ABBS 2005,38(02): Anti-HIV I/II activity and molecular cloning of a novel mannose/sialic acid-binding lectin from rhizome of Polygonatum cyrtonema Hua

*Corresponding authors:

Jin-Ku Bao: Tel,
86-28-85410672; Fax, 86-28-85417281; E-mail, [email protected]

Fang CHen: Tel,
86-28-85417281; Fax, 86-28-85417281; E-mail, [email protected]

Abstract        The anti-human immunodeficiency virus
(HIV) I/II activity of a mannose and sialic acid binding lectin isolated from
rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its
HIV infection inhibitory activity in MT-4 and CEM cells with that of other
mannose-binding lectins (MBLs). The anti-HIV activity of Polygonatum
cyrtonema Hua lectin (PCL) was 10- to 100-fold more potent than other
tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To
amplify cDNA of PCL by 3‘/5‘-rapid amplification of cDNA ends
(RACE), the 30 amino acids of N-terminal were determined by sequencing and the
degenerate oligonucleotide primers were designed. The full-length cDNA of PCL ­contained
693 bp with an open reading frame encoding a precursor protein of 160 amino
acid residues, consisting of a 28-residue signal peptide, a 22-residue
C-terminal cleavage peptide and a 110-residue mature polypeptide which
contained three tandemly arranged subdomains with an obvious sequence homology
to the monocot MBL. However, only one active mannose-binding site (QDNVY) was
found in subdomain I of PCL, that of subdomain II and III changed to HNNVY and
PDNVY, respectively. There was no intron in PCL, which was in good agreement
with other monocot MBLs. Molecular modeling of PCL indicated that its ­­three-dimensional
structure resembles that of the snowdrop agglutinin. By docking, an active
sialic acid-binding site was found in PCL. The instabilization of translation
initiation region (TIR) in mRNA of PCL benefits its high expression in
rhizomes.

Key words        Polygonatum cyrtonema HUA;
mannose-binding lectin; anti-human immunodeficiency virus (HIV) I/II; molecular
cloning; 3‘/5‘-rapid amplification of cDNA ends (RACE); sequence
alignment; molecular­ modeling; docking

Plant lectins are
reversible carbohydrate-binding proteins­ (or glycoproteins) of non-immuno
origin that agglutinate cells and/or precipitate glycoconjugates. They possess
at least one non-catalytic domain and are usually considered a heterogeneous
group of proteins because of the apparent­ differences in molecular structure,
sugar specificity and biological activities between individual lectins [1,2].
Recent­ advances in the biochemistry, molecular ­cloning and structural­
analysis of the lectins revealed the occurrence of seven families of
structurally and ­evolutionary related proteins which include the legume
lectins, the monocot mannose-binding lectins (MBLs), the chitin-binding lectins
composed of hevein domains, the type 2 ribosome inactivating­ proteins (RIPs
II) and ­relevant lectins, jacalin-related lectins (amaranthin lectin family)
and the cucurbitaceae phloem lectins [3]. Among them, the monocot­ MBLs (a
superfamily of strictly mannose-specific­ lectins) have been found exclusively
in a subgroup of the monocotyledonous plants. All monocot MBLs consist of
subunits with a similar sequence and overall ­three-dimensional­ structure. The
first monocot MBL was reported­ in 1987, when a lectin with an exclusive
specificity­ towards mannose was isolated from snowdrop (Galanthus nivalis)
bulbs [4]. Since then, related lectins have been found in various tissues of
monocot families: Alliaceae, Amaryllidaceae, Araceae, Bromeliaceae, Iridaceae,
Liliaceae and Orchidaceae. Biochemical analysis and molecular ­cloning clearly
indicated that all these lectins belong to a single superfamily of monocot
mannose-binding proteins, which were named according to their origin and ­specificity
[5,6]. At present, the monocot MBLs are still being ­studied intensively because
of their interesting
biological properties, such as potent inhibition towards retroviruses, which could
possibly be applied in crop
protection against insects­ and nematodes [3]. Two representative
monocot MBLs, Galanthus nivalis agglutinin (GNA) and Narcissus
pseudonarcissus agglutinin (NPA), have been previously reported to be
effective inhibitors of the infection of HIV and feline immunodeficiency virus
(FIV), respectively [7,8].

