28 DOI: 10.17344/acsi.2018.4465 Acta Chim. Slov.

2019, 66, 28–36

Scientific paper

β-Trefoil Protease Inhibitors Unique

to Higher Fungi
Jerica Sabotič,1,* Miha Renko2 and Janko Kos1,3
1 Department of Biotechnology, Jožef Stefan Institute, Ljubljana, Slovenia
2 MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
3 Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia

* Corresponding author: E-mail: jerica.sabotic@ijs.si;

Tel: +38614773754; Fax: +38614773594

Received: 05-14-2018
Dedicated to the memory of Prof. Dr. Igor Kregar

Abstract
The cysteine protease inhibitors, clitocypin and macrocypins, from higher fungi (mycocypins), together with the serine
protease inhibitors highly specific for trypsin cospin and cnispin from higher fungi (mycospins), display several char-
acteristics that distinguish them from protease inhibitors from other sources. Their high genetic heterogeneity affects
their functionality and/or stability and results in numerous protein variants with slightly different inhibitory profiles that
influence the type of protease inhibited and/or the strength of inhibition. They possess the β-trefoil fold that shows high
plasticity in their utilization of the 11 diverse loops for the inhibition of various families of proteases through different
mechanisms of inhibition. Their high versatility is also seen in their regulatory and defence functions and in their poten-
tial applications in biotechnology, crop protection and medicine.

Keywords: Protease inhibitor; regulation; defence; clitocypin; macrocypin; cospin

1. Introduction bitory specificity, provide new mechanisms of inhibition

and offer multiple possibilities in medical and biotechno-
Proteolytic enzymes, also termed proteases or pep- logical applications.
tidases, are degradative enzymes that catalyse the hydro- We decided to utilize higher fungi or mushrooms as
lysis of peptide bonds. They are ubiquitous and essential a new and promising source of protease inhibitors10 since
for the survival of all organisms, since they enable nutri- very little was known at the time about the proteolytic
ent acquisition, growth, proliferation and reproduction. systems in higher fungi. A first glimpse of the complexity
Furthermore, they are critical for defence against patho- of these systems was provided by an investigation of the
gens and parasites. Since their activity is so essential, it proteolytic activity of mushrooms using gelatin zymo-
needs to be tightly regulated and dysregulation often le- graphy combined with selected protease inhibitors. The
ads to disease.1–5 Because of their important roles in number and diversity of proteolytically active bands ob-
physiological and pathophysiological processes they are served was unexpectedly high. These proteases were classi-
paramount targets and tools in the search for new strate- fied into different catalytic classes, a large proportion of
gies with applications in medicine, pharmaceutical indu- them showing atypical properties. This indicated the great
stry and agriculture. potential for finding novel protease inhibitors in the prote-
The regulation of proteolytic enzymes is also vital, olytic systems of higher fungi.11
because hydrolysis of peptide bonds is irreversible. Speci- An overview is provided here of the protein protease
fic protease inhibitors play a very important role in their inhibitors that we have identified in higher fungi, together
regulation.6–9 For this reason, we undertook a search for with their unique features established by characterization
further protease inhibitors that could exhibit unique inhi- at the genetic, molecular, structural and functional levels.

