Molecular Characterization of a Galactose-Binding Lectin from Momordica
charantia Seeds and Its Expression in Tobacco Cells
This study examines the function and genetic structure of Momordica charantia lectin. A galactose-binding lectin (MCL1) was purified from M. charantia seeds. The MCL1 showed highest hemagglutinating activity toward human type-O(H) erythrocytes followed by A, B and Omh (para-Bombay phenotype, also known as H-deficient secretor) erythrocytes. Moreover, we observed that MCL1 inhibited the cell-free synthesis of luciferase in a rabbit reticulocyte lysate system. The N-terminal amino acid sequence of purified MCL1 was identified and used to design degenerate oligonucleotide primers. The 3' and 5' ends of the gene coding for this protein were amplified by rapid amplification of cDNA ends, cloned and sequenced. The coding region (1641 bp, 547 amino acid residues) consisted of a 23 amino acid N-terminal signal sequence preceding an A-chain of 263 amino acid residues encoding a ribosome-inactivating protein that was joined to the B-chain of 261 amino acid residues encoding a lectin. The transcript was detected only in embryos, but hemagglutinating activity was detected both in embryos and cotyledons. These results suggest that gene expression occurred only during embryogenesis and the product accumulated in embryos and cotyledons. The MCL1 was expressed in tobacco BY-2 cells and the supernatant fluid of disrupted cells showed higher hemagglutinating activity toward human type-O(H) erythrocyte than the other tested erythrocytes. Thus, transgenic tobacco suspension culture cells harboring the cloned cDNA encoding the lectin purified from M. charantia are expected to be useful for the production of MCL1.
Lectins are non-catalytic proteins or glycoproteins that are capable of recognizing
and binding reversibly to specific saccharide moieties of glycoconjugates (Goldstein
et al., 1980). Lectins have a variety of biological properties including
antifungal (Melo et al., 2005), antiproliferative
(Kaur et al., 2005) mitogenic (Wong
et al., 2006) and hemagglutinating activities. Some lectins possess
a domain with a distinct biological activity. Lectins belonging to the group
of type-2 Ribosome-Inactivating Proteins (RIP) consist of a carbohydrate binding
B-chain linked to an A-chain that has a highly specific rRNA-N-glycosidase activity
and are capable of catalytically inactivating ribosomes. Type-2 RIPs have been
grossly divided into two groups: toxic and non-toxic, based on the considerable
differences of their cytotoxicity and consequences of their toxicity to animals
(Stirpe and Battelli, 2006). On the other hand, type-1
RIP consists of a single, catalytically active subunit and has comparatively
weaker cytotoxicity than type-2, because type-1 cannot penetrate through cell
membranes. All type-1 RIPs tested so far have antiviral activity against plant,
fungal and animal viruses, whereas only a few type-2 RIPs have been reported
to be active (Chen et al., 2002; Stirpe
and Battelli, 2006; Xu et al., 2007).
Momordica charantia (bitter gourd), a non-leguminous climber belonging
to the Cucurbitaceae, has been widely used in the Orient as a foodstuff,
although the seeds are not consumed. Some lectins from M. charantia have
been isolated and characterized (Barbieri et al.,
1980; Wang and Ng, 1998; Toyama
et al., 2008) and some researchers have suggested that the lectin
has RIP activity. Amino acid compositions and/or partial sequences of these
lectins have been revealed in previous reports; however, the genes for these
lectins have not been cloned and sequenced yet. In the present study, we describe
the cloning and sequencing of cDNAs encoding a galactose-binding lectin from
the seeds of M. charantia and monitored gene expression in different
tissues using reverse transcription PCR (RT-PCR).
MATERIALS AND METHODS
The studies were conducted at University of Miyazaki during the period of 2004-2008.
Sample materials: Momordica charantia [L.] seeds were collected from industrial waste of a food-processing company (Ishihara Foods, Miyazaki, Japan). Fresh embryo (collected from immature fruits) and other tissues were harvested and used immediately or stored at -80°C in RNAlater (Applied Biosystems, Foster City, CA, USA).
Hemagglutination and inhibition of protein synthesis: The hemagglutinating
activity of lectin extracts were assayed by the twofold serial-dilution method
against 3% human erythrocyte suspensions (Ortho-Clinical Diagnostics, Tokyo,
Japan). A nonradioactive assay for inhibition of protein synthesis was performed
as described by Langer et al. (1996). Inhibition
of protein synthesis was determined quantitatively by measuring luciferase activity
in a cell-free rabbit reticulocyte lysate system (Promega, Madison, WI, USA).
The luciferase activities were measured using the Luciferase Assay System (Promega)
and a microplate reader (GENios; Tecan, Research Triangle Park, NC, USA).
