Download A new type of plant chitinase containing LysM domains from a fern

Glycobiology vol. 18 no. 5 pp. 414–423, 2008
doi:10.1093/glycob/cwn018
Advance Access publication on February 29, 2008
A new type of plant chitinase containing LysM domains from a fern (Pteris
ryukyuensis): Roles of LysM domains in chitin binding and antifungal activity
Shoko Onaga and Toki Taira1
Department of Bioscience and Biotechnology, Faculty of Agriculture, Ryukyu
University, Okinawa 903-0213, Japan
Received on October 26, 2007; revised on February 19, 2008; accepted on
February 20, 2008
Chitinase-A (PrChi-A), of molecular mass 42 kDa, was purified from the leaves of a fern (P. ryukyuensis) using several
column chromatographies. The N-terminal amino acid sequence of PrChi-A was similar to the lysin motif (LysM). A
cDNA encoding PrChi-A was cloned by rapid amplification
of cDNA ends and polymerase chain reaction. It consisted
of 1459 nucleotides and encoded an open-reading frame of
423-amino-acid residues. The deduced amino acid sequence
indicated that PrChi-A is composed of two N-terminal LysM
domains and a C-terminal catalytic domain, belonging to
the group of plant class IIIb chitinases, linked by proline,
serine, and threonine-rich regions. Wild-type PrChi-A had
chitin-binding and antifungal activities, but a mutant without LysM domains had lost both activities. These results
suggest that the LysM domains contribute significantly to
the antifungal activity of PrChi-A through their binding activity to chitin in the cell wall of fungi. This is the first report
of the presence in plants of a family-18 chitinase containing
LysM domains.
Keywords: antifungal activity/chitin binding/family 18
chitinase/LysM/plant chitinase
Introduction
Chitinases (EC 3.2.1.14) catalyze the hydrolysis of chitin, which
is a β-1,4-linked homopolymer or oligomer of N-acetyl glucosamine (GlcNAc). There are several types of chitinases in
seed plants. Based on their amino acid sequences, plant chitinases are divided into five classes (Beintema 1994): class I
chitinases, consisting of an N-terminal chitin-binding domain
and a catalytic domain; class II chitinases, which have only a
catalytic domain homologous to that of class I chitinases; class
IV chitinases, which share homology with class I chitinases
but are smaller as a result of four deletions; and class III and
V chitinases, which share no homology with class I, II, or IV
chitinases but have distant sequence similarity to bacterial and
fungal chitinases. Yamagami and Ishiguro (1998) have proposed
an additional subclass of chitinases, designated IIIb chitinases;
these constitute a subclass of class III chitinases, based on some
whom correspondence should be addressed: Tel: +81-98-895-8802;
Fax: +81-98-895-8802; e-mail: [email protected]
1 To
differences in structure and function between typical class III
and some bulb chitinases. According to the classification of glycosyl hydrolases by Henrissat and Bairoch (1993), enzymes of
classes I, II, and IV are included in family 19, whereas class III,
IIIb, and V are included in family 18.
Many seed plants synthesize various chitinases (Collinge
et al. 1993; Graham and Sticklen 1994). However, an endogenous substrate for plant chitinases has not yet been found. In
the absence of an endogenous substrate, plant chitinases may be
involved in the interaction between plants and microbes, which
produce chitin and chitin-related compounds. One of the physiological roles of these chitinases is to protect plants against
fungal pathogens by degrading chitin, a major component of the
cell wall of many fungi (Schlumbaum et al. 1986). However,
some chitinases do not show any antifungal activity (Taira et al.
2005). Plant chitinases are induced not only by pathogenesis but
also by abiotic stress. Several plant chitinases are constitutive,
developmentally regulated, and tissue and organ specific. It appears that the role of plant chitinases does not consist solely of
defense against pathogen attack (Kasprzewska 2003). In addition, their roles seem to differ among different plants.
This diversity in the structures and roles of plant chitinases
has developed during the course of plant evolution, and it is to be
expected that different plants in an evolutionary lineage would
have chitinases that differ in structure and role. Researchers
have primarily focused on the chitinases of seed plants. We are
interested in chitinases derived from ferns. Ferns are seedless
vascular plants and are clearly different from seed plants in their
evolutionary lineage (Pryer et al. 2001). We think that structural
and physiological research on chitinases in ferns will probably
lead to a better understanding of the fundamental aspects of
chitinases in the plant kingdom. However, to date there has
been no report of chitinases from ferns.
We screened various fern species for chitinase activity. Pteris
ryukyuensis showed high chitinase activity relative to the other
ferns assayed. Here, we describe the successful isolation, characterization, and cloning of a chitinase from P. ryukyuensis.
Surprisingly, sequence analysis showed that this chitinase is of
an entirely new type and is designated P. ryukyuensis chitinaseA (PrChi-A). This chitinase consists of two lysin motif (LysM)
domains and a catalytic domain of class IIIb chitinase. LysM
domains are found in a variety of peptidoglycan- and chitinbinding proteins (Bateman and Bycroft 2000). In the plant kingdom, LysM domains are found in receptors of chitooligosaccharide and related compounds. It has been reported that these
receptors are involved in the interaction between plants and microbes (Limpens et al. 2003; Radutoiu et al. 2003; Kaku et al.
2006). This is the first report of a plant family-18 chitinase having an extra LysM domain. Therefore, we have focused on the
functions of LysM domains in the chitin-binding and antifungal
activity of PrChi-A. In this study, mutational analyses showed
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414
LysM-containing novel plant chitinase from a fern
Table I. Purification of chitinase from Pteris ryukyuensis
Step
Total proteina (mg)
Total activityb (units)
Specific activity (units/mg)
Yield (%)
Fold
Crude
Butyl-Toyopearl 650 M
Sephadex G-75
Phenyl superose
Mono-Q
1890.00
65.44
11.84
0.73
0.25
1360
553
498
127
73
0.7
8.5
42.0
174.3
293.7
100.0
40.7
36.6
9.4
5.4
1
12
58
242
408
a Protein concentrations were measured by the bicinchoninic acid method.
b Chitinase activities were measured with glycolchitin as substrate. One unit
per min at 37◦ C.
of enzyme activity was defined as the amount of enzyme releasing 1 µmol of GlcNAc
Fig. 1. Molecular mass of P. ryukyuensis chitinase-A. SDS–PAGE was done
by the method of Laemmli. Lane 1, marker proteins and lane 2, purified
PrChi-A (from leaves of P. ryukyuensis). The gel was stained with Coomassie
brilliant blue R250.
that the LysM domains of PrChi-A bind to a chitin and contribute
significantly to the antifungal activity of PrChi-A through their
binding activity. In addition, the roles of LysM-containing chitinases in ferns are discussed.