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 Acta Chim. Slov. 2019, 66, 28–36 29
2. Protease Inhibitors from Higher their ability to unfold reversibly.16–20 Furthermore,
Fungi mycocypins are resistant to proteolytic digestion by the
highly non-specific proteinase K although they do not in-
Protease inhibitors are indispensable regulators of hibit its proteolytic activity.21
proteolytic enzymes and are present in all kingdoms of life. Family I66 comprises the trypsin-specific inhibitors
They can be classified according to their origin, inhibitory mycospins: cnispin22 (Cnp) from Clitocybe nebularis and
mechanism, structural similarity or their specificity. The cospin23 (PIC) from Coprinopsis cinerea, as well as a repre-
last is most often used to group inhibitors according to sentative from Lentinula edodes.24 These inhibitors are
those that inhibit a number of classes of peptidases, those small proteins, the molecular mass of cnispin being 16.4
that inhibit a single class of peptidases, those that inhibit kDa and that of cospin 16.7 kDa and both with a low isoe-
one or more families of peptidases or just a single peptida- lectric point around pH 5. They are both stable at extreme
se.6,12 The MEROPS database (https://www.ebi.ac.uk/me- pH and cospin is resistant to proteolytic digestion.21–23
rops/) provides the most extensive classification of protea-
se inhibitors into families, based on sequence homology,
and into clans, based on similarity of 3D structure. There
2. 1. Genetic Heterogeneity
are currently 79 families of protease inhibitors in the ME-
ROPS database (release 12.0; April 2018). Of these, 24 in- Mycocypins are encoded by small families of genes
clude members of fungal origin and only seven include whose members show sequence heterogeneity. Genes en-
members from higher fungi, of which only four families coding clitocypin in the C. nebularis genome are compo-
(I9, I48, I66 and I85) include members that have been cha- sed of four exons and three short introns. Nucleotide
racterized at the protein level.13 Family I9 comprises subti- substitutions are evenly distributed throughout the gene
lisin propeptide-like inhibitors isolated from oyster mu- sequence. The diversity of amino acid substitutions howe-
shroom (Pleurotus ostreatus)14,15 and the other three are ver is mainly conservative and the isogene sequences for
described in this review (Table 1). clitocypin share more than 90% identity at the level of the
Families I48 and I85 comprise mycocypins, the fun- deduced protein sequence.25 A family of clitocypin enco-
gal cysteine protease inhibitors clitocypin16 (Clt, family ding genes that show similar heterogeneity has also been
I48) from the clouded agaric (Clitocybe nebularis), and detected in the M. procera genome.17
macrocypin17 (Mcp, family I85) from parasol mushroom Genes encoding macrocypins in the M. procera ge-
(Macrolepiota procera). Mycocypins are small proteins nome are also composed of four exons and three introns.
with molecular masses between 16.6 kDa (clitocypin) and Their diversity is, however, much greater. The deduced
19.0 kDa (macrocypin 1) that have similar biochemical amino acid sequences are divided into five groups with
properties. They all exhibit isoelectric points around pH 75–86% sequence identity between groups and more than
4.8. An exceptional feature is their apparent resistance to 90% sequence identity within groups. Some of the variable
high temperature and to extremes of pH that results from codons have been subject to positive evolutionary selecti-

Table 1. Overview of β-trefoil protease inhibitors from higher fungi.

Mycocypins Mycospins
Protease inhibitor Clitocypin Macrocypin 1 Macrocypin 3 Macrocypin 4 Cnispin Cospin
Origin Clitocybe Macrolepiota Macrolepiota Macrolepiota Clitocybe Coprinopsis
nebularis procera procera procera nebularis cinerea
Abbreviation Clt Mcp1 Mcp3 Mcp4 Cnp PIC
MEROPS family I48 I85 I85 I85 I66 I66
Mass 16582 Da 19062 Da 18900 Da 18639 Da 16407 Da 16713 Da
Isoelectric
4.8 4.8 4.8 5.1 5.2 4.9
point (pH)
PDB entry 3H6R, 3H6S 3H6Q / / / 3N0K, 3VWC
Protease family
C1/C13 C1/C13 C1/C13 C1/S1 S1 S1
inhibited
Inhibitory β1–β2 and β1–β2 and β1–β2 and β1–β2 and β11–β12 β2–β3
loop β3–β4/β5–β6 β3–β4/β5–β6 β3–β4/β5–β6 β3–β4/β5–β6
Resistant to high high high
exposure to temperature temperature temperature alkaline pH extreme pH extreme pH
& extreme pH & extreme pH & extreme pH
Resistant to
proteolytic digestion yes yes yes yes no yes
by proteinase K

