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CLITOCYPIN, A NEW TYPE OF CYSTEINE PROTEINASE INHIBITOR FROM

FRUIT BODIES OF MUSHROOM Clitocybe nebularis *

Jože Brzin“¶**, Boris Rogelj“¶, Tatjana Popovič¶, Borut Štrukelj+¶, and Anka

Ritonja¶

From the ¶Department of Biochemistry and Molecular Biology, Jožef Stefan Institute,

Jamova 39, 1000 Ljubljana, Slovenia and +Department of Pharmaceutical Biology, Faculty

of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia

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Running title:

A novel inhibitor of cysteine proteinases from mushroom

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SUMMARY

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 HPLC. The active inhibitor has

an apparent molecular mass of about 34 kDa by gel filtration and by SDS-PAGE without

prior boiling of the sample. Boiling in 2.5 % SDS or incubation in 6M GdmHCl resulted in a

single band of 17 kDa, indicating homodimer composition with no intersubunit disulphide

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 towards 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 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 metastasising cancer (13), bone resorbtion

(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 characterised 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, Clitocybe nebularis were collected in their

natural habitat in forest in November, and frozen at –20°C or –70°C until use. A specimen is

deposited at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Ljubljana.

Chemicals and Enzymes Iodoacetate, Bz-Arg-NA1 were from Sigma (Germany),

CNBr-activated Sepharose 4B, Sephacryl S-200 were from Amersham Pharmacia Biotech

(Sweden). Z-Phe-Arg-MCA 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) 2x crystallised, 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, UK) and

was prepared as described (18). Endoproteinase Lys-C (EC 3.4.21.50) was from Boehringer

Mannheim (Germany). β-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 Clitocybe nebularis (500 g fresh

weight) were homogenised in 1000 ml of Tris/HCl buffer, pH 7.5, containing 0.5 M NaCl

(Buffer A). The homogenate was centrifuged at 8000 x g, for 30 min. The supernatant was

applied in aliquots of 300 ml to a column of CM-papain Sepharose (2.5 x 15 cm) prepared

according to manufacturer’s instructions, carboxymethylated as described in (21) and

equilibrated with Buffer A. Bound inhibitory fractions were eluted with 0.01 M NaOH,

pooled, neutralised with dilute HCl, concentrated on an Amicon UM-10 membrane and

chromatographed on a Sephacryl S-200 column (4 x 110 cm) washed with buffer A. For the

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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 x 250 mm), using a gradient of 0-80 % (v/v) acetonitrile in water containing 0.1 % (v/v)

of trifluoroacetic acid, over 25 min.

Isolation of RNA, RT-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 BamHI restriction site on its 5’ end. Both primers

contained inosine (I) in the place of 4-nucleotide degeneration. Reverse transcription was

performed in a reaction-mix containing 1 x PCR buffer II, 5 mM MgCl2, 1 mM of each

dNTP, 1 U/µl RNasin, 0.5 µg of CnR primer, and 2.5 U/µl of MuLV 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 x PCR buffer II, 5 mM MgCl2, 0.83 µg CnF

primer, 0.33 µg CnR primer, and 2.5 U AmpliTaq DNA polymerase (Perkin Elmer, USA) to

the final volume of 100 µl. After electrophoresis on 1.7 % agarose gel a band at about 400 bp

was excised, inserted into EcoRI/BamHI digested pUC19, and sequenced using the

Pharmacia T7 sequencing kit following the appropriate manufacturer’s protocol.

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Protein and Sugar Determination Protein concentration of the purified inhibitor

was determined by absorbance at 280 nm using a Perkin Elmer UV/VIS Spectrometer

Lambda 18. A molar absorbance coefficient of 22900 M-1cm-1 was calculated from the amino

acid sequence (25).

Clitocypin was assayed for potential glycosylation with incubation with N-

glycosidase F (Boehringer Mannheim), according to the manufacturer′s instructions. The

reaction was incubated overnight at 37°C and the products followed by SDS-PAGE. The

inhibitor was also tested with a phenol-sulfuric acid assay for hexoses and pentoses as

described (26).