Results
Fig. 2. HPLC analysis of the hydrolysis products of (GlcNAc)5 by PrChi-A,
class IIIb, and class III chitinase. S, Standards (GlcNAc)1–5 . (A) (GlcNAc)5
was reacted with PrChi-A for 30 min in a 4 mM glycine–HCl buffer (pH 3.0) at
25◦ C. (B) (GlcNAc)5 was reacted with TBC-1 (class IIIb chitinase) for 30 min
in a 4 mM sodium acetate buffer (pH 5.0) at 25◦ C. (C) (GlcNAc)5 was reacted
with PLC-B (class III chitinase) for 30 min in a 4 mM sodium glycine–HCl
buffer (pH 3.0) at 25◦ C. Standard (GlcNAc)1–5 and the reaction mixtures were
analyzed by HPLC, as described in Materials and methods. I to V indicate
(GlcNAc)1–5 and α and β indicate the α- and β-anomer, respectively.
Properties and the N-terminal amino acid sequence of purified
P. ryukyuensis chitinase-A
The purification procedures are summarized in Table I and some
chromatograms in the purification process of the enzyme are
shown in supplementary Figure 1. The enzyme was purified to
homogeneity, corresponding to a 408-fold increase compared
to the crude enzyme, and a 5.4% yield. Using sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in the
presence of 2-mercaptoethanol, the molecular mass of purified
PrChi-A was found to be 42 kDa (Figure 1). The isoelectric
point of PrChi-A was 3.5 (data not shown). The optimum pH
and the optimum temperature for PrChi-A were 3 and 50◦ C,
respectively, using glycolchitin as substrate (supplementary
Figure 2A and C). pH and temperature stability ranges of PrChiA were from pH 2.5 to pH 11 and from 4◦ C to 60◦ C, respectively
(supplementary Figure 2B and D).
The cleavage patterns of chitin oligomer produced by
PrChi-A and the anomeric form of the products were determined as described in Materials and methods (Figure 2). In the
case of PrChi-A, the major hydrolysis products from (GlcNAc)5
were (GlcNAc)3 and (GlcNAc)2 . The ratio of α:β in the reac-
tion product (GlcNAc)3 was 1:3.3; that is, the newly formed
trisaccharide is largely β. In contrast, the ratio of α:β for the
(GlcNAc)2 product was 1:0.6, similar to that in the standard
mutarotation equilibrium. The result indicates that PrChi-A is
a retaining enzyme that mainly hydrolyzed the third linkage
from the nonreducing end side of the substrate. This cleavage
pattern produced by PrChi-A was very similar to that produced
by tulip bulb chitinase-1 (TBC-1), a member of the plant class
IIIb chitinases (compare Figure 2A and B). The cleavage patterns produced by PrChi-A and TBC-1 were clearly different
from that produced by pokeweed leaf chitinase-B (PLC-B), a
member of the plant class III chitinases (Figure 2C). The results
suggest that PrChi-A has a class IIIb-like catalytic domain.
The N-terminal sequence of PrChi-A, which was reduced and
S-pyridylethylated, was analyzed up to the 20th position with
a protein sequencer. The N-terminal sequence of PrChi-A was
found to be DCTTYTVKSGDTCYAISQAN. This sequence is
not homologous to the sequence of any plant chitinase, but
shares homology with the LysM of other proteins from several
organisms.
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S Onaga and T Taira
The N-terminal sequence and cleavage pattern of the chitin
oligomer suggested that PrChi-A consists of LysM domain(s) at
the N-terminal end and a class IIIb-like catalytic domain at the
C-terminal end. These results suggest that PrChi-A is a novel
type of plant chitinase.
Cloning and sequence analysis of PrChi-A cDNA
The sequences of all primers are presented in Table II. A 540-bp
putative PrChi-A PCR product was obtained using degenerate
primers based on the N-terminal amino acid sequence of the
purified PrChi-A and the consensus amino acid sequence
around the catalytic region of plant class IIIb chitinases. Using
gene-specific primers based on the sequence of this fragment,
a 502-bp 5 -cDNA fragment and a 659-bp 3 -cDNA fragment
were then amplified by RACE and sequenced. The resulting
full-length PrChi-A cDNA consisted of 1459 nucleotides and
encoded an open-reading frame of 423-amino-acid residues
(Figure 3A). An amino acid sequence corresponding to the
N-terminal sequence (Asp1 to Asn20) of the purified protein
was found in the deduced amino acid sequence from the
open-reading frame of the cDNA. Thus, 28 amino acids prior to
the N-terminal amino acid of purified protein were predicted to
be a signal peptide. Analysis using computer program signalP
(http://www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al.
2004) supports the presequence of 28 amino acids is a signal
peptide. The remaining 395 amino acids (Asp1 to Ser395) were
considered to constitute the PrChi-A mature protein.