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30 Acta Chim. Slov. 2019, 66, 28–36

on, indicating their importance for the function of the pro- differences indicating regulatory complexity are seen in
tein.17 Gene sequences encoding macrocypins are also the different levels of clitocypin mRNA expression in diffe-
present in the C. nebularis genome, the degree of sequence rent parts of fruiting bodies and by variation of expression
identity to that of macrocypin 2 from M. procera being the in mycelium.17,25,26
highest.26 The complex expression pattern of macrocypin ge-
Deduced amino acid sequences of macrocypins and nes reflects and enhances the diversity of their gene sequ-
clitocypin show very low overall sequence identities of 17 to ences. They show tissue specific expression patterns at the
21% (Table 2 & Figure 1). Furthermore, macrocypin sequ- promoter, mRNA and protein levels that differ for different
ences contain sulphur containing amino acids, a cysteine macrocypin genes.26
residue being present in most macrocypin sequences and In addition to developmental regulation of mycocy-
several histidine and methionine residues are present in all pins, environmental factors have influenced their expressi-
macrocypins while they are absent in clitocypin sequences. on, as indicated by the clitocypin genes in L. bicolor myce-
On the other hand, the deduced amino acid sequences of lium, whose expression was upregulated specifically in the
both clitocypin and macrocypin contain high contents of presence of an antagonistic soil bacterium, Collimonas
proline and tyrosine but low contents of leucines.16,17,25 fungivorans.28
The low sequence identity between different families Mycospins are encoded by very small gene families,
of mycocypins hinders the search for their homologs in as indicated by the sequence diversity in natural isolates of
other fungal genomes. Indeed, BLASTP analysis, using cli- cnispin from C. nebularis fruiting bodies and confirmed in
tocypin sequence, across 145 fungal genomes revealed si- the C. cinerea genome for cospin, where four isogenes
milar protein encoding genes in only four, namely Botryo- were found. Cnispin genes are composed of four exons and
basidium botryosum (10 genes), Rhizoctonia solani (4 three short introns. The sequence identity between the de-
genes), Laccaria amethystina (13 genes) and Laccaria bico- duced amino acid sequences of cnispin and cospin is 26 to
lor (10 genes), all members of the class Agaricomycetes.27 30% (Table 2 & Figure 2).22,23
These genomes include small families of clitocypin analog Despite the low sequence identity among mycospins,
genes (indicated in brackets) that show low sequence iden- several homologous sequences have been found in diffe-
tity between organisms. Given this low sequence identity, rent species. For example, a four-gene family was identifi-
it is probable that mycocypins are much more widespread ed in L. bicolor with 17 to 30% amino acid sequence iden-
in higher fungi. tity to that of cospin.23 Furthermore, BLASTP analyses
The complex regulation of expression of mycocypins across 145 fungal genomes, using cospin sequence, revea-
at different levels is indicated by the different promoter led similar protein encoding genes in 21 basidiomycete
sequences as well as by differences in 5’UTR, 3’UTR and species from class Agaricomycetes and also in two
intron sequences and in their lengths. This was confirmed ascomycetes. Either one gene or small gene families, ran-
by the distinct expression levels of clitocypin and macro- ging from 2 to 12 genes, were identified.27 Furthermore,
cypin in both their origin mushrooms and in the model mycospins were identified and characterized at the protein
species C. cinerea.17,25,26 and functional levels in three additional basidiomycete
The expression of clitocypin appeared to be uniform species: Armillaria mellea, Macrolepiota procera and Ama-
at the protein level in both C. nebularis and M. procera fru- nita phalloides.29 Mycospins appear to be more widely pre-
iting bodies. The expression pattern guided by the clitocy- sent in fungi than mycocypins, although this could also be
pin promoter in the model species was very similar to that the consequence of more favourable search parameters
of the constitutive promoter gpdII of glyceraldehyde- arising from the higher sequence similarity among mycos-
3-phosphate dehydrogenase from Agaricus bisporus. Some pins.

