Proteins with antifungal properties and other medicinal applications from plants and mushrooms

Abstract

Living organisms produce a myriad of molecules to protect themselves from fungal pathogens. This review focuses on antifungal proteins from plants and mushrooms, many of which are components of the human diet or have medicinal value. Plant antifungal proteins can be classified into different groups comprising chitinases and chitinase-like proteins, chitin-binding proteins, cyclophilin-like proteins, defensins and defensin-like proteins, deoxyribonucleases, embryo-abundant protein-like proteins, glucanases, lectins, lipid transfer proteins, peroxidases, protease inhibitors, ribonucleases, ribosome-inactivating proteins, storage 2S albumins, and thaumatin-like proteins. Some of the aforementioned antifungal proteins also exhibit mitogenic activity towards spleen cells, nitric oxide inducing activity toward macrophages, antiproliferative activity toward tumor cells, antibacterial activity, and inhibitory activity toward HIV-1 reverse transcriptase. In contrast to the large diversity of plant antifungal proteins, only a small number of mushroom antifungal proteins have been reported. Mushroom antifungal proteins are distinct from their plant counterparts in N-terminal sequence. Nevertheless, some of the mushroom antifungal proteins have been shown to inhibit HIV-1 reverse transcriptase activity and tumor cell proliferation.

Full text

MINI-REVIEW
Proteins with antifungal properties and other medicinal
applications from plants and mushrooms
Jack H. Wong &T. B. Ng &Randy C. F. Cheung &X. J. Ye &H. X. Wang &S. K. Lam &
P. Lin &Y. S. Chan &Evandro F. Fang &Patrick H. K. Ngai &L. X. Xia &X. Y. Ye &
Y. Jiang &F. Liu
Received: 8 March 2010 / Revised: 17 May 2010 / Accepted: 17 May 2010 / Published online: 8 June 2010
#Springer-Verlag 2010
Abstract Living organisms produce a myriad of molecules
to protect themselves from fungal pathogens. This review
focuses on antifungal proteins from plants and mushrooms,
many of which are components of the human diet or have
medicinal value. Plant antifungal proteins can be classified
into different groups comprising chitinases and chitinase-
like proteins, chitin-binding proteins, cyclophilin-like
proteins, defensins and defensin-like proteins, deoxyribo-
nucleases, embryo-abundant protein-like proteins, gluca-
nases, lectins, lipid transfer proteins, peroxidases, protease
inhibitors, ribonucleases, ribosome-inactivating proteins,
storage 2S albumins, and thaumatin-like proteins. Some of
the aforementioned antifungal proteins also exhibit mito-
genic activity towards spleen cells, nitric oxide inducing
activity toward macrophages, antiproliferative activity
toward tumor cells, antibacterial activity, and inhibitory
activity toward HIV-1 reverse transcriptase. In contrast to
the large diversity of plant antifungal proteins, only a small
number of mushroom antifungal proteins have been reported.
Mushroom antifungal proteins are distinct from their plant
counterparts in N-terminal sequence. Nevertheless, some of the
mushroom antifungal proteins have been shown to inhibit HIV-
1 reverse transcriptase activity and tumor cell proliferation.
Keywords Antifungal .Medicinal .Plants .Mushrooms
Introduction
Pathogenic fungi cause considerable damage in humans,
farm animals, crops, and other organisms. Fungal infections
can be devastating with a serious effect on health or lead to
enormous economic losses. Living organisms have the
innate ability to combat fungal invasions by producing
antifungal proteins. In agriculture, genes encoding antifungal
proteins can be introduced into crops to boost their resistance
against fungal pathogens.
Different organisms may produce different antifungal
proteins for defense against fungi. Hence, there is a
diversity of antifungal proteins that exist in nature. The
J. H. Wong :T. B. Ng (*):R. C. F. Cheung :X. J. Ye :
S. K. Lam :P. Lin :Y. S. Chan :E. F. Fang
The School of Biomedical Sciences, Faculty of Medicine,
The Chinese University of Hong Kong,
Shatin, New Territories,
Hong Kong, China
e-mail: b021770@mailserv.cuhk.edu.hk
H. X. Wang (*)
State Key Laboratory of Agrobiotechnology,
Department of Microbiology,
China Agricultural University,
Beijing, China
e-mail: hxwang@cau.edu.cn
P. H. K. Ngai
Department of Biochemistry, Faculty of Science,
The Chinese University of Hong Kong,
Shatin, New Territories,
Hong Kong, China
L. X. Xia (*)
College of Medicine,
Shenzhen University,
Shenzhen, China
e-mail: xialixin@126.com
X. Y. Ye
College of Biological Science and Technology,
Fuzhou University,
Fuzhou, China
Y. Jiang :F. Liu
Department of Microbiology, College of Life Sciences,
Nankai University,
Tianjin, China
Appl Microbiol Biotechnol (2010) 87:1221–1235
DOI 10.1007/s00253-010-2690-4

scope of the present review is confined to antifungal
proteins produced by plants and mushrooms. Previously,
plant antifungal proteins have been reviewed (Aerts et al.
2008; De Lucca et al. 2005;Ng2004; Selitrennikoff 2001).
The intent of the present article is to provide a more update
review of plant and mushroom antifungal proteins.
Plant proteins with antifungal properties and other
medicinal applications
Chitinases and chitinase-like proteins
The antifungal action of chitinases is due to the
hydrolysis of chitin, a major component of fungal cell
wall which results in an impaired cell wall and
subsequently brings about cell lysis. However, some
plant chitinases and bacterial family 18 chitinases are
devoid of antifungal activity (Kawase et al. 2006).
Furthermore, the antifungal activity of a tobacco class I
chitinase was substantially augmented in the presence of a
chitin-binding domain (Iseli et al. 1993). These results
indicate that a large proportion of the antifungal activity
of chitinases was due to chitin-binding and not to
chitinase activity. The main constituent of the fungal wall,
chitin, is a polymer of N-acetylglucosamine. Chitinases
possess hydrolytic activity toward chitin and thus exert an
antifungal action (Graham and Sticklen 1994).
A 29-kDa chitinase with antifungal activity toward
Fusarium oxysporum and Rhizoctonia solani has been
isolated from bulbs of the Indian squill Urginea indica.
The lack of a Lys-rich N-terminal domain typical of class I
chitinases, the sequence similarity to Hordeum vulgare
chitinase (a class II chitinase), and the identical positions of
five of the cys residues to those H. vulgare chitinase, strongly
suggest that it is a class II chitinase (Shenoy et al. 2006).
The emperor banana fruit produces two 30-kDa chitinase-
like proteins which can be separated from one another by fast
protein liquid chromatography on Mono S and differ in their
antifungal potency toward F. oxysporum. Neither of them has
activity toward Mycosphaerella arachidicola, indicating a
specificity of antifungal action (Ho and Ng 2007).
A 42-kDa chitinase with two terminal lysine motif
domains that contribute significantly to antifungal activity
has been isolated from fern (Pteris ryukyuensis) leaves
(Onaga and Taira 2008).