Total RNA isolation and cDNA cloning: Total RNA was isolated and purified
from 70 mg of M. charantia embryos using an RNAqueous column with the
Plant RNA Isolation Aid (Applied Biosystems) according to the manufacturer's
instructions. The 3' and 5' ends of the transcripts were amplified by Rapid
Amplification of cDNA Ends (RACE) method using a GeneRacer Kit (Invitrogen,
Carlsbad, CA, USA). For 3' RACE, the forward primer P1 (5'-AAYGARCARTGYTCWCC-3')
corresponding to the N-terminal amino acid sequence NEQCSP and the GeneRacer
3' primer provided in the kit were used. For 5' RACE, the GeneRacer 5' primer
provided in the kit and a reverse primer P2 (5'-CATGCATTGAGTTCATGTGTGGATAAGC-3')
were used. The 5' and 3' RACE products were cloned into pCR4-TOPO with a TOPO
TA-Cloning kit (Invitrogen) and then sequenced. Sequence data were analyzed
using GENETYX-MAC software package (Software Development, Tokyo, Japan). Phylogenetic
trees were constructed from the aligned amino acid sequences using the MEGA
software package (Kumar et al., 2004).
Expression of M. charantia MCL1 in tobacco BY-2 cells: MCL1 cDNA
was expressed in tobacco BY-2 suspension cells (Nicotiana tabacum L.
cv. Bright Yellow-2) (Kato et al., 1991) using
the binary vector pH35CG (Inplanta Innovations, Yokohama, Japan) introduced
by the particle bombardment method (Finer et al.,
1992; Takeuchi et al., 1992; Akashi
et al., 2002). The vector contains MCL1 cDNA linked to the cauliflower
mosaic virus 35S promoter and the nopaline synthase (NOS) terminator. A hygromycin
phosphotransferase gene, conferring hygromycine tolerance, is linked to the
NOS promoter. The vector was coated onto gold particles (1.5-3 μm diameter;
Aldrich Chemical, Milwaukee, WI, USA) following the protocol described previously
(Akashi et al., 2002; Gondo
et al., 2005). Bombardment was carried out at a reduced air pressure
of -0.1 MPa, target distance of 9.6 cm, helium pressures of 5 kg cm-2
and single shot per plate-mode. Bombarded cells were placed on solidified LS
medium (Linsmaier and Skoog, 1965) containing 50 mg
mL-1 hygromycin and subsequently subcultured several times for 14
day periods on the same medium in the dark. After 50 day of subculture under
selective conditions, hygromycin-resistant cells were grown on a rotary shaker
(110 rpm) in 500 mL Erlenmeyer flasks containing 100 mL of LS medium at 27°C
in the dark for 10 day. The suspended cells were collected and disrupted by
using a Tissue Ruptor (Qiagen, Valencia, CA, USA) on ice. The disrupted cells
were centrifuged at 20,000x g for 10 min. The supernatant fluids were assayed
for hemagglutinating activities as described earlier.
Expression analysis: The expression of MCL1 mRNA in different tissues was measured by RT-PCR. Tissues, roots, shoots, leaves, cotyledons and embryos of M. charantia were collected and total RNA was extracted using TRIzol Reagent (Invitrogen). The first-strand cDNA was synthesized using a QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer's instructions. The analysis was performed based on a 99 bp amplicon generated using gene-specific primers P3 (5'-TTTGCGAACGCTTCCTTCTC-3') and P4 (5'-CACGATATGCTGCGAATCCA-3'). The expression of a housekeeping 18S rRNA of M. charantia (GenBank accession number AY900000) as an endogenous reference was carried out using a set of 18S rRNA primers (5'-CTCCGGCGCTGTTACTTTGA-3' and 5'-TCCCGAAGGCCAACAGAATA-3'). The thermal cycling program consisted of an initial step at 94°C for 60 sec, followed by 50 cycles of 94°C for 30 sec and 60°C for 30 sec. The PCR products were separated in 2% agarose gel and visualized by ENVISION DNA dye as a loading buffer (Amresco, Solon, OH, USA).
Nucleotide sequence accession number: The nucleotide sequence of the 1,698-bp region containing MCL1 gene will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession number AB373132.
RESULTS AND DISCUSSION
Purification and N-terminal amino acid sequences of MCL1 subunits: The
anti-H lectin was purified from M. charantia seeds as described by Toyama
et al. (2008). The protein showed higher hemagglutinating activity
toward human type-O erythrocyte followed by -A2, -A1,
-B and -Omh (para-Bombay phenotype, also known as H-deficient
secretor) erythrocytes. These observations indicated that the purified protein
has anti-H activity. In a previous study, anti-H lectin was dissociated into
four subunits, designated MCL1-a, -b, -c and -d, under reducing conditions (Toyama
et al., 2008). N-terminal amino acid sequencing identified the first
8 to 20 amino acid residues of each subunit: MCL1-a, NEQCSPQQRTT; MCL1-b, NEQCSPQQ;
MCL1-c, NLSLSQSXFSADTYKSFIKN; and MCL1-d, NLSLSQS.