Table 2. A sequence identity matrix of mycocypin and mycospin deduced from amino acid sequences. The percent sequence identity is given for
each pair. ID. identical.

Mcp1 Mcp2 Mcp3 Mcp4 Mcp5 Clt-Kras Clt-Vrh PIC Cnp

Mcp1 ID 82.8 78.6 79.8 76.3 17.4 18.0 10.7 10.9
Mcp2 ID 80.2 85.0 79.6 19.4 19.4 10.8 12.2
Mcp3 ID 80.8 78.4 21.1 21.7 10.8 11.6
Mcp4 ID 82.0 18.2 18.8 10.8 13.9
Mcp5 ID 18.9 18.9 10.2 11.6
Clt-Kras ID 91.4 12.6 13.9
Clt-Vrh ID 13.2 15.1
PIC ID 27.0
Cnp ID

Sabotič et al.: β-Trefoil Protease Inhibitors Unique to Higher Fungi

 Acta Chim. Slov. 2019, 66, 28–36 31

Cnispin and cospin are expressed in vegetative myce- changed and trypsin is inhibited instead of legumain. The
lium and in fruiting bodies and are not secreted. Much sequence heterogeneity in the clitocypin gene family has
(approximately 700 fold) higher expression in fruiting bo- no influence on the inhibitory activity while the greater
dies than in mycelium has been determined for cospin.22,23 sequence heterogeneity in macrocypin sequences is refle-
cted in their inhibitory profiles (Table 3).17,20,22,23,30
Mycospins are strong and highly specific inhibitors
2. 2. Inhibitory Specificity
of trypsin with values of Ki in the picomolar range for cos-
Mycocypins inhibit cysteine proteases of plant and pin and in the nanomolar range for cnispin. Both are also
animal origins but the strength of inhibition against diffe- weak inhibitors of chymotrypsin with Ki in the micromo-
rent proteases differs between members of clitocypin and lar range. Other serine proteases are very weakly or not at
macrocypin families (Table 3). They are all strong inhibi- all inhibited (Table 3).22,23
tors of papain-like proteases (family C1), with equilibrium Natural isolates of clitocypin and macrocypin display
constants for inhibition (Ki) ranging from picomolar to inhibitory profiles that differ slightly when (Table 4) com-
micromolar for various cysteine cathepsins and papain. pared with those of recombinant variants. This is the effect
Cathepsins with endopeptidase activities are strongly inhi- of the mixture of isoforms in the natural sample isolated
bited while cathepsins B and H, that exhibit both endopep- from mushrooms growing in the wild.16,17,20,25 Despite
tidase and exopeptidase activity, are not inhibited by clito- their sequence heterogeneity, natural isolates of cnispin,
cypins and only very weakly by macrocypins. They also CnSPIs, display the same inhibitory profile as cnispin and
inhibit legumain/asparaginyl peptidase with Ki in the na- are very strong inhibitors of trypsin, while inhibition of
nomolar range, albeit involving a different inhibitory acti- chymotrypsin is about 40 times weaker and elastase and
ve site (Figure 1 & 3). The latter is, in some macrocypins, thrombin are not inhibited at all.22

Figure 1. Alignment of mycocypin deduced amino acid sequences. Identical residues are highlighted in dark grey and similar residues in light grey.
Amino acid sequences of macrocypins (Mcp) and clitocypins (Clt) were aligned with the BLOSUM62 matrix. Residues forming inhibitory loops that
inhibit papain-like proteases are boxed. P1 residues, crucial for the inhibition of legumain or trypsin, are underlined and shown in bold. (Mcp –
macrocypin, Clt-Kras and Clt-Vrh are native sequences of clitocypin isolated from fruiting bodies collected in the two widely separated regions, Kras
and Vrh in Slovenia.)