A class III chitinase cDNA has been cloned using a
cDNA library from suspension-cultured bamboo (Bambusa
oldhamii) cells and subsequently transformed into Pichia
pastoris X-33 for expression. Two recombinant proteins, a
28.3-kDa unglycosylated chitinase and a 35.7-kDa glyco-
sylated chitinase with antifungal activity toward Scoleco-
basidium longiphorum have been purified. Both proteins
possess relatively high thermostability, but differ slightly
in optimum pH, optimum temperature, and chitinolytic
activity toward ethylene glycol chitin (Kuo et al. 2008).
Chitinase-like proteins with a molecular mass of about
30 kDa have been isolated from a variety of leguminous
seeds including chickpea (Cicer arietinum) (Vogelsang and
Barz 1993), rice bean (Delandia umbellata) (Ye and Ng
2002c), pinto bean (Phaseolus vulgaris cv. Pinto) (Ye and
Ng 2002a) and cowpea (Vigna unguiculata) (Ye et al.
2000). Some of them have stimulatory effects on macro-
phages and splenocytes. Monocots including chive (Allium
tuberosum) (Lam et al. 2000), leek (Allium porrum)
(Vergauwen et al 1998), garlic (Allium sativum) (Van
Damme et al. 1993), and maize (Zea mays) (Huynh et al.
1992) produce chitinase. The medicinal plant Panax
notoginseng produces a small 15-kDa and a large 35-
kDa chitinase-like antifungal protein (Lam and Ng 2001d,
2002) which possess both antifungal and HIV-1 reverse
transcriptase inhibitory activities.
Chitin-binding proteins
The inhibitory effect of chitin-binding proteins on fungal
growth is ascribed to their ability to bind to chitin. Since
nascent chitin of the hyphal apex, in which hyphal growth
and hence cell wall assembly occur, is easy accessible,
chitin-binding proteins are present along fungal cell walls
and accumulate at septa and hyphal tips (Koo et al. 1998).
This binding ensures in changes in morphology including
abnormal branching, hyphal swelling, and shorter hyphae
(Nielsen et al. 1997).
A 3,184-Da antifungal peptide from Amaranthus hypo-
chondriacus seeds is thermostable and protease-resistant. It
displays a Cys/Gly-rich chitin-binding domain and it also
degrades chitin. It potently inhibits Aspergillus candidus,
Candida albicans, Fusarium solani, Geotrichum candidum,
Penicillium chrysogenum, and Trichoderma sp. (Rivillas-
Acevedo and Soriano-García 2007).
“”Two forms of a hevein-type antifungal peptide with a
unique 10-Cys motif typical of chitin-binding domains of
cereal class I chitinases have been purified from Triticum
kiharae seeds. They are active against chitin-free and
chitin-containing pathogens alike (Odintsova et al. 2009).
Chitin-binding proteins with antifungal activity have
been purified from intracellular washing fluid from sugar
beet leaves (Nielsen et al. 1997; Kristensen et al. 2001), the
leaves of guilder rose (Yang and Gong 2002), and tobacco
(Ponstein et al. 1994).
Cyclophilin-like proteins
Cyclophilins, also known as immunophilins, peptidylprolyl
cis–trans isomerases, and cyclosporin A-binding proteins,
1222 Appl Microbiol Biotechnol (2010) 87:1221–1235

have been sequenced from a variety of organisms
including plants, yeast, fruit flies, parasites, and the rat
(Galat 1999). They show sequence homology to each
other. Cyclophilins catalyze cis–trans isomerization of
imide bonds in peptides and proteins and may be
implicated in protein folding and in distant interaction
between cells (Pliyev and Gurvits 1999). The prolyl
isomerase activity of cyclophilins is not crucial to their
immunosuppressive activity (Göthel and Marahiel 1999).
High-molecular-weight cyclophilins bind and activate
steroid receptors (Cunningham 1999; Silverstein et al.
1999). Cyclophilins promote the assembly of multiprotein
complexes that usually comprise a protein kinase or a
phosphoprotein phosphatase or both (Cunningham 1999).
Göthel and Marahiel (1999) suggested the involvement of
cyclophilins in protein folding, signal transduction, trafficking,
assembly, and cell-cycle regulation. Marivet et al. (1994)
suggested that cyclophilins might be stress-related proteins.
Fungal and viral invasions may constitute stresses. Thus,
some cyclophilin-like proteins have antifungal and antiviral
activities.
Cyclophilin-like antifungal proteins have been isolated
from black-eyed pea (Ye and Ng 2001), mungbean (Ye and
Ng 2000b), and chickpea (Ye and Ng 2002d). They have a
molecular mass of 18 kDa. The chickpea protein, but not its
mungbean counterpart, stimulates the mitogenic response of
murine splenocytes.
Defensins and defensin-like peptides
Plant defensins are small (45–54 amino adds) Cys-rich
proteins implicated in host defense against fungal
pathogens. Plant defensins are highly varied in their
primary amino acid sequences with only eight structure-
stabilizing Cys residues in common. Defensins cause
membrane permeabilization. They did not form ion-
permeable pores in artificial membrane but may act on
certain membrane-bound receptors (receptor-mediated
mechanism). They elicit Ca
2+
influx and K
+
efflux,
thereby inhibiting fungal growth (Thevissen et al. 1996).
Furthermore, they act on young developing hyphae but not
dormant conidia, indicating that the defensin receptors of
them might only appear in the early stage of the fungi (de
Lucca et al. 1999).
A 7-kDa defensin-like peptide with antifungal activity
toward Botrytis cinerea, F. oxysporum, and M. arachidicola
and antibacterial activity toward Escherichia coli, Bacillus
megaterium, Mycobacterium phlei, and Proteus vulgaris
has been isolated from seeds of Vigna sesquipedalis cv.
“ground bean”. The peptide, designated as sesquin, inhibits
proliferation of leukemia M1 cells and breast cancer MCF-7
cells and reduces the activity of HIV-1 reverse transcriptase
(Wong and Ng 2005).
Another defensin-like peptide with a similar molecular
mass(6.5kDa)andinhibitoryactivitytowardHIV-1
reverse transcriptase and translation in a cell-free rabbit
reticulocyte lysate system has been purified from the
shelf bean. It inhibits mycelial growth in B. cinerea, F.
oxysporum,andM. arachidicola with an IC
50
of 2.9, 2.1,
and 0.34 μM, respectively. It exhibits mitogenic activity
toward mouse splenocytes (Wong and Ng 2006).
Trichosanthes kirilowii defensin is a 47-amino acid
peptide that inhibits F. oxysporum with an IC
50
of
247 μg/ml (ca 48 μM). The defensin has been obtained
by cloning the gene encoding the protein expression in
bacteria, protein refolding, and purification (Da-Hui et al.
2007).
RsAFP2 is an antifungal peptide isolated from Raphanus
sativus seeds. It interacts with glucosylceramides in fungal
and yeast membranes, induces membrane permeabilization
and production of reactive oxygen species, resulting in
cessation of growth and cell death (Aerts et al. 2007). It has
prophylactic effectiveness against murine candidiasis but is
devoid of toxicity to human cells (Tavares et al. 2008).
French bean defensin has a molecular mass of 6 kDa. It
is thermostable, pH stable, and trypsin-stable (Leung et al.