SDS-PAGE 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 100oC for 5 min. For sample reduction, 2-

mercaptoethanol in 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

(Pharmacia). Gels were stained with 0.1 % (w/v) Coomassie Briliant Blue R 250.

Isoelectric focusing Samples were run on a Pharmacia Phast System using

commercial precast pH 3-9 gradient gels following instructions provided. pI values were

determined using the Pharmacia broad-pI calibration kit (pI range 3.65 - 9.30)

Electrospray-Ionisation Mass Spectrometry HPLC purified protein was dissolved

in water/methanol 1 : 1 (v/v) solution containing 1 % acetic acid and analysed 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 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

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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) TFA 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 GdmHCl and

incubated for 48 h at 37°C. After removal of GdmHCl 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 β-trypsin was carried out at 37oC in 0.5 M N-methyl morpholine, pH 8.2,

for 30 min at an enzyme to substrate ratio of 1 : 100 (w/w). Endoproteinase Lys-C at an

enzyme to 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 0-80 % 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 110oC for 24 h and analysis of the obtained

hydrolysates on an Applied Biosystems 421 amino acid analyser with precolumn PTH

derivatisation. 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 PTH

analyser from the same manufacturer. For sequence comparison of clitocypin with other

known protein sequences, databases were searched with ExPASy protein sequence similarity

search using the BLAST algorithm (27) and SAMBA (28).

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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 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 Bz-Arg-NA in DMSO and the

mixture was incubated for 10 min at 37°C. The reaction was stopped and A520 read, following

the procedure of Barrett (29).

Active Site Titrations Active concentrations of cathepsins B, L and papain were

determined by titration with Ep-475 (30). Residual activities were determined with Bz-Arg-

NA 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 concentrations stated 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 nM to 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 DTE and 1.5

mM EDTA, while cathepsin L was assayed in 0.4 M sodium acetate buffer, pH 5.5,

containing 2 mM DTE 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

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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 kdiss and kass 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 kass and kdiss (Ki =

kdiss/kass).

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 µM to 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 7-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 equation: Ki =

Ki,app/(1 + [So]/Km), where [So] is the initial substrate concentration and Km values of 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

(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, 0) and rabbit red

blood cells were extensively washed and suspended in buffered saline. 50 µl samples of crude

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Clitocybe nebularis extract and purified clitocypin (1mg/ml) and their serial dilutions were

mixed with 1 ml of 3 % (v/v) erythrocyte suspension in test tubes and incubated for 3 hours 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 as it specifically removed other non-inhibitory proteins, so that after gel

filtration the inhibitor was practically homogeneous (Fig. 1, 3). In a typical preparation we

obtained 2 mg of purified inhibitor from 100 g of fresh mushrooms.

The molecular mass of clitocypin under native conditions, as estimated by calibrated

gel filtration, as well as that obtained in non-reducing SDS-PAGE without boiling of the

sample was 34 kDa. The same Mr 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 two bands of 34 and

17 kDa on SDS-PAGE without boiling (Fig. 2). Fraction Cn1 is thus composed solely of

dimers (about 2/3 of the total) and Cn2 a mixture of dimers and monomers (about 1/3 of the

total ).

By ES mass spectrometry a single mass of 16863 Da was obtained for clitocypin from

gel filtration and from both HPLC peaks.

Isoelectric Focusing On IEF the inhibitory protein ran as a single band at pH value

of 4.4 (Fig. 3) which agrees well with the value of 4.42 calculated from the amino acid

composition.

Subunit Composition Analysis and Stability of Clitocypin Clitocypin proved to be

an extremely stable protein. When boiled in non-denaturing buffer (see details in the

Experimental procedures) it remained, as judged by inhibitory activity, elution volume in gel

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filtration and behaviour on SDS-PAGE, indistinguishable from the initial inhibitor (Fig. 1).

Incubation in denaturing buffer with GdmHCl 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 16854 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.

A primer pair designed to span from the previously determined N-terminus to the

apparent C-terminus was used for the RT-PCR amplification of mRNA isolated from

Clitocybe nebularis fruit bodies. A product of approximately 400 base pairs termed Cn c1

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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 7 amino acid residues that differ between the cDNA derived and

protein sequences are located in the C-terminal region of the protein.

