Similarity, in Molecular Structure and Function, between
the Plant Toxin Purothionin and the Mammalian
Pore-forming Proteins ’
Takashi Oka, * Yoshiaki Murata,t Toshihiro Nakanishi,$
Hajime Yoshizumi,$ Hidenori Hayashida,$ Yuji Ohtsuki, *
Kumao Toyoshima, I[ and Akira Hakura#
*Department of Pathology, Kochi Medical School; tDepartment of Oncological Pathology,
Cancer Center, Nara Medical College; *Suntory Institute for Biomedical Research,
Shimamoto-cho; $Department of Genetic Information Analysis, National Institute of
Genetics; 11
Department of Oncogene Research; and #Department of Tumor Virology,
Research Institute for Microbial Diseases, Osaka University
Many proteins containing domains of a cysteine-rich repeated motif, such as epidermal growth factor (EGF), have been reported. Here we report strong similarity
between the amino acid sequence of a plant toxin-i.e.,
purothionin and its homologues-and
with those of a domain found in mammalian pore-forming cytoplasmic proteins: components of complement and perforin of cytotoxic T-lymphocytes or natural killer-like cytotoxic cells. These similar sequences were found
to be identical to the so-called EGF-like cysteine-rich repeated motif itself. Electronmicroscopic observations indicated that, Iike complement and perforin, purothionin
forms pores in the cytoplasmic membrane of target cells, resulting in their death
within a few hours. On the basis of these sequence comparisons and physiological
functions, we propose a scheme for the evolution of proteins containing modules
of the cysteine-rich repeat motif.
Introduction
Purothionins
and their homologues
constitute
a set of low-molecular-weight
(molecular weight -5,000)
protein toxins ubiquitous both in monocotyledonous
and
dicotyledonous
plants (Ohtani et al. 1977; Vernon et al. 1985; Ponz et al. 1986 ). The
toxicity of these thionins to pathogenetic
fungi of plants and thionins’ synthesis, triggered by pathogens, suggested that thionins are naturally occurring, inducible proteins
involved in the mechanism of defense of plants against microbial infections (Bohlmann
et al. 1988). Thionins also lyse a wide variety of mammalian
cells (Nakanishi
et al.
1979). However, the molecular basis of the physiological
functions of these toxins is
unknown.
Epidermal growth factor (EGF)-like
proteins play an important
role in many
aspects of eukaryotic cell control, acting as signals for proliferation,
growth inhibition,
differentiation,
and defense. This family of proteins, initially identified in vertebrates,
includes, in addition to EGF itself (Gray et al. 1983 ) , TGF- 1 (transforming
growth
I. Key words: purothionin, perforin, pore-forming proteins (PF?), cystein-rich repeat motif, phylogeny.
Address for correspondence and reprints: Takashi Oka, Department of Pathology, Kochi Medical School,
Nankoku, Kochi 783, Japan.
Mol. Bid
Ed
9(4):707-715. 1992.
0 1992 by The University of Chicago. All rights reserved.
0737-4038/92/0904-0011.$02.00
707
708
Oka et al.
factor) (Marquardt et al. 1984), vaccinia virus p19 protein (Blomquist et al. 1984),
low-density-lipoprotein
(LDL) receptor (Hursh et al. 1987)) a variety of the components of blood coagulation (Furie and Furie 1988), and pore-forming proteins:
components of complement (Rao et al. 1987; Discipio et al. 1988) and perform (Shinkai et al. 1988; Lowrey et al. 1989). The most characteristic feature among EGF and
its family of proteins is the ordered array of cysteines and other conserved amino
acids. A regular spacing of disulfide bonds is known to afford an ordered and energetically stable structure to these proteins which may participate directly in ligand
binding and/or receptor aggregation (Carpenter 1987). This EGF-like motif has recently been found in invertebrate proteins. These include three encoded by the neurogenic loci Notch, Delta, and slit of Drosophila melanogaster (Wharton et al. 1985;
Vassin et al. 1987; Rothberg et al. 1988), by lin-12, a gene of Caenorhabditis elegans
(Greenwald et al. 1985 ) , and uEGF- 1, one of EGF homologues in sea urchins (Hursh
et al. 1987 ). Here, we report the similar molecular structure of this cysteine-rich motif
found in the plant toxin purothionin, indicating that the EGF-like cysteine-rich motif
is widely used in metazoa. Moreover, the plant toxin purothionin is found to kill
target cells by forming pores in the cytoplasmic membrane in a fashion similar to that
of mammalian pore-forming proteins: components of complement and perform containing the EGF-like cysteine-rich motif also. This suggests that the similar molecular
structure of the EGF-like cysteine motif is used for defense both in higher plants and
in animals, in addition to having similar killing function.