Figure 2. Alignment of deduced amino acid sequences of mycospins. Identical residues are highlighted in dark grey and similar residues in light grey.
Amino acid sequences of cnispin (Cnp) and cospin (PIC) are aligned using the BLOSUM62 matrix. The arrows indicate the trypsin reactive P1
residue in cospin (white) and in cnispin (black).

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32 Acta Chim. Slov. 2019, 66, 28–36

Table 3. Kinetic constants for the interaction of mycocypins and mycospins with various proteases.17,20,22,23,30 Standard deviations are given where
appropriate. rClt, recombinant clitocypin; rMcp1, recombinant macrocypin 1; rMcp3, recombinant macrocypin 3; rMcp4 recombinant macrocypin
4; Cnp, recombinant cnispin; PIC, recombinant cospin; n.i., no inhibition; ND, not determined.

Protease Ki (nM)
Protease
family rClt rMcp1 rMcp3 rMcp4 Cnp PIC
Papain C1 6.2 ± 0.55 0.95 ± 0.33 0.12 ± 0.05 0.19 ± 0.01 n.i. n.i.
Cathepsin L C1 0.02 ± 0.001 0.64 ± 0.22 0.31 ± 0.06 2.76 ± 0.92 ND ND
Cathepsin V C1 0.08 ± 0.03 0.69 ± 0.06 0.45 ± 0.01 1.44 ± 0.11 ND ND
Cathepsin S C1 2.2 ± 0.3 23.1 ± 1.2 5.1 ± 0.5 6.3 ± 0.6 ND ND
Cathepsin K C1 0.03 ± 0.002 170 ± 20 17.5 ± 1.2 21.8 ± 5.2 ND ND
Cathepsin B C1 > 1000 490 ± 18 > 1000 125 ± 10 ND ND
Cathepsin H C1 n.i. 100 ± 10 24 ± 5 32 ± 6 ND ND
Legumain C13 21.5 ± 2.81 3.38 ± 1.44 9.17 ± 1.09 > 1000 n.i. n.i.
Caspase 3, 6, 9 C14 n.i. ND ND ND ND ND
Trypsin S1 n.i. n.i. n.i. 160 ± 14 3.10 ± 0.66 0.022 ± 0.002
Chymotrypsin S1 ND ND ND ND 120 ± 20 116 ± 8
Kallikrein S1 ND ND ND ND > 1000 > 1000
Thrombin S1 ND ND ND ND n.i. n.i.
Subtilisin S8 ND ND ND ND > 1000 > 1000
Pepsin A1 n.i. n.i. n.i. n.i. n.i. n.i.