A search of the deduced amino acid sequence of PrChiA against conserved domain database (http://www.ncbi.nlm.
nih.gov/Structure/cdd/cdd.shtml) (Marchler-Bauer et al. 2005)
showed that it possessed two LysM domains at the N-terminal
end and a glycosyl hydrolases family-18 domain at the Cterminal end (Figure 3B). Multiple alignment of LysM domains
of PrChi-A and LysM-containing proteins was performed using
the ClustalX program (Thompson et al. 1997) and the result is
shown in Figure 4A. Two LysM domains in PrChi-A share
high-sequence homology to each other. The LysM domains
have the moderate level of homology to LysM domain proteins from fungi (Aspergillus fumigatus, 47% identity; A. clavatus, 47%; Neosartorya fischeri, 47%) and to peptidoglycanbinding proteins (PGB-P) and N-acetylmuramoyl-L-alanine
amidase (amidase) from bacteria (Alkaliphilus oremlandii, 45%;
A. metalliredigens, 41%; Bacillus pumilus, 43%). However,
the LysM domains show relatively low homology to putative chitinase/lysozyme from Volvox carteri (35%), chitinoligosaccharide elicitor-binding protein (CEBiP) from Oryza
sativa (27%), and Nod-factor receptor kinase-5 (NFR-5) from
Lotus japonicus (21%). Phylogenetic analysis barely shows that
the LysM domain of PrChi-A is located in the clade consisting of
LysM domain proteins from fungi and nematodes (supplementary Figure 4). Alignment of the catalytic domain of PrChi-A
with family-18 chitinases from plants is shown in Figure 4B.
The catalytic domain of PrChi-A had relatively high homology to tulip bulb chitinase-1 (TBC-1, 55%) and gladiolus bulb
chitinase-a (GBC-a, 50%), which are members of the plant class
IIIb chitinases. However, the catalytic domain showed very low
sequence homology to the plant class III chitinases (Hevamine,
16%; PLC-B, 15%; OsChib3a, 14%). In addition, the catalytic
domain of PrChi-A lacks cysteine residues, which form disulfide
bonds, at the conserved sites in class III chitinases (see boxed
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Table II. Primers for PCR, RACE, expression, and mutation
Primer
Sequence
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10a
P11a
P12a
P13a
P14b
5 -GAYTGYACNACNTAYAC-3
5 -TCRTARTCDATRTCDATNCC-3
5 -GGNGAYACNTGYTAYGC-3
5 -GAGCACCTATGTACTCTCTG-3
5 -GCCGTTAGAGCTCGAGGA-3
5 -CTTCATGGGTAGCCAATGCGG-3
5 -CCAATGCGGTTTCCTCGCTCA-3
5 -GGACAAGCGCCAAGTTCGTA-3
5 -CCAGAAGATTGGTGAGACAAG-3
5 -GGAATTCCATATGGATTGCACAACATACACT-3
5 -GGCGGATCCTTAACTAGTCAGCAGAGTTT-3
5 -GGAATTCCATATGAAGGTCTTCAGAGAGTAC-3
5 -GGCGGATCCTTATTTCGAAACACAAACAAC-3
5 -CGACATAGACTATC A ACATTTTGATCAGG-3
a Single underlines and double underlines indicate NdeI and BamHI
recognition sites, respectively.
b A broken line indicates a codon encode amino acid change.
C in Figure 4B). Phylogenetic analysis clearly shows that the
catalytic domain of PrChi-A is located in the clade of class IIIb
chitinases (supplementary Figure 5). Thus, PrChi-A consists of
two N-terminal LysM domains and a C-terminal catalytic domain that belongs to the group of plant class IIIb chitinases,
linked by proline, serine, and threonine-rich regions. No other
chitinase consisting of this domain combination is known among
the plant chitinases.
Preparation of recombinant PrChi-A and its mutants
Since PrChi-A is a new type of plant chitinase that has LysM
domains, we were interested in the roles of the LysM domains
and the catalytic domain in the chitin-hydrolytic, chitin-binding,
and antifungal abilities of PrChi-A. To clarify the functions of
these domains in PrChi-A, we prepared a recombinant PrChi-A
(Asp1 to Ser395) and its mutants, as described in the Materials
and methods (Figure 5A). Since the amino acid residue Glu247
was expected to be the catalytic residue of PrChi-A, based on
sequence alignments with hevamine and TBC-1, Glu247 was
selected for mutation in order to make a completely inactive
mutant. Deletion mutants—CatD (consisting of only a catalytic
domain) and wLysMD (consisting of two LysM domains with a
linker)—were also prepared (Figure 5A). Isolation of recombinant PrChi-A (wild type) and its mutants was successful
(Figure 5B).
Chitin-hydrolytic and -binding activities of PrChi-A
and its mutants
As shown in Table III, recombinant PrChi-A (wild type) and
CatD exhibited nearly the same chitinase activity as native
PrChi-A for the soluble substrate glycolchitin. The mutants
E247Q and wLysMD did not exhibit any chitinase activity. As
shown by the time course of hydrolysis of an insoluble substrate, chitin beads, the efficiency of CatD was lower than that
of the wild type (Figure 6). These results indicate that the LysM
domains were not essential for catalytic activity, but that the
domains contributed to the hydrolysis of an insoluble substrate
by PrChi-A.
Wild-type PrChi-A, E247Q, and wLysMD were adsorbed
to a chitin column and then eluted with a 0.1 to 1 M, 0.1 to
LysM-containing novel plant chitinase from a fern
Fig. 3. Primary structure of PrChi-A. (A) Nucleotide sequence of PrChi-A cDNA with its deduced amino acid sequence. The putative signal peptide at the
N-terminal is indicated by a dotted underline. The N-terminal sequence of the purified protein from leaves is indicated by a solid underline. The putative poly(A)
additional signal is indicated by a double underline. (B) Schematic representation of PrChi-A. PST indicates proline, serine, and threonine. GH indicates glycosyl
hydrolase.
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S Onaga and T Taira
Fig. 4. Alignment of amino acid sequences from domains of PrChi-A with those from corresponding regions of several proteins. (A) Alignment of sequences from
LysM domains of PrChi-A with those of LysM-containing proteins. (B) Alignment of the sequence from a catalytic domain of PrChi-A with that from class IIIb and
class III chitinases. Identical residues are shown in white with a black background and dashes indicate gaps. An underline indicates the consensus region around the
catalytic site in family-18 glycosyl hydrolase. An arrow indicates an expected catalytic residue. Boxed C indicates conserved cysteine residues, which formed
disulfide bonds in plant class III chitinases. PrChi-A 1st and 2nd indicate the first LysM domain and the second LysM domain, respectively, from the N-terminal of
PrChi-A. PrChi-A, P. ryukyuensis chitinase-A (NCBI accession no. BAE98134); Af LysM, LysM domain protein from Aspergillus fumigatus (NCBI accession no.