2008).
Cowpea (V. unguiculata) defensin potently inhibits α-
amylases of weevils, (Acanthoscelides obtectus and Zabrotes
subfasciatus), weakly inhibits mammalian α-amylases, but
it's devoid of inhibitory action toward α-amylases from
Aspergillus fumigatus and Callosobruchus maculatus.The
N-terminal region of the defensin binds with the active sites
of weevil α-amylases (Pelegrini et al. 2008).
Vitis vinifera defensin-(VrAMP1) is a 5,495-Da peptide
expressed only in berry tissue at the time of ripening and
henceforth. It has remarkable thermostability and a broad
spectrum of antifungal action with high potency against F.
oxysporum and Verticillium dahliae. It exerts its antifungal
activity by changing the permeability of fungal membranes
as evidenced by results of the propidium iodide uptake
assay (de Beer and Vivier 2008).
Games et al. (2008) reported a 47-amino acid defensin
PvD1 that inhibits the growth of a variety of yeasts
including C. albicans, Candida guilliermondii, Candida
parapsilosis, Candida tropicalis, Kluyveromyces marxian-
nus,andSaccharomyces cerevisiae and a number of
phytopathogenic fungi including Fusarium lateritium, F.
oxysporum, F. solani, and R. solani.
A 6.8-kDa defensin, with antiproliferative activity against
human hepatoma cells Bel-7402 and neuroblastoma cells
SHSY5Y and thermostable antifungal activity against Alter-
naria alternata,B. cinerea, and F. solani has been isolated
from the large lima bean (Phaseolus limensis)legumes.
However, it lacks antibacterial activity toward Salmonella sp.
and Staphylococcus aureus (Wang et al. 2009a,b).
Appl Microbiol Biotechnol (2010) 87:1221–1235 1223

The seeds of P. vulgaris cv. “Purple Pole Bean”produce
a 5,443-Da defensin with antifungal, antiproliferative, and
HIV-1 reverse transcriptase inhibitory activities (Lin et al.
2009a). It inhibits growth in C. albicans, F. oxysporum,
Helminthosporium maydis, M. arachidicola, R. solani,
Setosphaeria turcica, and V. dahliae with an IC
50
below
1μM. It causes membrane permeabilization in C. albicans
as revealed by Sytox green uptake and chitin accumulation
at hyphal tip in M. arachidicola as indicated by Congo red
staining. It inhibits proliferation of a variety of tumor cell
lines including MCF-7 breast cancer cells, SiHa cervical
cancer cells, HT29 colon cancer cells, and HepG2
hepatoma cells but does not affect human embryonic liver
WRL68 cells. It exhibits highly potent HIV-1 reverse
transcriptase inhibitory activity (IC
50
=0.5 μM).
Corn defensin (PDC1) consists of 47 amino acids and
possesses eight Cys residues. It has antifungal activity
against Fusarium graminearum. The peptide expressed in
P. pastoris has less unordered random structure and more
β-sheets than that expressed in E. coli (Kant et al. 2009).
Gymnin is a 6.5-kDa defensin-like peptide from Gym-
nocladus chinensis seeds. It demonstrates antiproliferative
activity toward tumor cells and inhibitory activity toward
HIV-1 reverse transcriptase (Wong and Ng 2003).
Deoxyribonucleases
Deoxyribonucleases probably act by hydrolyzing DNA of
invading foreign organisms. The 30-kDa asparagus DNase
exhibits antifungal activity against B. cinerea and inhibits
translation in cell-free rabbit reticulocyte system, but it does
not inhibit HIV-1 reverse transcriptase (Wang and Ng
2001a).
Embryo-abundant protein-like proteins
Embryo-abundant proteins, also known as late embryo-
genesis abundant proteins, are stress proteins which are
hydrophilic in nature. These mitochondrial proteins are
plentiful in seeds and accumulate in desiccation-tolerant
organisms (Tolleter et al. 2007).
A 13-kDa antifungal protein demonstrating an N-
terminal sequence with striking homology to white
spruce embryo-abundant protein has been isolated from
Ginkgo biloba seeds. The protein, designated ginkbilobin,
exhibits a strong antifungal action against B. cinerea,
Coprinus comatus, F. oxysporum, M. arachidicola, and R.
solani. It shows a moderate antibacterial action against E.
coli, Pseudomonas aeruginosa, and S. aureus.Itreduces
the activity of HIV-1 reverse transcriptase and inhibits
proliferation of murine splenocytes (Wang and Ng 2000a).
An antifungal protein with about 85% identity to Picea
embryo-abundant proteins has been purified from G. biloba
seeds. It exerts antifungal activity toward C. albicans, F.
oxysporum, and Trichoderma reesei. It is slightly inhibitory
against the aspartic protease pepsin (Sawano et al. 2007).
Glucanases
Glucans are the second predominant component of the
fungal cell wall. The mechanisms of antifungal activity of
glucanases consist of a direct one and also an indirect one.
The direct antifungal activity accounts for the digestion of
β1,3-glucans in fungal cell walls, which produces a
weakened cell wall and cell lysis. The indirect antifungal
effect of glucanases contributes to partial digestion of
glucans and chitin. Glycan is a component of fungal cell
wall. Glucanases act against fungi by hydrolyzing glycan in
fungal wall.
Vo g e l s a n g a n d B a r z ( 1993)isolateda36-kDabasic
from chickpea (C. arietinum L.) cell-suspension cultures.
Expression of beta-1,3-glucanase activity appeared to be
regulatedbyauxininthecellcultureandintheintact
plant.
Leah et al. (1991) isolated a 26-kDa chitinase, a 30-kDa
ribosome-inactivating protein, and a 32-kDa (1-3)-beta-
glucanase from barley (H. vulgare L.) seeds which
synergistically inhibit the growth of fungi. The glucanase
mRNA is present at low levels during seed development
and at elevated concentrations in aleurone and seedling
tissues during germination.
Treatment with mercuric chloride, salicylic acid, or
riboflavin induced beta-1,3-glucanase in three wheat
varieties, i.e., 331, Kangdao 680, and Lumai 23. A beta-
1,3-glucanase with a molecular weight of 52.0–53.6 kDa
was isolated from leaves of variety 331 exposed to
mercuric chloride for 24 h. It inhibited mycelial growth
in Alternaria longipes and Rhizoctonia cerealis and germ
tube elongation and spore germination in V. d a h l i a e and
Fusarium oxysporum f.sp cucumerinum (Sun et al. 2004).
The product of wheat beta-1,3-glucanase gene (TaGluD)
inhibited A. longipes, Phytophthora capsici, R. cerealis,
and R. solani. Its transcript induction was considerably
higher in a resistant wheat line than in a susceptible wheat
line, after infection with R. cerealis (Liu et al. 2009).
Lectins
Plant lectins are not capable of binding to glycoconjugates
on the fungal membranes or penetrating the cytoplasm due
to the barrier formed by the cell wall. Thus, it is not likely
that lectins directly inhibit fungal growth by changing the
structure and/or permeability of the fungal membrane.