Table 4. Inhibition of various proteases by natural mycocypins. The either side of the active site cleft with several hydrogen
kinetic data are as reported.17 Standard deviations are given where bonds, occluding the catalytic cysteine. The binding is as-
appropriate. nMcp, natural macrocypin; nClt, natural clitocypin;
sociated with a glycine-glycine peptide-bond flip (Gly-
n.i., no inhibition.
24-Gly25 in clitocypin) that occurs before or concurrently
Protease Ki (nM) with inhibitor docking.31 The mode of cysteine protease
Enzyme inhibition, as revealed by the cathepsin V – clitocypin
family nClt nMcp
complex, is unique in utilizing two loops to achieve inhibi-
Papain C1 2.5 ± 0.94 5.04 ± 0.98 tion, while other known modes of inhibition by cysteine
Cathepsin L C1 0.03 ± 0.002 3.81 ± 1.66
protease inhibitors, like those of cystatins and thyropins,
Cathepsin V C1 0.14 ± 0.01 12.6 ± 3.77
utilize three loops to achieve active site occlusion.31,32
Cathepsin S C1 3.2 ± 0.3 47.1 ± 3.1
Cathepsin K C1 0.02 ± 0.005 4.5 ± 0.5 Inhibition of asparaginyl peptidase/legumain by
Cathepsin B C1 > 1000 515 ± 36 mycocypins is achieved via a second inhibitory active site
Cathepsin H C1 n.i. 370 ± 11 in the β5–β6 loop. Site-directed mutagenesis has con-
Legumain C13 7.1 ± 1.12 110 ± 23 firmed that residues Asn72 in macrocypins and Asn69 in
Trypsin S1 n.i. n.i. clitocypin mediate inhibition of asparaginyl peptidase.31
Pepsin A1 n.i. n.i. Furthermore, the same inhibitory active site mediates the
inhibition of trypsin when Asn replaces Lys or Arg in di-
fferent macrocypins but not in clitocypin.31 The inhibitory
2. 3. Structural Plasticity mechanism of the cysteine protease asparaginyl peptidase
(family C13) and of the serine protease trypsin (family S1)
Mycocypins and mycospins both possess a β-trefoil appears to involve a similar substrate-like binding inhibiti-
fold,23,31 thus classifying them to clan IC in the MEROPS on.32
classification, together with Kunitz serine protease inhibi- Cospin and cnispin are classic canonical inhibitors
tors from plants (family I3).13 that bind to the active site in a substrate-like manner and
The β-trefoil fold consists of a β-barrel composed of form a tight and stable complex with trypsin. The trypsin
three pairs of antiparallel β-strands. An additional three pairs -cospin complex is stable for weeks at 37 °C while the
of β-strands cover the β-barrel. The strands are connected by trypsin-cnispin complex is degraded within 24 h. This is
loops of different shapes and compositions (Figure 3). The also reflected in the stronger inhibition of trypsin by cos-
large surface area of the loops, that accounts for approxima- pin as opposed to that by cnispin (Table 3). Inhibition of
tely 70% of the protein’s total solvent accessible area, enables trypsin by mycospins is achieved through the different in-
these proteins to interact with many different binding par- hibitory reactive sites in cospin and cnispin. The reactive
tners, including proteins, carbohydrates and DNA.23,31,32 site residue P1 of cospin is Arg27 in the β2–β3 loop, while
For the inhibition of papain-like proteases, mycocy- Lys127 in the β11–β12 loop of cnispin fulfils the same role
pins utilize two loops (β1–β2 and β3–β4) that bind to (Figure 2 & 3).23,30 Inhibitory activity can be mediated by

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 Acta Chim. Slov. 2019, 66, 28–36 33

Figure 3. Structures of fungal cysteine protease inhibitors. Ribbon diagrams of the cysteine protease inhibitors macrocypin 1 (PDB code 3H6Q) in
orange and clitocypin (PDB code 3H6S) in magenta are shown next to that of the serine protease inhibitor cospin (PDB code 3N0K9) in cyan. Loops
with the following inhibitory reactive sites are marked with arrows: light grey arrows indicate asparaginyl protease/legumain inhibition, dark grey
arrows indicate papain-like protease inhibition while white and black arrows indicate trypsin inhibition. A schematic representation of the β-trefoil
fold loops involved in protease inhibition is shown bottom right. The protease family inhibited by individual loops of the β-trefoil fold in these fun-
gal inhibitors is indicated by coloured arrows as follows: the asparaginyl protease/legumain with orange, papain-like proteases with magenta and
trypsin with cyan.