Polygonatum cyrtonema Hua is a
typical representative of the monocot Liliaceae family and is also an important
traditional Chinese herbal medicine. The rhizomes of this plant have been used
as a tonic, which benefits the spleen, lung and kidney, and as a traditional
medicine for hypertension and diabetes without side effects in Chinese medicine
science for about 2000 years. The polysaccharide extracted from Polygonatum
cyrtonema has an active effect on the immune system of mice [9] and the
mannose/sialic acid-binding protein from the rhizome has ­multi-biological
activities, such as hemagglutination, mitogen and calcium channel block
[10,11]. In addition, anti-tumor studies on Polygonatum cyrtonema lectin
(PCL) indicated that the lectin exhibited a potent suppressive activity on
gastric tumor cell lines SGC and HSC [12].

In this paper, we
describe the anti-HIV-I/II activity of lectin and the molecular cloning of the
lectin gene from rhizome of Polygonatum cyrtonema Hua. More
bioinformatic analysis based on the sequence of PCL is also presented which may
enable us to further understand its mechanism at the molecular level.

Materials and methods

Polygonatum cyrtonema Hua plant

The young rhizomes of Polygonatum
cyrtonema Hua were collected from

Purification of PCL and
mass spectrometry analysis

PCL was purified
according to a previous study [13]. Matrix-assisted laser
desorption/ionization-time of flight (MALDI-TOF) mass spectrometry was
performed using a Voyager-RP mass spectrometer (PerSeptive,

Antiviral assay for HIV

PCL was evaluated for
its inhibitory effect against HIV-I/II-induced cytopathicity in CEM and MT-4
cells,
and other four monocot MBLs, GNA [14], Lycoris radiata agglutinin
(LRA), Listera ovata agglutinin (LOA) and Scilla
campanulata agglutinin (SCA) [15–17], were
tested as controls. The method of the anti-HIV assay has been described
previously [18]. Briefly, CEM and MT-4 cells (4.5´105 cells/ml) were suspended in fresh culture
medium and infected with HIV-I and HIV-II at 100 CCID50 per milliliter of cell suspension (1 CCID50 being the dose infective for 50% of cell
cultures). Then, 100 ml of the infected
cell suspension was transferred to microplate wells, mixed with 100 ml of the appropriate dilutions of the test lectins
(i.e. final concentrations of 200, 40, 8, 1.6, 0.32, and 0.062 mg/ml respectively) and further incubated at 37 ºC.
After 4–5 d, syncytium
formation was examined microscopically in the infected CEM and MT-4 cell
cultures. Antiviral activity was expressed as EC50, the 50% effective concentration
corresponded to the lectin concentration required to inhibit HIV-induced
syncytium formation by 50% in the ­virus-infected CEM or MT-4 cell cultures.
The 50% cytotoxicity (CC50) corresponded to the lectin concentration required to reduce
the viability of CEM or MT-4 cells by 50%.

Analysis of N-terminal
amino acid sequence

The N-terminal amino acid
sequence of PCL was determined by automated Edman degradation. The purified PCL
was subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and electrotransferred to a PVDF membrane
(Millipore,

RNA and DNA isolation

The total RNA and DNA
were extracted using the RNA and DNA extraction kit (TaKaRa Bio,

3‘/5‘-RACE
of Polygonatum cyrtonema lectin gene

cDNA synthesis was
performed with the 3‘-RACE kit (TaKaRa Bio). First, RNA was reversely
transcribed with a cDNA synthesis primer and primer 1 (5‘-TCGGATCCCCSAACTCCCTSTTCACYGGSCA-3‘)
was designed according to the N-terminal amino acid sequence of PCL. The 3‘-RACE
was performed essentially according to the manufacturer’s instructions.
Polymerase chain reaction (PCR) was performed under the following conditions:
cDNA was denatured at 94 ºC for 5 min, followed by 30 cycles of amplification
(94 ºC for 30 s, 55 ºC for 30 s and 72 ºC for 2 min) and 7 min at 72 ºC. The
PCR product was purified and cloned into pUC18-T vector (TaKaRa Bio) for
sequencing.

Based on the sequence of
the 3‘-RACE product, the specific primers 2 (5‘-GACCACATTGCGGTCTTGCT-3‘)
and 3 (5‘-GCCTGAGTCGTACAACACAAA-3‘) were designed to amplify the
5‘ end of PCL. According to the manufacturer’s instructions in the
SMART-RACE cDNA amplification kit (Clontech,

Generation of Polygonatum
cyrtonema lectin ­full-length DNA sequence

Based on the nucleotide
sequence of the 3‘/5‘-RACE products, gene-specific primers 4 (5‘-GAAGCATGCGCAGCTAGTAGTAGTCCAATC-3‘)
and 5 (5‘-AGGGTCGACATAGATAGATAGTAGTGGTG-3‘) were designed for the
amplification DNA of PCL from genomic DNA. The thermal cycling program was the
same as that utilized for 3‘/5‘-RACE.