However, there may be indirect effects produced by the
binding of lectins to carbohydrates on the surface of the
fungal cell wall. Chitinase-free chitin-binding stinging
1224 Appl Microbiol Biotechnol (2010) 87:1221–1235

nettle (Urtica dioica) lectin inhibited fungal growth. Cell
wall synthesis was affected as a consequence of impaired
chitin synthesis and/or deposition (Van Parijs et al. 1991).
The effects of nettle lectin on fungal cell wall and hyphal
morphology indicate that the nettle lectin is involved in the
control of colonization of the rhizomes by endomycorrhiza.
Severa1 other plant lectins have antifungal activity. The
first group comprises small chitin-binding merolectins with
a single chitin-binding domain, e.g., hevein from rubber-
tree latex (Van Parijs et al. 1991) and chitin-binding
polypeptide from Amaranthus caudatus seeds (Broekaert
et al. 1992). The only plant lectins that can be regarded as
fungicidal proteins are the chimerolectins belonging to the
class I chitinases. However, the antifungal activity of these
proteins is attributed to their catalytic rather than
carbohydrate-binding domain.
The 14.5-kDa mannose-binding lectin from Dendrobium
findlayanum exhibits antifungal activity against A. alter-
nata and Colletotrichum sp. (Sattayasai et al. 2009).
The lectin from P. vulgaris cv. “Flageolet Bean”exerts
antifungal activity against M. arachidicola but is inactive
against B. cinerea and F. oxysporum. It exhibits anti-
proliferative activity against leukemia cells (IC
50
=4 μM),
but there is no inhibitory activity against HIV-1 reverse
transcriptase or mitogenic activity toward mouse spleno-
cytes (Xia and Ng 2005).
The lectin from roots of the Chinese herb Astragalus
mongholicus is a 66-kDa homodimeric glycoprotein with
specificity for D-galactose and lactose. It has inhibitory
activity against B. cinerea, Colletotrichum sp., Drechslera
turia, and F. oxysporum but is inactive on M. arachidicola
and R. solani (Yan et al. 2005).
A 14-kDa lectin with antifungal and insecticidal activ-
ities has been reported by Boleti et al. (2007) from Pouteria
torta seeds. It inhibits the yeast S. cerevisiae and the fungi
C. musae and F. oxysporum.
Phaseolus coccineus lectin is a homodimeric 30-kDa
protein with cytotoxicity toward L929 cells and inhibitory
activity toward phytopathogenic fungi. It shows specificity
toward sialic acid (Chen et al. 2009). A 30-kDa lectin with
sequence similarity to ConA and antifungal activity against
major phytopathogens has been isolated from leaves of the
medicinal herb Withania somnifera (Ghosh 2009). A
dimeric 62-kDa lectin with antifungal activity against Valsa
mali (IC
50
=18 μM), antiproliferative activity against
HepG2 and MCF-7 cells (IC
50
=2 μM), HIV-1 reverse
transcriptase inhibitory activity (IC
50
=0.28 μM), and
mitogenic activity toward mouse splenocytes has been
isolated from caper seeds. It is specific toward D-galactose,
lactose, rhamnose, and raffinose (Lam et al. 2009).
A homodimeric 67-kDa lectin from the seeds of P.
vulgaris cv. “Red Kidney Beans”inhibits mycelial growth
in F. oxysporum and R. solani (Ye et al. 2001b).
Lipid transfer proteins
Lipid transfer proteins (LTPs) retard the growth of fungal
pathogens. However, the mechanism of action has not
been elucidated (Kader 1996). It is possible that LTPs
permeabilize membranes by inserting into membranes,
and the central hydrophobic cavity forms a pore, which
permits outflow of intracellular ions thus leading to cell
death. How this is related to their lipid transfer activity is
unknown (Selitrennikoff 2001). Using the hydrophobic
cavity, LTPs transfer acyl monomers necessary for the
synthesis of cutin, which covers aerial surfaces of plants.
This extracellular lipophilic coating may protect the plants
from pathogens. The synthesis of cutin and LTPs is
increased by pathogen infection (Kader 1996).
Brassica campestris seeds produce a 9,412-Da LTP
with potent antifungal activity against M. arachidicola
(IC50=4.5 μM) and F. oxysporum (IC
50
=8.3 μM). It
displays dose-dependent binding to the phospholipid
lyso-α-lauroyl phosphatidylcholine (Lin et al. 2007a). B.
campestris LTP and mungbean LTP are thermostable, pH
stable, and protease-stable. The former LTP also has
antiproliferative activity against HepG2 and MCF-7
cells and HIV-1 reverse transcriptase inhibitory activity
although it lacks antibacterial activity. On the other
hand, the latter LTP manifests antibacterial activity but
has no antiproliferative and HIV-1 reverse transcriptase
inhibitory activities (Lin et al. 2007b).
Ace-AMP from onion seeds is a 10-kDa protein with
antifungal and antibacterial activities. Its sequence
resembles those of plant lipid transfer proteins (Cammue
et al. 1995).
Peroxidases
Lignin forms an extensive network of aromatic structures
with cross-links in plant cell walls. It confers mechanical
strength to cell walls. Peroxidases reinforce the plant cell
wall by catalyzing deposition of lignin. Based on their
affinity for cinnamyl alcohols and expression in lignified
tissue, peroxidases contribute to lignin synthesis by
catalyzing the polymerization of cinnamyl alcohols into
lignin and utilizing phenolic acids to form covalent cross-
links between lignin via hydroxyl groups. Peroxidase-
catalyzed lignification is increased during fungal infection
and wounding (Lagrimini 1991). Reactive oxygen species
like H
2
O
2
released during cell wall lignification by
peroxidases are toxic to pathogens (Thordal-Christensen et
al. 1997) and can act as intracellular messengers to trigger
other defense responses such as synthesis of other
pathogenesis-related proteins (Levine et al. 1994).
A 34-kDa peroxidase with antifungal activity toward F.
solani (IC
50
=76 μM), M. arachidicola (IC
50
=103 μM),
Appl Microbiol Biotechnol (2010) 87:1221–1235 1225

and Pythium aphanidermatum (IC
50
=119 μM), has been
isolated from lima bean seeds (Wang et al. 2009a, b).
French bean legumes produce a 37-kDa peroxidase with
inhibitory activity on mycelial growth of B. cinerea, F.
oxysporum,andM. arachidicola (Ye and Ng 2002e).
Protease inhibitors
Fungal hyphae may penetrate the plant cell wall by
secreting lytic enzymes and then ramify throughout the
leaves to absorb nutrients. Protease inhibitors inhibit the
fungal proteases and thus increase the resistance of plants to
fungal pathogens. The antifungal mechanism of protease
inhibitors has not been fully elucidated. Phytopathogenic
fungi secrete proteases (Clark et al. 1997). Plant pathogenicity
appears to be related to secreted proteases since protease-
deficient mutants lack the capability to induce lesions in
plants.
Ribeiro et al. (2007) have isolated a protein with sequence
homology to protease inhibitors from seeds of the chili
pepper Capsicum annuum. It potently inhibits S. cerevisiae.
Three Kunitz-type serine protease inhibitors (APTIA,
APTIB, and APTIC) have been isolated from Acacia plumosa
seeds. They are characterized by a molecular mass of
20 kDa, pronounced pH stability, and thermostability and
antifungal activity towards Aspergillus niger, Colletotrichum
sp., and Thielaviopsis paradoxa (Lopes et al. 2009).