Amino Acid Sequence Comparison In a search for homologous proteins in

databases 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 just to 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 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 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, (kass), and

dissociation, (kdiss), rate constants and equilibrium constants (Ki) are presented in Table I.

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Both kass and kdiss 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 µM range. Under the same

conditions no inhibition of cathepsin H was observed, even at 100-fold excess of the

inhibitor.

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

Clitocybe 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 Clitocybe nebularis contains a specific inhibitor of cysteine

proteinases - clitocypin. It was obtained in high amount 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

stabilised by non-covalent 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

aminoacid sequence agrees well with that obtained by ES mass spectrometry, indicating that

no post-translational modification occurs. Taken together, this data show that this inhibitor

consists of only one kind of polypeptide chain of 16854 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

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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 to 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 which 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 Pleurotus

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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 characterised 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

Clitocybe nebularis lectin as an oligomeric protein, with subunit molecular mass in the range

of 15 to 20 kDa, isoelectric point around pH 4.4, no cysteines, lacking or containing 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 Clitocybe 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 3-D 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

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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

difference in the levels of expressed proteins at the different developmental or environmental

conditions.

The physiological target of clitocypin in Clitocybe nebularis is not known. The

inhibitor is expressed at very high levels, characteristic of proteins with roles that are more

structural, as opposed to signalling, catalysis or control. In relation to the control, although

no cysteine proteinase activity has been detected in mushroom juice, it may well be localised

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

characterised 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).

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Aknowledgements We are grateful to Dr. Roger Pain for his advice regarding the

manuscript and Dr. Bogdan Kralj for his invaluable help with ES mass spectrometry

measurements.

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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

were defrayed in part by the payment of page charges. This article must therefore be hereby

marked “advertisement” in accordance with 18 U.S.C. Section 1734, solely to indicate this

fact.

“ 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

The protein sequence reported in this paper has been submitted to the Swiss Protein

Database under Swiss-Prot accession number P82314 and the nucleotide sequence in the

GenBank under accession number AF230360.

1 The abbreviations used are: Bz, benzoyl; CM, carboxymethyl; DTE, dithioerythritol;

Ep-475, L-3-carboxy-trans-2,3-epoxypropylleucylamido-(3-guanidino)butane; GdmHCl,

guanidine hydrochloride; MCA, 7(4-methyl)-coumarylamide; NA, 2-naphthylamide; PAGE,

polyacrylamide gel electrophoresis; SDS, sodium dodecyl-sulphate; TFA, trifluoroacetic

acid; Z, benzyloxycarbonyl;

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FIGURE LEGENDS

FIG. 1. Analysis of the purification of clitocypin from Clitocybe nebularis by SDS-

PAGE. Samples were run as follows: Clitocybe 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 non-denaturing buffer and eluted from gel filtration (lane 9) and GdmHCl

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 analysed in the presence of 5 % 2-

mercaptoethanol are indicated as ME. The gel was Coomassie stained.

FIG. 2. HPLC chromatography of clytocypin. The elution profile of clitocypin

obtained from gel filtration (solid line). Linear gradient from 0-80 % of acetonitrile in 0.1 %

TFA (dashed line).

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

Clitocybe nebularis extract (lane 3).

FIG. 4. Summary of the determination of the complete amino acid sequence of

clitocypin. The sequenced regions are marked by 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.

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

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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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bt rt rt bt bt rt rt bt rt rt

1 2 3 4 5 6 7 8 9 10

94

67

30

14.4

ME MEMW

43

20.1

8

btbt

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Cn2

Cn1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

-5 0 5 10 15 20 25

time (min)

abs

orba

nce

(215

nm)

0

20

40

60

80

acet

onitr

ile (%

)

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8.658.45

8.15

7.35

6.856.55

5.85

5.20

4.55

3.50

1 2 3

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10 20 30 40LEDGIYRLRAVTTHNPDPGVGGEYATVEGARRPVKAEPNT

N ----------------------------------------A ----------------------------------------T ----------------------------------------G ----------------------------------------L ----------------------------------------