Isolation and characterization of the gene encoding MCL1: We have isolated
cDNA clones encoding a precursor protein (MCL1) of 547 amino acid residues with
a calculated molecular weight of 60,993 Da (Fig. 1). The N-terminal
amino acid sequences of the mature protein purified from seeds of M. charantia
matched with the deduced amino acid sequences of Asn-24 to Asn-30 of MCL1-c
and/or -d and Asn-287 to Thr-297 of MCL1-a and/or -b. Wang
and Ng (1998) purified a lectin from M. charantia and identified
the sequence of the first 50 N-terminal amino acids.
||Nucleotide sequence of the MCL1 gene and deduced amino acid
sequence. The non-coding sequences are shown as lower-case letters. Amino
acid sequences underlined by a thick line are identical to those found in
the purified protein. Potential sites for N-linked glycosylation
are underlined. Five QxW repeats are double underlined. The positions of
gene-specific primers, P1 to P4, are indicated by horizontal arrows above
The first 50 residues share 76% sequence similarity with MCL1, suggesting that
the two proteins are different. According to the described method of von
Heijne (1986) for predicting signal sequence cleavage sites, the signal
peptide of MCL1 was most probably cleaved between Cys-23 and Asn-24, a finding
in agreement with the N-terminal amino acid sequences of MCL1-c and -d of this
study as described above. Proteolytic processing of this polypeptide showed
the A- and B-chains of mature MCL1 and implicated more proteolytic cleavages.
Since MCL1-a and -b started with the sequence NEQCSPQQ as described above, cleavage
must have taken place between Asn-286 and Asn-287 of the precursor after cleavage
of the signal peptide to yield an A-chain of 263 amino acid residues encoding
a RIP domain with a calculated molecular weight of 29,473 and a B-chain of 261
amino acid residues encoding a lectin domain with a calculated molecular weight
of 28,991. To determine if protein synthesis could be inhibited by the purified
lectin, luciferase activity was measured in the absence of the protein and then
normalized to 100% translational activity. As shown in Fig. 2,
we observed that MCL1 was able to effectively reduce the yield of luciferase
and a presumed IC50 value for MCL1 was 1.9 nM. These results indicate
that MCL1 could possibly be classified as a type-2 RIP.
||Ribosome-inactivating activity of MCL1. Ribosome-inactivating
activity was determined by measuring the degree of inhibition of luciferase
translation in a rabbit reticulocyte lysate system. The luciferase activity
was normalized to 100% in the absence of MCL1. Results are given (Mean±SE)
for 3 experiments consisting of duplicate samples
There were nine potential N-linked glycosylation sites (Asn-X-Ser/Thr)
in the deduced amino acid sequence (Fig. 1). Five QxW repeats
were observed in the B-chain of MCL1, which may be involved in its tolerance
to accommodate greatly differing amino acids at most positions in the structure
(Hazes, 1996). To check whether MCL1 has introns, genomic
DNA from M. charantia was amplified by PCR using oligonucleotide primers
corresponding to the N- and C-terminal sequences of the primary translation
product of MCL1 (5'-ATGAGAATGAGAGTGTTA-3' and 5'-ATAGAACACCGTCCATTG-3'). Analysis
of PCR products confirmed that the 1.7 kbp amplified fragment was the same size
as a fragment amplified by using the cDNA template. Moreover, the sequence of
this amplicon was identical to the cDNA encoding MCL1. These results suggest
that no intron exists in the coding region of MCL1, a result that is similar
to many other reported type-2 RIPs (Halling et al.,
1985; Wood et al., 1991; Kaku
et al., 1996; Peumans et al., 1998).
Sequence comparisons with other lectins: A BLASTP search for MCL1 in
the protein sequence database found significant degrees of identity to the following
type-2 RIPs: 40 and 39% for Sambucus nigra SNAI' and SNAV (Van
Damme et al., 1996, 1997), respectively;
Abrus precatorius Abrin-c (36%, Wood et al.,
1991) and Ricinus communis Ricin (34%, Halling
et al., 1985). The alignment of some representatives of these homologous
sequences is shown in Fig. 3. These results show that MCL1
was different from other reported lectins. Although Glu177 and Arg180 play an
important role in the rRNA-N-glycosidase activity of ricin A-chain (Katzin
et al., 1991) and were highly conserved in this study, the Arg186
residue was replaced by Lys186 in MCL1. Moreover, Katzin
et al. (1991) also reported several polar residues involved in the
catalysis. Tyrosines 80 and 123 in ricin were replaced by nonpolar Gly95 and
Phe134, respectively. The phylogenetic trees that were built based on the amino
acid sequences of A- and B-chains of type-2 RIPs from these different species
indicate that the A-chain of MCL1 is comparatively more distantly related to
these species, whereas the B-chain is evolutionary closer to that of Sambucus
lectins (Fig. 4). MCL1 seemed to be phylogenetically distant
from the other type-2 RIPs, because evolutionary relationships between A- and
B-chain are similar in this class of RIPs, except for MCL1.