Broad bean (Vicia faba) trypsin–chymotrypsin inhibitor
manifests antifungal, mitogenic, and HIV-1 reverse tran-
scriptase inhibitory activity (Ye et al. 2001a) Pearl millet
cysteine protease inhibitor (Joshi et al. 1998) and Clausena
lansium sporamin-type protease inhibitor (Ng et al. 2003)
have antifungal activity. The latter also has antiproliferative
and HIV-1 reverse transcriptase inhibitory activities.
Ribonucleases
Ribonucleases probably act by hydrolyzing RNA from
intruding foreign organisms. Antifungal ribonucleases have
been isolated from roots of Panax ginseng (Chinese ginseng),
P. notoginseng (sanchi ginseng), and Panax quinquefolius
(American ginseng). The RNases of both Chinese and
American ginseng are homodimeric and demonstrate HIV-1
reverse transcriptase inhibitory activity that can be enhanced
by succinylation (Wang and Ng 2000b). Sanchi ginseng
RNase is heterodimeric (Lam and Ng 2001c).
Ribosome-inactivating proteins
The ribosome-inactivating activity of ribosome-inactivating
proteins (RIPs) is attributed to RNA N-glycosidase activity.
For instance, α-sarcin, a type-1 RIP, specifically cleaves one
phosphodiester bond of the 28S rRNA. RIPs also demonstrate
synergistic effects with other antifungal proteins. For example,
Lam and Ng (2001a–d,) isolated a fungal RIP together with
an antifungal protein and showed that the two proteins act
synergistically.
Barley RIPs inhibit protein synthesis by virtue of their
N-glycosidase activity. Type-1 RIPs have a molecular mass
of approximately 30 kDa and exhibit antifungal activity
(Roberts and Selitrennikoff 1986). Hairy melon RIP with a
molecular mass of 21 kDa (Ng and Parkash 2001) and the
small RIP luffacylin from Luffa cylindrica seeds (Parkash et
al. 2002) have antifungal activity.
Thaumatin-like proteins
Some plant thaumatin-like proteins (TLPs) have been
demonstrated to have β1,3-glucanase activity (Grenier et al.
1999). Their amino acid sequences resemble that of
thaumatin, a sweet-tasting protein isolated from the katemfe
fruit (Thaumatococcus daniellii) (van der Wel and Loeve
1972). Several TLPs capable of acting on fungal membranes
(Roberts and Selitrennikoff 1990) or binding to actin
(Takemoto et al. 1997) have been reported. The discovery
of TLPs with β-1,3-glucanase activity may hint at additional
antifungal mechanisms which are more widespread between
other glucanases. Hydrophobic TLPs may interact with
components of fungal cell membranes leading to structural
disruption of membranes and formation of transmembrane
pores and rapid release of cytoplasmic content (Woloshuk et
al. 1991).
TLPs are different from thaumatin in possessing antifungal
activity.TLPswithantifungalactivityhavebeenisolatedfrom
intercellular washing fluid of chickpea (Hanselle et al. 2001),
French bean legumes (Ye et al. 1999), Diospyros texana
fruits (Vu and Huynh 1994), chestnut Castanopsis chinensis
(Chu and Ng 2003a)andCastanea mollisima (Chu and Ng
2003b), and kiwi fruits (Wang and Ng 2002; Wurms et al.
1999).
A 23-kDa thaumatin-like protein with antifungal activity
against Candida species has been purified from Cassia
didymobotrya cell culture (Vitali et al. 2006).
A 20-kDa thaumatin-like protein with antifungal activity
against F. oxysporum and M. arachidicola and slight HIV-1
reverse transcriptase inhibitory activity has been isolated by
Ho et al. (2007) from banana fruits. The protein has no
mitogenic or antiproliferative activity. Some TLPs have β-
1,3-glucanase activity (Grenier et al. 1999), while other TLPs
work via a mechanism involving mitogen-activated protein
kinase leading to changes in fungal wall (Yun et al. 1998).
Storage 2S albumins
The 2S albumins are storage proteins in various dicots that
may be implicated in some allergic reactions. Two 11 kDa
1226 Appl Microbiol Biotechnol (2010) 87:1221–1235

and one 5 kDa proteins from Passifora edulis and peptides
from Malva parviflora and Brassica species, all of which
resemble 2S albumin, have been found to possess
antifungal activity (Agizzio et al. 2003;Wangetal.2001).
A 5-kDa antifungal peptide with high-sequence homology
to storage 2S albumins has been isolated from seeds of the
passion fruit Passiflora edulis. It demonstrates antifungal
activity against A. fumigatus, F. oxysporum, and Tric ho-
derma harzianum with an IC
50
of 8, 6.8, and 6.4 μM,
respectively. However, there is no activity against C. albicans,
Paracoccidioides brasiliensis,andR. solani (Pelegrini et al.
2006).
A protein with high-sequence homology to 2S albumins
has been isolated from chili pepper (C. annuum) seeds
(Ribeiro et al. 2007).
Novel antifungal proteins
There are other antifungal proteins that do not fit nicely into
any of the aforementioned categories. Some of them are as
follows.
A 32-kDa antifungal protein with inhibitory activity
against B. cinerea, F. oxysporum, M. arachidicola, and P.
piricola has been isolated from ginger rhizomes (Wang and
Ng 2005a).
Coconut antifungal peptide has a molecular mass of
10 kDa. It inhibits HIV-1 reverse transcriptase (IC
50
=
52.5 μM) and mycelial growth of M. arachidicola (IC
50
=
1.2 μM), F. oxysporum, and P. piricola (Wang and Ng
2005b).
An 11-kDa antifungal protein with inhibitory activity
against M. arachidicola (IC
50
-36 μM) and F. oxysporum
but inactive toward tumor cell and HIV-1 reverse
transcriptase has been isolated from red lentil seeds (Wang
and Ng 2007).
The Chinese cabbage B. campestris produces an
antifungal protein C-FKBP identical to human FK506-
binding protein. It has no antibacterial activity, but inhibits
C. abicans, B. cinerea, R. solani, and Trichoderma viride
(Park et al. 2007).
Buckwheat antifungal peptide has a molecular mass of
4 kDa and antifungal activity against F. oxysporum (IC
50
=
35 μM) and M. arachidicola (IC
50
=40 μM). Its antifungal
activity has marked pH stability and thermostability. It
exerts antiproliferative activity toward HepG2, MCF-7, and
leukemia cells with an IC
50
near 30 μM and displays HIV-1
reverse transcriptase inhibitory activity (IC
50
=5.5 μM).
However, there is no mitogenic activity toward splenocytes.
Neither does it stimulate macrophages to produce nitric
oxide (Leung and Ng 2007).
A 98-kDa urease from cotton (Gossypium hirsutum)
demonstrates low ureolytic activity but high inhibitory
activity against a number of phytopathogenic fungi. An
irreversible inhibitor of the enzyme fails to inhibit its
antifungal activity (Menegassi et al. 2008).