50 60 70 80PPFFEQQIWQVTRNADGQYTIKYQGLNTPFEYGFSYDELE

N ----------------------------------------A ----------------------------------------T ----------------------------------------G ----------------------------------------L ----------------------------------------

90 100 110 120PNAPVIAGDPKEYILQLVPSTADVYIIRAPIQRIGVDVEE

N ----------------------------------------A ----------------------------------------T ----------------------------------------G ----------------------------------------L ----------------------------------------

130 140 150GGQQNTLTYKFFPVDGSGGDRPAWRFTREE

N ------------------------------A ------------------------------T ------------------------------G ------------------------------L ------------------------------

A1 A2

T1

G1

N1

G1

L1

L1

G2

A4A3

L2

A5A4

A3A2

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A1

11

21

31

41

51

61

71

clitocypin

LEDGIYRLRA

VTTHNPDPGV

GGEYATVEGA

RRPVKAEPNT

PPFFEQQIW.Q

VTRNA.DGQYT

IKYQGLNTPF

EYGFSYDELE

Cn

c1

---

----------

----------

---------.-

-----.-----

----------

----------

PC-LRP

SDAFMLRAS-FV--

--M---GQ-Q

GN-IIVARLG

...MQ--L-VE

AQP-KIG-DDA

VAIFSKDARL

TWK-T....-

81

91

101

111

121

131

141

clitocypin

PNAPVIAGDP

KE....YILQLV.PS

TA.DVYIIRAP

IQRIGVD.VEE

GGQQNTLTYK

FFPVDGSGGD

RPAWRFTREE

Cn

c1

----------

--....------.--

--.--------

-------.-VV

–V-G---V--

-------S--

----S-

PC-LRP

-GR--TLTES

EQPSLW--RRV-K-E

DGQE-VQ-V-K

TDLL-ATWYAD

V-PD-MIVI-

SI--APPFTP

G-V-Q

BP

GV

GG

EY

AT

VE

GA

RR

PV

KA

ECCGGGGGTGGGGGGGGAGTACGCTACCGTAGAAGGAGCTCGCCGACCCGTCAAGGCCGAA

60

PN

TP

PF

FE

QQ

IW

QV

TR

NA

DG

CCTAACACACCTCCCTTCTTTGAGCAACAAATCTGGCAGGTCACTCGGAATGCCGACGGC

120

QY

TI

KY

QG

LN

TP

FE

YG

FS

YD

CAATACACCATCAAATATCAAGGGTTGAACACCCCTTTTGAGTACGGATTTTCTTACGAT

180

EL

EP

NA

PV

IA

GD

PK

EY

IL

QL

GAGCTTGAGCCGAATGCACCCGTCATCGCTGGAGACCCAAAGGAATACATTCTTCAGCTT

240

VP

ST

AD

VY

II

RA

PI

QR

IG

VD

GTCCCTTCTACTGCTGATGTTTACATCATCAGGGCCCCTATACAGCGTATTGGCGTAGAC

300

VV

VG

VQ

GN

TL

VY

KF

FP

VD

GS

GTTGTCGTTGGTGTACAGGGGAACACTCTTGTTTATAAATTTTTCCCTGTTGATGGTTCT

360

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TABLE I

Kinetic data for the interaction of clitocypin with different cysteine proteinases

Kinetic and equilibrium constants for the inhibition of papain and cathespin L were

determined under pseudo first-order conditions in continous 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 in “Experimental

Procedures”. Interactions were performed at 25oC.

Enzyme 10-6 x kass 104 x kdiss Ki

M-1s-1 s-1 nM

Papain 1.6 ± 0.20 9.5 ±1.71 0.59 ± 0.15

Cathepsin L 0.8 ± 0.15 3.3 ± 0.79 0.41 ± 0.14

Cathepsin B ND ND 480 ± 90

Bromelain ND ND 160 ± 70

a ND is not determined.

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Joze Brzin, Boris Rogelj, Tatjana Popovic, Borut Strukelj and Anka RitonjaFROM FRUIT BODIES OF MUSHROOM Clitocybe nebularis

CLITOCYPIN, A NEW TYPE OF CYSTEINE PROTEINASE INHIBITOR

published online March 23, 2000J. Biol. Chem. 

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