Tissue distribution of MCL1 mRNA: RT-PCR and real-time PCR were performed
to determine the tissue-specific expression of MCL1 mRNA. The RT-PCR products
were cloned and sequenced to confirm the specificity of RT-PCR amplification
and identity of the expected sequences. Xu et al.
(2007) reported that the RIP coding gene cloned and sequenced by Lee-Huang
et al. (1995) from M. charantia was constitutively expressed
in seeds, flowers, roots and stems, whereas the MCL1 transcript was detected
only in the embryos (Fig. 5).
||Alignment of amino acid sequences of M. charantia MCL1
and other homologous type-2 RIPs. The alignment was performed using the
ClustalW program. Dashes indicate gaps introduced during the alignment process.
Asterisks indicate identity and single and double dots indicate semi-conservative
and conservative replacements, respectively. Numbering of the amino acids
starts at the N-termini of the proteins. Cysteine residues potentially involved
in disulfide-bridges are indicated by bold letters. The boxed regions are
described in the text. MCL1, Momordica charantia (this study); SNAV,
Sambucus nigra (U41299); Ricin, Ricinus communis (X03179);
AbrinC, Abrus precatorius (X55667)
Moreover, similar results were obtained using real-time PCR (data not shown).
The hemagglutinating activity was detected in embryos and cotyledons, but was
not detected in leaves or stems. The results of this study show that MCL1 gene
expression occurs only during embryogenesis and the product accumulates in embryos
and cotyledons. The translation product of MCL1 may play an important role in
the protection of seeds against insects and microorganisms.
Expression of M. charantia MCL1 in tobacco BY-2 cells: In order
to prove the identity of MCL1 as a lectin gene, the expression of MCL1
cDNA in tobacco BY-2 suspension cells was studied using crude extracts of the
cells. The supernatant fluid of the disrupted cells showed higher hemagglutinating
activity toward human type-O(H) erythrocyte than the other tested erythrocytes,
whereas the control transformant without the MCL1 gene showed no detectable
activity. Moreover, the carbohydrate-binding specificity of the lectin was studied
by carrying out using saccharide inhibition assays. The following saccharides
did not inhibit hemagglutination at concentrations up to 100 mM: L-fucose, D-mannnose,
D-fructose and sucrose.
||Phylogenetic relationships between M. charantia MCL1
and homologous type-2 RIPs. The phylogenetic trees were built up based on
the amino acid sequences of A- and B-chains of type-2 RIPs from different
species. The values represent the percentage of 1,000 bootstrap replications.
MCL1, Momordica charantia (this study); LPRSN1, SNAI and SNAV, Sambucus
nigra (U58358, U27122, U41299); Ebulin1, Sambucus ebulus (AJ400822);
PMRIPt, Polygonatum multiflorum (AF213984); Viscumin1, Viscum
album (AY377890); Ricin, Ricinus communis (X03179); AbrinC, Abrus
precatorius (X55667); Cinnamomin, Cinnamomum camphora (AY039801).
Panel A, phylogenetic tree of A-chain; panel B, phylogenetic tree of B-chain
||Tissue distributions of MCL1 mRNA expression in various tissues
analyzed by RT-PCR. 18S rRNA was used as the internal control. Total RNA
extracted from individual tissues was reverse transcribed and used for PCR
analysis of MCL1 and 18S rRNA mRNA expression
Human blood type-H antigen trisaccharide showed the strongest inhibition of
all tested reagents. These results suggest that MCL1 was encoding a galactose-specific
anti-H lectin and the binding specificity resembles the purified lectin (Toyama
et al., 2008).
In conclusion, we have cloned an MCL1, coding anti-H lectin, from the seeds
of M. charantia and successfully expressed this type-2 RIP protein in
tobacco BY-2 cells for the first time. The transgenic tobacco cells and the
expressed MCL1 are expected to be useful in blood typing (Toyama
et al., 2008), cell activator (Huang et al.,
2008) and other applications.
We wish to acknowledge Dr. Yoshihiko Tani and Ms. Junko Takahashi at the Osaka Red Cross Blood Center, Japan for useful discussions and technical advice. Tobacco BY-2 cells were provided by RIKEN BRC, which is participating in the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology, Japan. This research was supported by the Japan Science and Technology Agency (JST) Practical Application Research program.
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