Material
and Methods
A3 1 cells, a recloned mouse fibroblast cell line from BALB/ 3T3, were cultured
on l-cm* round glass cover slips placed on 5-cm* petri dishes in Eagle’s minimal
essential medium (Eagle 1959) supplemented with 5% fetal calf serum, and 10 ug
purothionin-A/ml
was added. Purothionin-A was obtained from flour of wheat ( Triticum vulgare, Manitoba no. 3) as described elsewhere (Ohtani et al. 1977). The cells
on the cover slips were fixed at 1,4, and 20 h in glutaraldehyde buffered with 50 mM
Na-cacodylate ( pH 7.4) for 4 h, following dehydration in a series of increasing concentrations of alcohol. These specimens were transferred to isoamylacetate. The cover
slips with the cells were mounted on aluminum stands by double-faced adhesive tape,
were dried with liquid CO2 in a critical point dryer (HCP- 1; Hitachi), and were coated
with gold 40 nm thick at DC 0.7 kV, 8 mA, 0.1 Torr for 5 min by ion-spattering
equipment (FC- 1100; JEOL, Tokyo). The specimens were at 15 kV, scanning angle
20”, horizontal scanning speed 40 ms/line, frame speed 100 s/frame, and total scanning-line number 2,500 and were scanned in the scanning electron microscope (JSM35; JEOL).
A search for similar amino acid sequences was performed on the PIR protein
sequence Database and on the SWISS-PROT protein sequence Database by using the
GENETYX-CD Bio-Database Retrieval Software. Gaps were introduced manually
into the sequences, to increase their aligned similarity. Hydropathy profiles were calculated according to the method of Kyte and Doolittle ( 1982). Phylogenetic analyses
were performed with the “Phylogeny Inference Package” by the neighbor-joining (NJ)
method (Saitou and Nei 1987). Nucleotide substitutions per site (evolutionary distances) were corrected by Jukes and Cantor’s ( 1969) formula. After exclusion of the
common gaps, each remaining gap is counted as one mismatch, regardless of its length.
710
Differentiation controlling
Cellular matrix
rd.EGFP Growth factors
712
Oka et al.
features of cell killing are like those of the pore-forming cytolytic proteins: mammalian
complement and perform of cytotoxic T-lymphocytes (Rao et al. 1987; Shinkai
et al. 1988).
Moreover, the central region of the purothionin molecule (amino acid residues
10-42) consists of an EGF-like cysteine-rich motif similar to cysteine-rich domains
that occur also in terminal complement proteins and perform, suggesting that the
cytolytically effective region of purothionin consists of a single domain of this motif.
The hydropathic profiles show that purothionins have the same amphipathic properties
as do the cysteine-rich domain of the ninth component of complement and perform.
Thus, both higher animals and plants have a similar molecular method of killing for
defense.
Many groups of proteins contain the EGF-type cysteine-rich motif. The amino
acid sequences of the EGF-like motifs of some of these proteins are aligned in the top
panel of figure 2. These proteins form a superfamily possessing various cellular functions, throughout the plant and animal kingdoms. What functional properties do the
sequences examined have in common that might suggest the function of this cysteinerich motif? The ordered array of cysteines and of other amino acids in this motif may
result in a similar structural conformation that could participate directly in cellular
recognition by ligand/receptor interactions. Perform does not lyse cytotoxic T-lymphocytes themselves (Lowrey et al. 1989), and purothionin is reported to kill only
cells in the DNA-synthesis phase (Nakanishi et al. 1979), suggesting that specific
interaction between cellular surface molecules and pore-forming proteins is essential.