XP 731484); Ac LysM, LysM domain protein from A. clavatus (NCBI accession no. XP 001272530); Nf LysM, LysM domain protein from Neosartorya fischeri
(NCBI accession no. XP 001263987); Ao PGB-P, peptidoglycan-binding protein from Alkaliphilus oremlandii (NCBI accession no. YP 001513478); Am PGB-P,
peptidoglycan-binding protein from A. metalliredigens (NCBI accession no. YP 001318427); Bp amidase, N-acetylmuramoyl-L-alanine amidase from Bacillus
pumilus (NCBI accession no. YP 001486402),Vc Chi, lysozyme/chitinase from Volvox carteri (NCBI accession no. AAC13727); Os CEBiP, chitooligosaccharide
elicitor-binding protein from Oryza sativa (NCBI accession no. Q8H8C7); Lj NFR-5, Nod-factor receptor kinase-5 from Lotus japonicus (NCBI accession no.
Q70KR1); TBC-1, plant class IIIb chitinase from Tulipa bakeri (NCBI accession no. BAA88408); GBC-a, plant class IIIb chitinase from Gladiolus gandavensis
(NCBI accession no. Q7M1R1); Hevamine, plant class III chitinase from Hevea brasiliensis (NCBI accession no. P23472); PLC-B, plant class III chitinase from
Phytolacca americana (NCBI accession no. AAB34670); OsChib3a, plant class III chitinase from Oryza sativa (NCBI accession no. BAA77605).
1 M, and 1 to 5 M acetic acid solution, respectively (Figure
7A, 7B, and 7D). The results indicate that the chitin-binding
ability of wLysMD is higher than that of the wild type, and that
the mutation of Glu247 had no effect on the chitin-binding of
PrChi-A. Under the same condition, CatD was found in the flowthrough fraction (Figure 7C). These results imply that the LysM
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domains contribute significantly to the chitin-binding activity of
PrChi-A.
Antifungal activity of PrChi-A and its mutants
Antifungal activity was determined by using the hyphal extension inhibition assay on agar plates with Trichoderma viride as
LysM-containing novel plant chitinase from a fern
Fig. 7. Elution profiles of wild-type PrChi-A and its mutants in affinity
chromatography on a chitin column. Protein was applied to a chitin column
equilibrated with a 10 mM phosphate buffer, pH 7.0. After washing out the
unadsorbed protein with the same buffer, the adsorbed protein was eluted
successively with 1 M NaCl, 0.1 M, 1 M, and 5 M acetate. A, wild type; B,
E247Q; C, CatD; D, wLysMD.
Fig. 5. Construction of recombinant PrChi-A and its mutants. (A) Schematic
representation of wild-type PrChi-A and its mutants: E247Q (Glu247→Gln
mutation); CatD; and wLysMD. (B) SDS–PAGE analysis of recombinant
PrChi-A and its mutant proteins. Homogeneity of the recombinant PrChi-A
and its mutant proteins was confirmed by SDS–PAGE as described by
Laemmli. Lane M, marker protein; lane 1, native PrChi-A; lane 2, recombinant
PrChi-A (wild type); lane 3, E247Q; lane 4, CatD; and lane 5, wLysMD.
Table III. Molar specific activities of native PrChi-A, recombinant PrChi-A
(wild type), and its mutants
Proteins
Molar-specific activity (units/mol)a
Native PrChi-A
Recombinant PrChi-A
E247Q
CatD
wLysMD
1.23 × 1010
1.32 × 1010
ND
1.24 × 1010
ND
Fig. 8. Antifungal activity of wild-type PrChi-A and its mutants against
Trichoderma viride. Samples to be tested were placed in wells in 10 µL
distilled water. 1, blank (distilled water); 2, wild type (100 pmol); 3, E247Q
(100 pmol); 4, CatD (100 pmol); 5, wLysMD (100 pmol); and 6, mixture of
CatD (100 pmol) and wLysMD (100 pmol).
a Chitinase
activities were measured using glycolchitin as a substrate.
ND: not detected.
the test fungus. Wild-type PrChi-A inhibited hyphal extension,
but E247Q, CatD, and wLysMD did not (Figure 8, wells 2 to
5). A mixture of CatD and wLysMD also did not exhibit antifungal activity (Figure 8, well 6). These results indicate that
the LysM domains, which by themselves exhibit no antifungal
activity, contributed significantly to the antifungal activity of
PrChi-A. And the catalytic activity of PrChi-A was essential for
antifungal activity.
Discussion
Fig. 6. Hydrolysis of insoluble substrate by wild type and CatD of PrChi-A.
Reaction mixtures contained 1.25 mg substrate and 1 pmol enzyme. Reactions
were performed at 37◦ C and the amount of reducing sugar generated was
monitored. 䊉; wild type; 䊊; CatD.
Primary structure of PrChi-A
In this study we demonstrate the existence of a new type of plant
chitinase, PrChi-A, found in a fern. The new type of chitinase
consists of two N-terminal LysM domains and a C-terminal
catalytic domain of family-18 chitinases.
LysM is a protein domain of about 45 amino acids found initially in several bacterial autolysin proteins (Joris et al. 1992).
These lysin motifs have now been shown to occur in various
proteins from bacteria and some eukaryotes but are not present
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S Onaga and T Taira
in Archaea (Bateman and Bycroft 2000). They may recognize peptidoglycan, chitin, and/or related molecules. In the
plant kingdom, LysM domains are found in receptors for
chitooligosaccharide and related compounds. Chitinases in seed
plants have been well studied, but LysM-containing chitinase
has not been found. In addition, a gene for LysM-containing
chitinase is also not found in complete genome sequences
of the seed plants Arabidopsis thaliana and Oryza sativa.
Pryer et al. (2001) showed that there are three monophyletic
groups of extant vascular plants: lycophytes, seed plants, and
ferns (seedless vascular plants without lycophytes). This finding
implies that ferns are not the ancestors of seed plants. More recently, we found PrChi-A-like LysM-containing chitinase from
another fern (unpublished data). Because the evolutionary lineage of fern plants is different from that of seed plants, it is not
surprising that LysM-containing chitinase exists in ferns and
not in seed plants. There is only one report of LysM-containing
lysozyme/chitinases in the plant kingdom, from the green alga
Volvox carteri. In this species, Amon et al. (1998) detected two
clones by differential screening of a cDNA library from sexually induced females, and one of the clones encoded putative
lysozyme/chitinase containing LysM domains. Except for the
LysM domain regions, the amino acid sequence of the protein has no homology with that of any lysozyme or chitinase.