A 5,907-Da antifungal peptide from kale (Brassica
alboglabra) seeds inhibits mycelial growth in F. oxysporum,
H. maydis,andM. arachidicola with an IC
50
below 5 μM
and V. m a l i with an IC
50
of 0.15 μM. Its antifungal activity is
extremely thermostable and pH stable. It exhibits antiproli-
ferative activity toward HepG2 cells and MCF cells with an
IC
50
below 4 μM and inhibits HIV-1 reverse transcriptase
with an IC
50
of about 4 μM(LinandNg2008).
A 4.7-kDa Cys-rich antifungal peptide designated as
hevein has been isolated from the non-rubber constituents
of rubber latex of Hevea brasiliensis. It is active against
various Candida spp. including C. albicans, Candida
krusei, and C. tropicalis (Kanokwiroon et al. 2008).
From caper (Capparis spinosa) seeds, a 38-kDa
antifungal protein with some sequence resemblance to
imidazole glycerol phosphate synthase and antifungal
activity against V. m a l i has been isolated. It inhibits the
activity of HIV-1 reverse transcriptase and the proliferaton
of HepG2, HT29, and MCF-7 tumor cells with an IC
50
of
0.23, 1, 40, and 60 μM, respectively (Lam and Ng 2009a).
The seeds of passion fruit produces a 67-kDa
dimeric protein with antifungal activity against R.
solani (IC
50
=16 μM) and antiproliferative activity
against MCF-7 cells (IC
50
=15 μM). Its N-terminal
sequence bears striking similarity to β-lactoglobulin.
However, the protein, designated as passiflin, is immuno-
logically distinct from β-lactoglobulin since it does not
react with an anti-β-lactoglobulin antiserum. The much
smaller size of β-lactoglobulin and the lack of antipro-
liferative and antifungal activities of the milk protein,
indicate that passiflin and β-lactoglobulin are distinct
proteins (Lam and Ng 2009b).
The 11-kDa antifungal peptide from pomegranate
peels, designated as pomegranin, possesses an N-
terminal sequence similar to rice disease resistance NB-
S-LRR-like protein. It exhibits antifungal activity against
B. cinerea and F. oxysporum with an IC
50
of 2 and 6 μM,
respectively (Guo et al. 2009).
A 14.8-kDa noncytotoxic and thermostable antifungal
protein from pumpkin rinds demonstrates a novel N-
terminal sequence. It is active against a number of fungal
species including B. cinerea, Colletotrichum coccodes, F.
oxysporum, F. solani, and T. harzianum (Park et al. 2009).
Amaryllin is a 15-kDa antifungal protein from Amaryllis
belladonna bulbs with activity against Aspergillus flavus and
F. o x y s p or u m . Crystallization and preliminary crystallograph-
ic studies of the protein have been reported (Kumar et al.
2009).
Juncin is an 18.9-kDa antifungal protein with a novel N-
terminal sequence isolated from seeds of the Japanese
takana (Brassica juncea var. integrifolia). The protein
Appl Microbiol Biotechnol (2010) 87:1221–1235 1227

inhibits mycelial growth in the phytopathogenic fungi F.
oxysporum, H. maydis, and M. arachidicola with IC
50
values of 13.5, 27, and 10 μM, respectively. It exerts
antiproliferative activity toward hepatoma (HepG2) and
breast cancer (MCF7) cells with IC
50
values of 5.6 and
6.4 μM, respectively, and the activity of HIV-1 reverse
transcriptase with an IC
50
of 4.5 μM. It has neither
mitogenic activity toward splenocytes nor nitric oxide
inducing activity toward macrophages (Ye and Ng 2009).
Many of the aforementioned plant antifungal proteins are
unabsorbed on anion exchangers but adsorbed on cation
exchangers and the affinity chromatography media Affi-gel
blue gel. The vast majority of them are monomeric.
However, they display a wide range of molecular masses
from a few thousand daltons in case of defensins to over
60 kDa in case of lectins.
Among the enormous number of plant lectins
published, antifungal activity has been described as a
characteristic of just some of them while the bulk does
not display such activity. A similar statement holds true
for plant ribonucleases. Likewise, only certain plant
RIPs have been shown with antifungal activity. After
browsing through the voluminous literature on lectins,
ribonucleases and RIPs from plants, one would be left
with the impression that plant lectins are endowed with
mitogenic activity toward splenocytes, cytokine-inducing
activity toward cells of the immune system, antiproli-
ferative activity toward tumor cells, and inhibitory
activity toward HIV (Balzarini 2007) and HIV-1 reverse
transcriptase (Cheung et al. 2009). Plant RIPs have
antimitogenic (Wang and Ng 2001b), immunosuppressive
(Yeung et al. 1987), anticancer (Tsao et al. 1990), and
antiviral (Ng et al. 2002) activities. Only some plant
RNases are known to exhibit antiproliferative activities
(Lam and Ng 2001c). A number of leguminous antifungal
proteins manifest inhibitory activity toward HIV-1 reverse
transcriptase, protease, and integrase (Ng et al. 2002).
Mechanism of antifungal action
The mechanisms of action of some antifungal actions
have been elucidated. Chitinases hydrolyze chitin, an N-
acetylglucosamine polymer that is a main constituent of
the fungal wall (Graham and Sticklen 1994). Some
thaumatin-like proteins evoke a mitogen-activated protein
kinase signal translation mechanism that results in changes
in the fungal wall and enhanced toxicity (Yun et al. 1998).
Other thaumatin-like proteins are capable of hydrolyzing
β-1,3-glucans (Grenier et al. 1999).
Plant defensins can be divided into a morphogenic
group and a non-morphogenic group. Both retard hyphal
elongation in filamentous fungi. However, only the former
group causes pronounced morphological distortions as
evidenced by an increase in hyphal branching. Unlike
their mammalian and insect counterparts, plant defensins
have not been demonstrated to create ion-permeable pores
in artificial phospholipid membranes or to alter their
electrical characteristics, indicating that plant defensins
do not directly interact with plasma membrane phospho-
lipids. The exact mechanism of action of plant defensins
has yet to be elucidated (Aerts et al. 2008).
Mushroom proteins with antifungal properties
and other medicinal applications
Antifungal proteins have been isolated from a few
Pleurotus species comprising Pleurotus eryngii, Pleurotus
ostreatus, and Pleurotus sajor-caju, two other related
species (Hypsizigus marmoreus and Lyophyllum shimeiji),
the medicinal mushroom Ganoderma lucidum,thewild
mushroom Polyporus alveolaris and two edible mush-
rooms (Tricholoma giganteum and Agrocybe cylindracea).
The proteins are unadsorbed on the anion exchanger
DEAE-cellulose but adsorbed on the affinity chromato-
graphic media, Affi-gel blue gel and cation exchanger
such as CM-sepharose, S-sepharose, and mono S.