both β2–β3 and β11–β12 loops in the two inhibitors; engi- the low number of cysteine residues and lack of glycosyla-
neering of various P1 residues in the aforementioned lo- tion in natural samples, no secretion of cnispin and clito-
ops yielded a strong or weak trypsin inhibitor, a chymot- cypin has been detected from cultivated mycelium.22,23,25
rypsin-specific inhibitor, a double headed trypsin inhibitor An obvious biological role for protease inhibitors is
or a double-headed trypsin and chymotrypsin inhibitor. the regulation of the endogenous proteolytic system. Acti-
Other serine proteases are only weakly or not at all inhibi- vity of several cysteine proteases from fruiting bodies of
ted. Cysteine and aspartic proteases were not inhibited and different basidiomycetes is inhibited by clitocypin.11 Simi-
asparaginyl peptidase/legumain inhibition was not achie- larly, cnispin and cospin inhibit the activity of various seri-
ved by introducing Asn as the P1 residue.30 ne proteases from their origin species as well as from other
basidiomycetes.11,22,23 Regulation of the complex develo-
pmental and temporal expression of both mycocypins and
2. 4. Functional Variability
mycospins indicates potentially different biological roles
The proposed function of mycocypins and mycos- for different inhibitors.22,6
pins is to regulate both the endogenous proteolytic system Another possible role for protease inhibitors, that is
and the defence against predators and pathogens. Mycocy- indirectly but strongly supported, is in defence against va-
pins and mycospins are cytoplasmic proteins that are not rious antagonists. The strong toxicity of cnispin and cospin
secreted. In addition to the absence, in all the identified against Drosophila melanogaster larvae, their cytoplasmic
genes, of a signal sequence for the classical secretion and to localization and the higher expression in fruiting bodies

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34 Acta Chim. Slov. 2019, 66, 28–36

compared to that in mycelium indicates their role in the nity chromatography. Active cysteine proteases of families
defence of the spore-bearing fruiting body against dipte- C1 (papain-like) and C13 (legumain/asparaginyl protease)
ran larvae that hatch and feed on fruiting bodies.22,23 Pro- have been isolated from plant and animal sources using
tection of reproductive tissues from pests and diseases has macrocypin affinity chromatography. Their superior cha-
also been suggested as a defensive role of mycocypins. racteristics in terms of stability to pH and temperature
Thus, cysteine proteases are predominant digestive protea- make them ideal candidates for use as affinity chromato-
ses in many insects and slugs and could be targeted, like graphy ligands. They withstand the harsh conditions du-
trypsin-like digestive proteases in dipteran insects are by ring immobilization procedures. For example, following
mycospins. Furthermore, cysteine proteases are important 24h incubation at neutral pH and 45 °C for covalent bin-
virulence factors of different pathogenic bacteria, parasites ding to a monolithic disk, the unoccupied groups are ina-
and mycoviruses. These could be targeted by different ctivated by incubation for 1 h in 0.5 M H2SO4 at 50 °C and
mycocypins, as indicated by their different expression pro- macrocypins then retain their inhibitory activity through
files. Clitocypin, which is constitutively expressed in large several elution cycles of extreme pH changes.39
amounts in fruiting bodies, represents one line of defence, The distinct inhibitory profiles of mycocypins and
while the specific pattern of expression localized to the ou- the highly specific inhibitory profile of mycospins makes
ter layer of the developing fruiting body of some macrocy- them valuable tools in medical research, since many cell
pins constitutes another defence strategy for protection processes depend on appropriately regulated proteolytic
against external attacks by predators or pathogens. The activity. Protease inhibitors are being studied as promising
inducible expression of clitocypin analogs in L. bicolor therapeutic drugs for many types of disease by targeting a
when challenged by an antagonistic soil bacterium variety of deregulated proteases, including those involved
supports their role in defence. A defensive role is further in cancer and autoimmune, neurodegenerative, inflamma-
supported by the high thermal and pH stability and the tory, and cardiovascular diseases; secreted bacterial, fun-
resistance to proteolytic degradation of these proteins, as gal, and parasite proteases; and viral polyprotein pro-
well as by their high sequence diversity and versatile inhi- cessing proteases.7,40
bitory profile.17,22,23,25,26,28
Another layer of regulation of the β-trefoil fungal
protease inhibitors mycocypins and mycospins, is indicated 3. Conclusions and Future
by their interaction in vitro with β-trefoil lectins MpL and
CNL from the same species, which are also expressed intra- Perspectives
cellularly and involved in fruiting body defence.21,27, 33–36 Protein protease inhibitors from a number of higher
fungi exhibit a variety of unique characteristics when com-
pared to similar protease inhibitors from plant, animal or
2. 5. Diverse Applications
microbial sources. Distinct inhibitory specificity profiles,
Based on their unique characteristics indicating their coupled with structural plasticity of the β-trefoil fold, af-
function in defence, as described in the previous section, ford these inhibitors their diverse functions. Further, their
mycocypins have been evaluated for their potential in pro- noteworthy stability enables wide-ranging application of
tecting plants against herbivores. A major pest, Colorado mycocypins and mycospins. The uniqueness of these pro-
potato beetle (Leptinotarsa decemlineata), that utilizes tease inhibitors lies in the combination of the mentioned
cysteine proteases for protein digestion, has been used as a exceptional features in one protein family promoted by
model. Clitocypin and macrocypins were shown to exhibit lack of homologues outside of the fungal kingdom.
adverse effects on Colorado potato beetle larvae, both The protease inhibitors from higher fungi described
when expressed as proteins in potato leaves and when here just scratch the surface of the immense potential of
recombinant proteins produced in a bacterial expression the fungal proteolytic defence system hidden in forests in
system were added to the diet. Clitocypin and macrocypins Slovenia and in forests worldwide.
reduced the weight gain of larvae and delayed their develo- In addition to the cysteine protease inhibitors repre-
pment. The effect was linked to inhibition of the special sented by mycocypins and the serine protease inhibitors
adaptive cysteine proteases, intestains, in larval guts. More- represented by mycospins, there are most probably prote-
over, dietary mycocypins did not induce the expression of ase inhibitors waiting to be identified from higher fungi,
known adaptation-related genes of digestive enzymes in possibly with the β-trefoil fold, that inhibit other catalytic
guts of Colorado potato beetle larvae, as was the case with classes of proteases, including aspartic and metallo-prote-
all other dietary inhibitors from other sources.37,38 ases. Another direction for research, involving the β-trefo-
The potential of mycocypins and mycospins in bio- il fold proteins with other functionalities such as carbo-
technological applications has been confirmed by their use hydrate binding, lies via the β-trefoil fold lectins that have
as ligands in affinity chromatography for isolating protea- been shown to be versatile and to support unique chara-
ses from various sources. Trypsin was isolated from a com- cteristics. It will be interesting to find more novel multi-
plex, partially purified, trypsin sample using cnispin-affi- functional proteins with both carbohydrate binding and