Furthermore, there is no direct evidence that the protein has
chitinase activity. In the plant kingdom, at least, PrChi-A is the
only family-18 chitinase that contains LysM domains.
Catalytic domain of PrChi-A
Yamagami and Ishiguro (1998) proposed that there is a subclass of class III chitinases, based on the following findings.
TBC-1 and GBC-a share the consensus sequence DXXDXDXE
found in class III chitinases. TBC-1 and GBC-a have no disulfide bonds, but class III chitinases have three conserved disulfide bonds; TBC-1 and GBC-a do not lyse Micrococcus luteus
cell walls, but hevamine and PLC-B, typical class III chitinases, lyse these cell walls. Furthermore, the hydrolysis of
(GlcNAc)5 by TBC-1 and GBC-a yields mainly (GlcNAc)3 and
(GlcNAc)2 ; however, PLC-B hydrolyzes (GlcNAc)5 to yield
mainly (GlcNAc)4 and GlcNAc. Therefore, they propose that
chitinases such as TBC-1 and GBC-a should be placed in subclass IIIb. As shown in Figures 2 and 4, enzymatic characteristics
and structure of PrChi-A were similar to TBC-1 and GBC-a. In
addition, PrChi-A exhibits no lytic activity for Micrococcus luteus cell walls (supplementary Figure 3), and is clearly located
in the clade of class IIIb chitinase by phylogenetic analysis (supplementary Figure 5). These all results indicate that the catalytic
domain of PrChi-A belongs to the class IIIb of plant chitinases.
Roles of LysM domains in chitin-hydrolytic and -binding
activities of PrChi-A
PrChi-A is a new type of plant chitinase that possesses LysM
domains. We were particularly interested in the roles of LysM
domains in the chitin-hydrolytic and chitin-binding activities of
PrChi-A. There have been no published enzymatic studies of
LysM-containing chitinase and no biochemical studies of the
chitin-binding activity of LysM. CatD (LysM-domain deletion
mutant) had the same chitinase activity as wild-type PrChi-A for
a soluble substrate, but the mutant exhibited lower hydrolytic activity than the wild type for an insoluble substrate (Table III and
420
Figure 6). Chitin-binding assay showed clearly that the LysM
domains of PrChi-A have strong chitin-binding activity and that
almost chitin-binding activity of PrChi-A depends on the LysM
domains (Figure 7). In light of these results, we consider that
the LysM domains in PrChi-A contribute to the hydrolysis of
insoluble substrates by PrChi-A through their binding activity.
Roles of LysM domains and a catalytic domain in antifungal
activity of PrChi-A
Several plant chitinases exhibit antifungal activity in vivo and/or
in vitro (Schlumbaum et al. 1986; Broglie et al. 1991). Taira
et al. (2002) showed that basic class I chitinase more effectively
inhibited fungal growth than basic class II chitinase did, as a
result of the high binding ability of the chitin-binding domain to
the fungal cell walls. We investigated whether the LysM domains
contributed to the antifungal activity of PrChi-A. Wild-type
PrChi-A clearly had antifungal activity but E247Q, CatD, and
wLysMD did not (Figure 8; compare well 2 with other wells).
Considering chitin binding and hydrolytic activity of PrChiA and its mutants, the result indicates that catalytic activity of
PrChi-A is essential for the antifungal activity and that the LysM
domains, which by themselves exhibit no antifungal activity,
contribute significantly to the antifungal activity of PrChi-A
through its binding activity. We infer that PrChi-A first binds to
chitin in the fungal cell wall mainly through LysM domains and
then it degrades the chitin by hydrolytic action. As a result, the
fungal cell wall is disrupted and fungal growth is inhibited.
Physiological roles of LysM-chitinase
Considering the combination of LysM domains and chitinase,
there are several possible roles for PrChi-A in vivo. The first
possible role is defense. In this study, the in vitro antifungal assay showed that PrChi-A inhibited fungal growth. In nature this
action could help defend the host plant against fungal pathogens.
Family-19 chitinases have been found in many seed plants and
some bacteria, and many of them exhibit antifungal activity.
However, many family-18 chitinases from plants and many bacteria do not exhibit antifungal activity (Taira et al. 2005; Kawase
et al. 2006). Interestingly, family-18 chitinases containing LysM
were found in some fungi. The Kluyveromyces lactis-type killer
toxin, consisting of α-, β-, and γ-subunits, inhibits the growth of
other yeast cells (Magliani et al. 1997). The α-subunit, a LysMcontaining chitinase, together with the β-subunit plays a role in
binding and translocation of the toxin, allowing the γ-subunit
to enter the cell, which leads to cell cycle arrest. Transcription
of Chi18-10, a LysM-containing chitinase from Trichoderma
reesei, was triggered upon the growth on cell walls of Rhizoctonia solani and in the plate confrontation test with R. solani
(Seidl et al. 2005). These findings suggest that LysM-containing
chitinases in fungi contribute to host defense against other fungi
and may have antifungal activity. To date, we have not found
any family-19 chitinases in fern species, including P. ryukyuensis. Thus, we speculate that fern plants have acquired LysMcontaining family-18 chitinase as their main defensive protein
during their evolution, instead of family-19 chitinases.
Another possible role of LysM-chitinase is in symbiosis.
Some family-18 chitinases in Medicago truncatula have been
reported to be specifically transcribed during interactions with
mycorrhizal fungi (Salzer et al. 2000). Many fern plants can
also establish symbiosis with mycorrhizal fungi as well as
LysM-containing novel plant chitinase from a fern
seed plants (Harrison 1999) and some ferns can do so with
bacteria (Nierzwicki-Bauer and Haselkorn 1986). PrChi-A may
contribute to symbiosis or the defense system by trimming or
abolishing chitin-related signal molecules. The physiological
roles of LysM-containing chitinases in ferns should be clarified
by future structural and physiological studies on fern chitinases
containing PrChi-A.