Mushroom antifungal proteins exhibit a range of molecular
masses. Pleurostrin from P. ostreatus, agrocybin from A.
cylindracea, eryngin from P. eryngii,ribonucleasefromP.
sajor-caju, trichogin from T. giganteum, ganodermin from G.
lucidum,hypsinfromH. marmoreus, Lyophyllum antifungal
protein from Lyophyllum shimeiji and alveolarvin from P.
alveolaris display a molecular mass of 7, 9, 10, 12, 27, 15,
20, 14, and 28 kDa, respectively. All except alveolarin is
monomeric. Aveolarin is dimeric. Mycelial growth of the
fungi F. oxysporum, M. arachidicola,andPhysalospora
pyricola is inhibited by pleurostrin, trichogin, and alveolarin.
Eryngin is inhibitory to F. oxysporum and M. arachidicola,
F. oxysporum is inhibited by ganodermin, hypsin and P.
sajor-caju ribonuclease with an IC
50
of 12.4, 14.2, and
9.5 μM, respectively. P. pyricola is inhibited by hypsin,
ganodermin, P. sajor-caju RNase, and Lyophyllum antifungal
proteinwithanIC
50
of 2.5, 18.1, 72, and 70 μM,
respectively. Hypsin and ganodermin impede mycelial
growth in B. cinerea, and hypsin and P. s a j o r - c a j u
ribonuclease inhibit M. arachidicola with an IC
50
of 0.06,
15.2, 2.7, and 72 μM, respectively. Lyophyllum antifungal
protein inhibits M. arachidicola and P. pyricola but has no
inhibitory action on C. comatus, Colletotrichum gossypii,
and R. solani, illustrating the specificity of its antifungal
action.
Agrocybin lacks inhibitory activity toward bacteria. P.
sajor-caju ribonuclease inhibits P. aeruginosa and S.
aureus out of the 12 bacterial species tested. Trichogin,
Lyophyllum antifungal protein and hypsin, inhibits HIV-1
1228 Appl Microbiol Biotechnol (2010) 87:1221–1235

reverse transcriptase with an IC
50
of 83 nM, 5.2 nM, and
8μM, respectively. Agrocybin exhibits some HIV-1
reverse transcriptase inhibitory activity. P. sajor-caju
ribonuclease inhibits proliferation of HepG2 hepatoma
cells and L1210 leukemia cells with an IC
50
of 0.22 and
1μM, respectively. Hypsin displays antiproliferative
activity against the same tumor cells while agrocybin is
inactive. Agocybin exhibits weaker mitogenic avtivity than
ConA toward mouse splenocytes whereas P. sajor-caju
ribonuclease inhibits mitogenic response with an IC
50
of
65 nM. Translation in a rabbit reticulocyte lysate system
is inhibited by P. sajor-caju ribonuclease. Lyophyllum
antifungal protein and hypsin with an IC
50
of 65 nM,
70 μM, and 7 nM, respectively.
Only a few isolated cases of mushroom ribonucleases
(Ngai and Ng 2004) and ribosome-inactivating proteins
(Lam and Ng 2001a,b) exhibit antifungal activity. No
mushroom lectins isolated to date have been shown to have
antifungal activity.
In addition to antifungal proteins, mushrooms produce
a number of other proteins with potential medicinal
application but sometimes devoid of antifungal activity.
These proteins comprise lectins, ribosomes-inactivating
proteins (RIPs), ribonucleases, and ubiquitin-like peptides.
Various mushroom lectins display mitogenic activity on
splenocytes, antiproliferative activity toward tumor cells
and HIV-1 reverse transcriptase inhibitory activity, but do
not suppress mycelial growth in fungi. The RIPs velutin,
flammulin, velin, and flammin from Flammulina velutipes,
pleuturegin from Pleurotus regium are devoid of antifungal
activity, unlike hypsin and lyophyllin from H. marmoreus
and L. shimeiji.TheRNAsefromP. sajor-caju is the only
mushroom RNase that has been reported with antifungal
activity. Ubiquitin-like peptides do not have antifungal
activity. Some of the mushroom RIPs, RNases, and
ubiquitin-like peptides have HIV-1 reverse transcriptase
inhibitory activity, antiproliferative activity toward tumor
cells, and mitogenic/antimitogenic activities.
Because of the much smaller number of antifungal
proteins reported from mushrooms to date, compared with
their counterparts from plants, they are not divided into
various groups according to structure or function.
Discussion
The foregoing account summarizes proteinaceous constitu-
ents of plants and mushrooms with activities useful to
mankind. In addition, plants and mushrooms also produce
non-peptidic biomolecules such as polysaccharides (Rop et
al. 2009; Qi et al. 2010) and small molecules including
phytoalexins with antipathogenic activities (González-
Table 1 N-terminal sequences of some plant antifungal proteins
Chitinase (Pinto bean phasein A)a,b
Chitinas e (Field b ean dolic hin)a,b
Chitinase (Cowpea -antifungal protein)a,b
Cyclophilin-like protein (Mungbean mungin)a,b,c
Cyclophilin-like protein (Black-eyed pea unguilin)a,b
Cyclophilin-like protein (Chickpea)a,b,c
Defensin ( Radish)
Defensin (Pea)
Defensin (Petunia)
Defensin (Clitora ternatea)
Defensin (Vigna unguiculata)
Defensin (Pisum sativum)
Defensin (Vigna radiatea)
Defensin (Phaseolus lunatus L)a,b,c
Defensin (Vicia faba)
Embryo abundant protein (G. biloba ginkbilobin)a,b
Lectin (Red kidney bean)a,b
Peroxidase (French bean)a,b
RNase (Panax notoginseng 29-kDa subunit)
RNase (Panax notoginseng 27-kDa subunit)
Riboso me ina ctivat ing prot ein (Lily bu lb lilin)
Ribosome inactivating protein (Sponge gourd luffacylin)
RNase-Ribosome inactivating protein (American Ginseng quinqueqinsin)
Ribosome inactivating protein (sugar snap sativin)
Thaumatin-like protein (Castanopsis)
Thaumat in-like protein ( French bean)
Trypsin in hibitor (Wampee spora min-like protein)
Identical amino acid residues in the plant antifungal proteins are shaded
a
Antifungal activity
b
Anti-HIV-1 reverse transcriptase activity
c
Antiproliferative activity (cancer cells)
Appl Microbiol Biotechnol (2010) 87:1221–1235 1229

Lamothe et al. 2009). These have not been covered in the
present review.
According to Van Loon and Van Strien (1999) and
Selitrennikoff (2001), there are 14 families of pathogenesis-
related proteins (PR proteins). The aforementioned β-1,3-
glucanases belong to PR-2 family. Most chitinases belong
to PR-3 family. A few chitinases belong to PR-4, PR-8, and
PR-11 families. Thaumatin-like proteins belong to PR-5
family. Protease inhibitors belong to PR-6 family. Peroxidases
belong to PR-9 family. Ribonucleases belong to PR-10 family.
Defensins belong to PR-12 family, and lipid transfer proteins
belong to PR-4 family. The PR proteins not covered in the
present review include PR-1 (protein induced in tobacco), PR-
7 (endoproteases), and PR-13 (thionins) families.