Molecules containing EGF motif all appear to interact with membrane lipids or proteins. The EGF motif seems to be part of an interacting protein structure with a loose
dynamic coupling (in contrast to the tight coupling of antibody-antigen, high-affinity
bindings).
Recent evidence in several fields has revealed many rearrangements of exon units,
FIG. 2.-Protein sequences homologous to EGF-like repeats, and phylogenetic tree constructed from
the sequences used in this alignment. Page 710, Comparison of homologous sequences from invertebrates,
vertebrates, and plants containing the characteristic EGF-like motif. The numbers before each sequence
indicate the positions of the start and end of the EGF-like repeat within the protein sequence. Shading
indicates that all sequences of at least three groups of sequences possess the same amino acid. To clarify the
sequence similarity between two groups, boxes are used to indicate amino acids that are a majority in at
least one of the two plant thionin groups or in the pore-forming molecule group. Sequences include the
following: 1, plant thionin group containing related plant toxins (Mellstrand et al. 1974; Mak and Jones
1976; Nakanishi et al. 1979; Thunberg 1983; Vernon et al. 1985; Ponz et al. 1986; Bohlmann et al. 1988);
2, the pore-forming cytolytic molecule group [perform (Shinkai et al. 1988; Lowrey et al. 1989) and terminal
complement proteins C6, C7, CI-alpha, CI-beta, and C9 (Rao et al. 1987; Discipio et al. 1988)]; 3, the
blood coagulation factor group [factors IX, X, and XII (Cool et al. 1985), tissue-specific plasminogen
activator (tPA), and urokinase (UK) (Ny et al. 1984)]; 4, the differentiation-controlling cell-surface molecule
group (Notch) (Wharton et al. 1985), Delta (Kopczynski et al. 1988), and lin-12 (Greenwald 1985)]; 5,
the cellular matrix-constructing and substrate-adhesion molecule group (laminin Bl ) (Sasaki et ai. 1987),
cytotactin (Jones et al. 1988), and entactin (Durkin et al. 1988)]; 6, the receptor- and homing-related
molecule group (ELAM-I ) (Bevilacqua et al. 1989), mLHRc (Siegelman et al. 1989), and LDL-receptor
(Hursh et al. 1987)]; and 7, the growth-factor group (rat TGF-I ) (Marquardt et al. 1984), EGF-precursor
(Gray et al. 1983), sea urchin EGF-1 (Hursh et al. 1987), and vaccinia virus p19 (Blomquist et al. 1984)].
Page 711, Matrix of sequence distances calculated as number of mutations per site, from the alignment
shown in the top panel. The matrix was used to determine the phylogenetic tree by means of the NJ method
( Saitou and Nei 1987). The actual branch lengths, given in corrected differences, are 0.0 1 of those shown.
Similarity between Plant Toxin and Mammalian Perforin
7 13
rearrangements that result in “mosaic” proteins (Gilbert 1985). In most cases “new”
proteins have evolved from “old” ones by gene duplication, but some are the result
of either exon shuffling or gene conversion (Gilbert 1978). Most proteins involving
the EGF-type motif have been shown to be mosaic molecules joined with several
modules (Gray et al. 1983; Yamamoto et al. 1984; Lowrey et al. 1989). However,
purothionins and their homologues consist of single modules of the EGF-type motif,
suggesting that members of the plant thionin family are direct progeny of an ancestral
EGF-type motif. Perhaps an ancestral gene for proteins involved in membrane attachment, membrane insertion, and the polymerization of perform and terminal complement proteins provided this domain to generate cytolytic molecules of plant thionins.
Acknowledgment
The authors thank Dr. H. Furutani of The Center of Medical Information Science,
Kochi Medical School, for excellent technical assistance.
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Received
January
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December
editor
23, 199 1; revision
27, 199 1
received
December
27, 199 1