Materials and methods
Materials
Leaves of P. ryukyuensis were collected on the campus of
Ryukyu University. Chitin and chitin beads were purchased
from Seikagaku kogyo Co. (Tokyo, Japan) and New England
BioLabs (Ipswich, MA), respectively. E. coli BL21(DE3) cells
and expression vector pET-22b were purchased from Novagen
(Madison, WI). Tulip bulb chitinase-1 (TBC-1) and pokeweed
leaf chitinase-B (PLC-B) were provided by Dr. Yamagami of
Kyusyu University. All other reagents were of analytic grade.
Protein measurement
Protein concentrations were measured by the bicinchoninic acid
method (Smith et al. 1985), using bovine serum albumin as the
standard.
Assay of chitinase activity
Chitinase activity was assayed colorimetrically using glycolchitin or chitin beads. Ten microliters of the sample solution
was added to 250 µL of 0.2% (w/v) glycolchitin and 1% (w/v)
chitin beads in a 0.1 M buffer. After incubation at 37◦ C for
15 min, the reducing power of the reaction mixture was measured using ferri-ferrocyanide reagent by the method of Imoto
and Yagishita (1971). The chitinase activity of each chromatographic fraction was expressed by the absorbance of the reaction
mixture at 420 nm (A420). One unit of enzyme activity was
defined as the amount of enzyme releasing 1 µmol of GlcNAc
per min at 37◦ C.
Purification of chitinase from leaves of P. ryukyuensis
All procedures were performed in a cold room. Absorbance
was measured at 280 nm to monitor the proteins, and
chitinase activity was measured at pH 3 during chromatographic separation. Leaves (100 g) of P. ryukyuensis
were homogenized with 500 mL of deionized water and
then left for 4 h at 4◦ C. After centrifugation, the supernatant was collected as crude chitinase. The crude chitinase was mixed with an equal volume of 3 M ammonium
sulfate solution, and the mixture was blended with 10 mL of
butyl-Toyopearl 650 M (Tosoh, Tokyo, Japan) resin. After 4 h,
the resin with adsorbed proteins was collected with a glass filter, and then packed into a 1.6 × 5 cm column. The adsorbed
proteins were eluted with a 50 mM sodium acetate buffer, pH
5.0. The fractions containing chitinase activity were pooled and
applied to a 2 × 140 cm Sephadex G-75 column (GE Healthcare UK Ltd.) previously washed with a 50 mM sodium acetate
buffer, pH 5.0, and developed using the same buffer. The fractions containing chitinase activity were pooled and mixed with
an equal volume of 1.5 M ammonium sulfate solution. The
mixture was applied to a 0.5 × 5 cm Phenyl Superose column
(Pharmacia, UK) previously equilibrated with a 25 mM sodium
acetate buffer, pH 5.0, containing 0.75 M ammonium sulfate.
The column was washed with the same buffer and then the adsorbed proteins were eluted with a linear gradient of ammonium
sulfate from 0.75 to 0 M in the same buffer. Several peaks that
showed chitinase activity were obtained, and the most abundant
peak was collected as PrChi-A, which was further purified. The
PrChi-A fraction was dialyzed against a 10 mM sodium acetate
buffer, pH 5.0. The dialysate was applied to a 0.5 × 5 cm MonoQ column (GE healthcare,) equilibrated with the dialysis buffer.
The column was washed with the same buffer, and adsorbed
proteins were eluted with a linear gradient of NaCl from 0 to 0.2
M in the same buffer. The active fraction was collected as PrChiA. The purified PrChi-A gave a single band having a molecular
mass of 42 kDa on SDS–PAGE (Figure 1, lane 2). One hundred
grams of P. ryukyuensis leaves yielded 0.25 mg of PrChi-A.
Electrophoresis
SDS–PAGE was done by the method of Laemmli (1970) using a 15% acrylamide gel. Proteins on the gel were stained
with Coomassie brilliant blue R250. The molecular mass was
measured in the presence of 2-mercaptoethanol using a molecular weight marker kit (Apro Science, Tokushima, Japan). The
isoelectric point of chitinase was measured by electrofocusing
on a PhastGel IEF 3–9 with the PhastSystem electrophoresis
system (GE Healthcare). Proteins on the gel were detected by
silver staining. The isoelectric point was determined with an
isoelectric point calibration kit (GE Healthcare).
N-terminal amino acid sequence analysis
N-terminal amino acid sequence analysis was done by Edman degradation with a protein sequencer (Model PPSQ-23A;
Shimadzu, Kyoto, Japan).
HPLC analysis of the hydrolysis products of (GlcNAc)5 by
chitinases
The products of hydrolysis of (GlcNAc)5 by chitinases were
analyzed by HPLC on a 0.46 × 25 cm TSK-Gel amide-80
column (Tosoh) using the method of Koga et al. (1998). The
reaction mixture, consisting of 20 pmol of chitinase and 10
nmol of (GlcNAc)5 in 100 µL of 4 mM glycine–HCl buffer, pH
3, after incubation for 30 min at 25◦ C, was immediately cooled
in an ice bath, and 5 µL of reaction mixture was applied to the
column. Hydrolysis products were eluted with 70% acetonitrile
at a flow rate of 0.7 mL/min.
cDNA cloning
The sequences of all primers are presented in Table II. Total
RNA was isolated from leaves of P. ryukyuensis using a RNeasy
kit (Qiagen, Hilden, Germany). First-strand cDNA synthesis
was performed on 5 µg of total RNA using a GeneRacer kit
(Invitrogen, Carlsbad, CA) with an oligo(dT) adapter primer.
The resulting cDNA was used as a template for polymerase
chain reaction (PCR) amplification with degenerate primers.
