirmed by three-dimensional structure
determination, it is our proposal that the members of this structurally
related family that are, like clitocypin, inhibitors, should be classed
as belonging to a new superfamily of inhibitors of cysteine
proteinases, named mycocypins. In this context, biochemical and genetic
studies of mushroom inhibitors are in progress.
The observed differences between the protein- and
nucleotide-derived sequence could point to the presence of several
homologous clitocypin-encoding genes, the apparent absence of fragments
encoding homologous proteins in the protein sequence determination
reflecting a difference in the levels of expressed proteins at the
different developmental or environmental conditions.
The physiological target of clitocypin in C. nebularis is
not known. The inhibitor is expressed at very high levels,
characteristic of proteins with roles that are more structural, as
opposed to signaling, catalysis, or control. In relation to the
control, although no cysteine proteinase activity has been detected in mushroom juice, it may well be localized in separate discrete structures or masked with excess inhibitor. Besides an endogenous physiological role, the other possible function may be protection of
the mushroom from pathogen infection or predation by insects, as shown
for several plants (50).
In conclusion, we have isolated a novel proteinase inhibitor designated
clitocypin and characterized its activity and primary and oligomeric
structure. The present study clearly establishes clitocypin as a new
and potent inhibitor that shares no structural or functional features
with previously known cysteine proteinase inhibitors but instead
appears to be related to a group of fungal lectins. Further studies are
needed to establish the precise physiological functions of this new
inhibitor in mushrooms. Finally, the discovery of clitocypin should
broaden the spectrum of specific cysteine proteinase inhibitors
available for potential use in human and veterinary medicine and in
agricultural crop protection to fight the remarkable adaptation of
insects to plant endogenous inhibitors (51).
| |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Roger Pain for advice
regarding the manuscript and Dr. Bogdan Kralj for invaluable help with
ES mass spectrometry measurements.
| |
FOOTNOTES |
*
This work was supported by the Ministry of Science and
Technology of the Republic of Slovenia and by INCO-Copernicus Grant ERBIC 15CT960921.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The amino acid sequence reported in this paper has been submitted
to the Swiss Protein Database under Swiss-Prot accession no.
P82314.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF230360.
The first two authors contributed equally to this work.
¶
To whom correspondence should be addressed. Tel.: 386 61 1773474; Fax: 386 61 273594; E-mail: joze.brzin@ijs.si.
Published, JBC Papers in Press, March 23, 2000, DOI 10.1074/jbc.M001392200
| |
ABBREVIATIONS |
The abbreviations used are:
Z, benzyloxycarbonyl;
Ep-475, L-3-carboxy-trans-2,3-epoxypropylleucylamido-(3-guanidino)butane;
MCA, 7(4-methyl)-coumarylamide;
PAGE, polyacrylamide gel
electrophoresis;
PCR, polymerase chain reaction;
HPLC, high pressure
liquid chromatography.
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ABSTRACT
INTRODUCTION