A bird's eye view of the foregoing account creates an
impression that there is a spectacular array of antifungal
proteins produced by plants and mushrooms (Tables 1,2,
and 3). New varieties of antifungal proteins continue to be
discovered. Those produced by plants (Tables 1and 2)
bear little structural resemblance to their mushroom
Table 2 N-terminal sequences of some plant novel antifungal proteins
Novel antifungal protein (Peanut hypogin)
a,b
KSPYYQKKTENPQAQRQLQSDDQEPAKLK
Novel antifungal protein (Chickpea cicerin)
a,b
ARCENFADSYRQPPISSSQT
Novel antifungal protein (Chickpea arietin)
a,b
GVGYKVVVTTTAAADDDDVV
Novel antifungal protein (Cowpea β-antifungal protein)
a,b
MTTGQVQGNLAQQIIGFLQNGIVVPPAANN
Novel antifungal protein (Miraculin-like protein from pea, sativin) APEVAPAVGHADLRA
Novel antifungal protein (Garland chrysanthemum chrysancorin) RVDQKAQNLKCCQQHRFNCHCERVCVFQDQ
Novel antifungal protein (Ceylon spinach α-basrubrin)
a
GADFQECMKEHSQKAHQHQG
Novel antifungal protein (Ceylon spinach β-basrubrin)
a
KIMAKPSKFYEQLRGR
Novel antifungal protein (Bamboo shoot dendrocin)
a
TTLTLHNLCPYPVWWLVTPNNGGFPIIDNTPVVLG
Novel antifungal protein (Pinto bean phasein B) GARKDDHAKLVFLLKDIEYQ
Novel antifungal protein (Ginger)
a
NGPAAQAAENNLA
Novel antifungal protein (Coconut)
a
EQCREEE DDR
Novel antifungal protein (Red lentil seed)
a
TETNSFSITKFSPDGNKLIFQGDGYTTKGK
Novel antifungal protein (Chinese cabbage)
a
MRSSASSAMKAAGVLVLLAV
Novel antifungal protein (Buckwheat seeds)
a
AQCGAQGGGATCPGG
Novel antifungal protein (Cotton seeds urease) IFKADIGVKNGCIVGLGKAG
Novel antifungal protein (Kale seed)
a,b
PEGPFQGPKATKPGDLAXQTWGGWXGQTPKY
Novel antifungal protein (Latex hevein) EQCGRQAGGKLCPNNLCCSQ
Novel antifungal protein (Caper seed)
a,b
SYDTQAEAAL
Novel antifungal protein (Passion fruit seed passiflin)
a,b
AFLDIQKVAGTWYSLA
Novel antifungal protein (Pumpkin rind) QGIGVGDNDGKRGKR
Novel antifungal protein (Japanese Takana seeds juncin)
a,b
GVEVTRELRSERPSGKIVTI
a
Antifungal activity
b
Anti-HIV-1 reverse transcriptase activity
Table 3 N-terminal sequences of mushroom antifungal peptides and proteins
Agrocybin (9 kDa)a
Alveolarin (28 kDa)a
Eryngin (10kDa)a
Ganodermin (15 kDa)a
Hypsin (20 kDa)a,b,c
Lyophyllin (20 kDa)a,b,c
Lyophyllum antifungal protein (14 kDa)a,b,c
Lentin (27.5 kDa)a,b
Pleurostrin (7 k Da)a,b
Pleurotus sajor-caju RNase (12 kDa)a,b,c
Identical amino acid residues in the mushroom antifungal proteins are shaded
a
Antifungal activity
b
Anti-HIV-1 reverse transcriptase activity
c
Antiproliferative activity (cancer cells)
1230 Appl Microbiol Biotechnol (2010) 87:1221–1235

counterparts (Table 3) as judged from their N-terminal
amino acid sequences. This is not surprising since other
mushroom proteins such as lectins, ribosome-inactivating
proteins, proteases, and ribonucleases are structurally
disparate from their corresponding plant proteins (Ng
2004). Even among the plants and again among the
mushrooms, there is a large variety of proteins with
antifungal activity. In some cases, such as chickpea seeds,
(Vogelsang and Barz 1993), French bean seeds (Ye and Ng
2009,2002e), and L. shimeji fruiting bodies (Lam and Ng
2001a), there is a duo or trio of antifungal proteins. One
antifungal protein may synergize with another antifungal
proteininaction.
Antifungal proteins/lectins display a broad range of
molecular weights and a diversity of amino acid sequences.
Antifungal proteins/lectins of the same class resemble each
other in molecular weight, amino acid sequence and
mechanism of action, but antifungal proteins/lectins of
different classes differ in these characteristics. It is interesting
that despite these differences, they exhibit similar biological
activities. It is likely that they have common intracellular
effectors. It probably deserves mention that the hormones
epinephrine, glucagon, and adrenocorticotropin show gross
structural differences and have their respective membrane
receptor in adipocytes. Yet, they all stimulate membrane-
bound adenyl cyclase and elevate intracellular cyclic AMP
level, eventually leading to an increase in lipolysis (Hadley
2000). A similar picture may explain the aforementioned
observation on antifungal proteins and lectins from plants
and mushrooms.
In addition to an inhibitory action on fungal growth,
some of the antifungal proteins may have as their other
attributes antibacterial, anti-HIV-1 reverse transcriptase,
antiproliferative (toward cancer cells), and mitogenic (toward
spleen cells) activities. The mechanism of antifungal action
has been elucidated for only a small number of antifungal
proteins. It has been shown that transgenic plants expressing
antifungal proteins are more robust and that some of the plant
antifungal proteins are active against human pathogens and
thus of potential value for antifungal therapy in humans. As
many natural products of botanical and microbial origins find
therapeutic applications in humans, it is possible that some of
the aforementioned plant and antifungal proteins can be
successfully exploited not only in agriculture but also in
medicine in the future.
It has been demonstrated that plant defensins have
antifungal activity against some human pathogens such as
C. albicans (Aerts et al. 2009) and that the plant lectin,
mistletoe lectin, can be used in cancer patients to improve
their quality of life (Semiglazov et al. 2006). In view of
the development of microbial resistance and the untoward
side reactions elicited by some of the currently available
drugs, in the foreseeable future, some of the plant and
mushroom proteins with antifungal, antibacterial, and
HIV-1 reverse transcriptase activities can be developed to
serve as alternative therapeutics. However, for the mean-
time, the mechanisms of antifungal and anticancer action
of these proteins have to be elucidated to ascertain if the
mechanisms bear any resemblance to the mechanisms that
have been worked out, e.g., the apoptotic mechanism of
French bean hemagglutinin on breast cancer cells (Lam
and Ng 2010). For those proteins with mitogenic activity
toward splenocytes and nitric oxide inducing activity
towards macrophages, further investigations should be
conducted to find out if they can stimulate production of
cytokines such as tumor necrosis factor, γ-interferon, and
interleukin-2. If so, they may be candidates for developing
into immune-enhancing agents used in the treatment of
immunosuppressed conditions.
The plant and mushroom proteins described above are
defense proteins or antipathogenic proteins in the sense that
they protect plants and mushrooms from diseases caused by
intruding pathogens. It is interesting to find that these
proteins have activity against pathogens that attack humans
and human diseases. As more and more of these proteins
are discovered and researchers know more and more about
them, man will make better use of them in the prevention of
human and plant diseases.
Acknowledgments The award of a direct grant from the Medicine
Panel, CUHK Research Committee is gratefully acknowledged.
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