The first PCR was performed with primers P1 (a forward primer
designed on the basis of the N-terminal amino acid sequence of
purified PrChi-A, Asp1-Thr6) and P2 (a reverse primer designed
on the basis of the published amino acid sequence of TBC-1,
Gly119-Glu125), and nested PCR with P3 (a forward primer
designed on the basis of the internal amino acid sequence of
purified PrChi-A, Gly10-Ala15) and P2. The resulting nested
421
S Onaga and T Taira
PCR products were cloned into a pGEM-T vector (Promega,
Madison, WI) and sequenced using the ABI Prism 310 Genetic
analyzer (Applied Biosystems, CA). To obtain the full-length
cDNA of PrChi-A, both 5 and 3 rapid amplification of cDNA
ends (RACE) were performed using a GeneRacer kit according
to the manufacturer’s instructions. The gene-specific primers
P4 (first PCR) and P5 (nested PCR) were used for the 5 RACE
and the gene-specific primers P6 (first PCR) and P7 (nested
PCR) for the 3 RACE. Finally, a cDNA fragment containing
the entire coding region of PrChi-A cDNA was amplified using
the forward primer P8 (designed from the 5 RACE products)
and reverse primer P9 (designed from the 3 RACE products).
The sequences of the resulting PCR products were analyzed
using the above-mentioned procedure.
Plasmid construction
We tried to express recombinant PrChi-A (wild type; Asp1Ser395, an open-reading frame without a putative signal sequence), the catalytic domain of PrChi-A (CatD; Lys125Ser395, consisting only of the catalytic domain), and the double
LysM domain of PrChi-A (wLysMD; Asp1-Lys107, consisting
of two LysM domains with a linker region between them) in
E. coli. The corresponding cDNA regions were amplified by
PCR using PrChi-A cDNA as the template, with the primers P10
and P11 for the wild type, P12 and P11 for CatD, and P10 and
P13 for wLysMD (P10 and P12 are forward primers containing
the NdeI recognition site, and P11 and P13 are reverse primers
containing the BamHI recognition site). The amplified DNA
fragment was digested with NdeI and BamHI and ligated onto
an expression vector, pET-22b (Novagen), previously digested
with the same enzymes. The resulting recombinant plasmids
were designated pET-PrChi-A, pET-CatD, and pET-wLysMD
for wild type, CatD, and wLysMD, respectively.
Site-directed mutagenesis was performed using the
QuickChange site-directed mutagenesis kit (Stratagene, La
Jolla, CA). The primer used for site-directed mutagenesis was
P14 for the Glu247→Gln mutation (E247Q). A mutation was
introduced into pET-PrChi-A and the resulting plasmid was designated pET-E247Q.
Expression and purification of recombinant PrChi-A and its
mutants in E. coli
The corresponding plasmid was introduced into E. coli
BL21(DE3). E. coli cells harboring the plasmid were grown to
A600 = 0.6 before induction with 1 mM IPTG. After induction,
growth was continued for 24 h at 15◦ C. The cells were harvested
by centrifugation, suspended in a 20 mM Tris–HCl buffer (pH
7.5), and disrupted with a sonicator. After cell debris was removed by centrifugation (10,000 × g, 10 min), the supernatant
was dialyzed against a 10 mM sodium acetate buffer, pH 4.0.
After dialysis, the resulting insolubilized proteins were eliminated by centrifugation at 10,000 × g for 15 min. The supernatant was dialyzed against a 10 mM sodium acetate buffer, pH
5.0. In the case of wild type and wLysMD, the dialysate was
applied to a 0.5 × 5 cm Mono-Q column equilibrated with the
dialysis buffer. The column was washed with the same buffer,
and adsorbed proteins were eluted with a linear gradient of
NaCl from 0 to 0.3 M in the same buffer. In the case of CatD
and E247Q, the dialysate was mixed with an equal volume of
2 M ammonium sulfate solution. The mixture was applied to a
422
0.5 × 5 cm Phenyl Superose column equilibrated with a 20 mM
sodium acetate buffer, pH 5.0, containing 1 M ammonium sulfate. The column was washed with the same buffer, and the adsorbed proteins were then eluted with a linear gradient of ammonium sulfate from 1.0 to 0 M in the same buffer. After confirmation of the purity of the corresponding fraction by SDS–PAGE,
the fraction was collected as purified recombinant protein.
Chitin-binding assay
The chitin-binding ability of the chitinase and its mutant was
examined using a 0.7 × 2.5 cm chitin column, as described by
Yamagami and Funatsu (1996). Twenty-five micrograms of the
chitinase was dissolved in a 10 mM sodium phosphate buffer,
pH 7.0, and applied to a chitin column previously equilibrated
with the same buffer. After washing the column with the same
buffer, the chitin-binding protein was eluted first with 1 M NaCl
in the same buffer, secondly with 0.1 M acetic acid, thirdly with
1 M acetic acid, and lastly with 5 M acetic acid.
Antifungal assay
Antifungal assay was done according to Schlumbaum et al.
(1986) with modification. An agar disk (6 mm in diameter) containing the fungus Trichoderma viride, prepared from actively
growing fungus that had previously been cultured on a potato
dextrose broth with 1.5% (w/v) agar (PDA), was placed in the
center of a Petri dish containing PDA. The plates were incubated
at room temperature for 12 h. Wells were subsequently punched
into the agar at a distance of 15 mm from the center of the
Petri dish. The samples to be tested were placed into the wells
containing 10 µL of sterile water. The plates were incubated for
24 h at room temperature and then photographed.
Footnotes
The nucleotide sequence for the PrChi-A gene has been deposited in the GenBank database under GenBank accession
number AB247328. The amino acid sequence of this protein
can be accessed through NCBI protein database under NCBI
accession number BAE98134.
Acknowledgements
We greatly appreciate the gift of TBC-1 and PLC-B enzymes
from Dr. Takeshi Yamagami (Kyusyu University). We thank
Dr. Etsuko Katoh and Dr. Takayuki Ohnuma (National Institute
of Agrobiological Sciences, Japan) for their practical advice
and discussions, Dr. Takakazu Shinzato (Ryukyu University)
for the identification of fern plants, and Miho Tobita (Ryukyu
University) for technical assistance.
Conflict of interest statement
None declared.