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 Acta Chim. Slov. 2019, 66, 28–36 35

inhibitory activities. Knowledge of the different functiona- (Eds.): Proteolytic enzymes: A practical approach, IRL Press,
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Povzetek
Inhibitorji cisteinskih proteaz, klitocipin in makrocipini iz višjih gliv (mikocipini), skupaj s serinskima proteaznima
inhibitorjema, ki sta zelo specifična za tripsin, kospin in knispin iz višjih gliv (mikospini), kažejo številne značilnosti, ki
jih razlikujejo od inhibitorjev proteaz iz drugih virov. Visoka genetska raznolikost ima vpliv na funkcionalnost in / ali
stabilnost proteinov in privede do številnih proteinskih variant z nekoliko različnimi inhibitornimi profili, ki vplivajo na
vrsto tarčne proteaze in / ali moč inhibicije. Imajo β-triperesno zvitje, ki kaže visoko plastičnost pri uporabi 11 različnih
zank za inhibicijo različnih družin proteaz z različnimi mehanizmi inhibicije. Njihova vsestranskost se kaže tudi v regu-
latorni in obrambni funkciji ter široki potencialni uporabi v biotehnologiji, kmetijstvu in medicini.

Sabotič et al.: β-Trefoil Protease Inhibitors Unique to Higher Fungi