Abbreviations
CBB, Coomassie brilliant blue; GBC-a, gladiolus bulb
chitinase-a; GlcNAc, N-acetyl glucosamine; LysM, lysin
LysM-containing novel plant chitinase from a fern
motif; PDA, potato dextrose broth with agar; PLC-B, pokeweed
leaf chitinase-B; PrChi, Pteris ryukyuensis chitinase; RACE,
rapid amplification of cDNA ends; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; TBC-1, tulip
bulb chitinase-1.
References
Amon P, Haas E, Sumper M. 1998. The sex-inducing pheromone and wounding
trigger the same set of genes in the multicellular green alga Volvox. Plant
Cell. 10:781–789.
Bateman A, Bycroft M. 2000. The structure of a LysM domain from E. coli
membrane-bound Lytic Murein Transglycosylase D (MltD). J Mol Biol.
299:1113–1119.
Beintema JJ. 1994. Structural features of plant chitinases and chitin-binding
proteins. FEBS Lett. 350:159–163.
Bendtsen JD, Nielsen H, von Heijne G, Brunak S. 2004. Improved prediction
of signal peptides: SignalP 3.0. J Mol Biol. 340:783–795.
Broglie K, Chet T, Holliday M, Cressman R, Biddle P, Knowlton S, Mauvais CJ,
Broglie R. 1991. Transgenic plants with enhanced resistance to the fungal
pathogen Rhizoctonia solani. Science. 254:1194–1197.
Collinge DB, Kragh KM, Mikkelsen JD, Nielsen KK, Rasmussen U, Vad K.
1993. Plant chitinases. Plant J. 3:31–40.
Graham LS, Sticklen MB. 1994. Plant chitinases. Can J Bot. 72:1057–1083.
Harrison MJ. 1999. Molecular and cellular aspects of the arbuscular mycorrhizal
symbiosis. Annu Rev Plant Physiol Plant Mol Biol. 50:361–389.
Henrissat B, Bairoch A. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J.
293:781–788.
Imoto T, Yagishita K. 1971. A simple activity measurement of lysozyme. Agric
Biol Chem. 33:1154–1156.
Joris B, Englebert S, Chu CP, Kariyama R, Daneo-Moore L, Shockman GD,
Ghuysen JM. 1992. Modular design of the Enterococcus hirae muramidase2 and Streptococcus faecalis autolysin. FEMS Microbiol Lett. 70:257–264.
Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N,
Takio K, Minami E, Shibuya N. 2006. Plant cells recognize chitin fragments
for defense signaling through a plasma membrane receptor. Proc Natl Acad
Sci USA. 103:11086–11091.
Kasprzewska A. 2003. Plant chitinases: Regulation and function. Cell Mol Biol
Lett. 8:809–824.
Kawase T, Yokokawa S, Saito A, Fujii T, Nikaidou N, Miyashita K, Watanabe
T. 2006. Comparison of enzymatic and antifungal properties between family
18 and 19 chitinases from S. coelicolor A3(2). Biosci Biotechnol Biochem.
70:988–998.
Koga D, Yoshioka T, Arakane Y. 1998. HPLC analysis of anomeric formation
and cleavage pattern by chitinolytic enzyme. Biosci Biotechnol Biochem.
62:1643–1646.
Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the
head of bacteriophage T4. Nature. 227:680–685.
Limpens E, Franken C, Smit P, Willemse J, Bisseling T, Geurts R. 2003. LysM
domain receptor kinases regulating rhizobial Nod factor-induced infection.
Science. 302:630–633.
Magliani W, Conti S, Gerloni M, Bertolotti D, Polonelli L. 1997. Yeast killer
systems. Clin Microbiol Rev. 10:369–400.
Marchler-Bauer A, Anderson JB, Cherukuri PF, DeWeese-Scott C, Geer LY,
Gwadz M, He S, Hurwitz DI, Jackson JD, Ke Z, et al. 2005. CDD: A
conserved domain database for protein classification. Nucleic Acids Res.
33:D192–D196.
Nierzwicki-Bauer SA, Haselkorn R. 1986. Differences in mRNA levels in Anabaena living freely or in symbiotic association with Azolla. EMBO J.
5:29–35.
Pryer KM, Schneider H, Smith AR, Cranfill R, Wolf PG, Hunt JS, Sipes SD.
2001. Horsetails and ferns are a monophyletic group and the closest living
relatives to seed plants. Nature. 409:618–22.
Radutoiu S, Madsen LH, Madsen EB, Felle HH, Umehara Y, Grønlund M, Sato
S, Nakamura Y, Tabata S, Sandal N, et al. 2003. Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases. Nature. 425:585–592.
Salzer P, Bonanomi A, Beyer K, Vögeli-Lange R, Aeschbacher RA, Lange
J, Wiemken A, Kim D, Cook DR, Boller T. 2000. Differential expression
of eight chitinase genes in Medicago truncatula roots during mycorrhiza
formation, nodulation, and pathogen infection. Mol Plant Microbe Interact.
13:763–777.
Schlumbaum A., Mauch F, Vögeli U, Boller T. 1986. Plant chitinases are potent
inhibitors of fungal growth. Nature. 324:365–367.
Seidl V, Huemer B, Seiboth B, Kubicek CP. 2005. A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases.
FEBS J. 272:5923–5939.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano
MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. 1985. Measurement
of protein using bicinchoninic acid. Anal Biochem. 150:76–85.
Taira T, Ohnuma T, Yamagami T, Aso Y, Ishiguro M, Ishihara M. 2002. Antifungal activity of rye (Secale cereale) seed chitinases: The different binding
manner of class I and class II chitinases to the fungal cell walls. Biosci
Biotechnol Biochem. 66:970–977.
Taira T, Toma N, Ishihara M. 2005. Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf. Biosci
Biotechnol Biochem. 69:189–196.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The
CLUSTAL_X windows interface: Flexible strategies for multiple sequence
alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876–4882.
Yamagami T, Funatsu G. 1996. Limited proteolysis and reductioncarboxymethylation of rye seed chitinase-a: Role of the chitin-binding domain in its chitinase action. Biosci Biotechnol Biochem. 60:1081–1086.
Yamagami T, Ishiguro M. 1998. Complete amino acid sequences of chitinase-1
and -2 from bulbs of genus Tulipa. Biosci Biotechnol Biochem.
62:1253–1257.
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