Bioinformatic analysis
of PCL sequence and structure

DNA sequence and the
associated molecular information were analyzed by DNA Tools 6.0 (

Results

Antiviral assay for HIV

All four
mannose-specific plant lectins (i.e. GNA, LRA, LOA and SCA) were proved highly
effective at inhibiting HIV-I- and HIV-II-induced cytopathicity in MT-4 and CEM
cells. Their EC50 values ranged from 0.43 mg/ml to 9.00 mg/ml (Table 1) without significant cytotoxicity
towards MT-4 and CEM cells, and the CC50 values ranged in 6.50–83.00 mg/ml. PCL
exhibited inhibition on HIV-I at EC50 of 0.08 mg/ml in MT-4 cells and 0.05 mg/ml in CEM cells, and on HIV-II at EC50 of 0.08 mg/ml in MT-4 cells and 0.10 mg/ml in CEM cells. These results mean that the anti-HIV
potency (EC50)
of PCL was 10- to 100-fold higher (more potent) than those of the other four
lectins. The cytotoxicity of PCL against MT-4 and CEM cells was much lower than
its EC50 towards HIV-I/II, and the CC50 value was 73.00 mg/ml and 74.00 mg/ml,
respectively. This was 10-fold less toxic than two mannose-specific plant
lectins in the total four investigated ones, as their CC50 values ranged from 6.50–8.60 mg/ml. The CC50 values of PCL were almost 1000-fold higher
than its EC50 values. Therefore, PCL was at least 10- to
100-fold more inhibitory against HIV-I and HIV-II in MT-4 and CEM cells.

Molecular mass and
N-terminal amino acid sequence of PCL

Upon SDS-PAGE, purified
PCL gave a single band corresponding to a molecular mass of approximately 12
kDa (data not shown). Similarly, MALDI-TOF mass spectrum showed that the
molecular mass of PCL was 11.92 kDa (Fig. 1).

Using automated Edman
degradation, 30 amino acids at N-terminal were sequenced as follows: VNSLSSPNSLFTGHSLEVGPSYRLIMPGDC.
Therefore, the degenerate primer was synthesized according to the underlined
amino acid sequence.

Cloning of PCL
full-length cDNA

Based on the primer 1
designed for the amplification of the 3‘ end of PCL cDNA, a 550-bp
fragment was obtained. Two specific primers (primer 2 and 3), which were
designed according to the 3‘-RACE fragment, were used for the
amplification of 5‘-PCL cDNA and a 290-bp fragment was obtained by
nested PCR. Finally, full-length cDNA and its deduced amino acid sequence were
obtained by analysis of cDNA 3‘ and 5‘ end sequence. The
full-length cDNA of PCL (Fig. 2) was 693 bp and contained a 480-bp open
reading frame (ORF) encoding a 160-aimino acid protein. Using the gene specific
primers 4 and 5, the full-length DNA and deduced amino acid sequences were
obtained by PCR on genomic DNA, which was extracted from young rhizomes of Polygonatum
cyrtonema Hua. By comparison, it was completely the same among the
nucleotide sequence and amino acid sequence of PCL from cDNA and genomic DNA.
Comparison of the nucleotide acid sequence and the amino acid sequence of PCL
from cDNA and genomic DNA revealed completely same among them. This result
indicated that there was no intron in PCL, which was in good agreement with
those of other Amaryllidaceae species, such as Galanthus nivalis.
According to the rules of predicting lectin signal peptide [19] and the
cleavage site of C-terminal [20], a 28-amino acid signal peptide (cleavage site
between A28 and V29) and a 22-amino acid C-terminal
cleavage peptide (cleavage site between A138 and V139) were identified from the PCL
full-length cDNA sequence. So the precursor protein of PCL consists of a
28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a
110-residue mature polypeptide with a molecular weight of 11.92 kDa, and a
predicted isoelectric point of 7.0. These results are consistent with the
accurate molecular weight of 11.920451 kDa by MALDI-TOF mass spectrometry (Fig. 1).

Sequence comparison
between various lectins

The alignment of the
amino acid sequences encoding the PCL, Orchidaceae lectin, Liliaceae
lectin, Alliaceae lectin and Amaryllidaceae lectin clearly
indicated that PCL belonged to the monocot MBL superfamily. Homolog analysis
showed the identity between PCL and GNA, Cymbidium hybrid agglutinin
(CA), Allium sativum agglutinin (ASA) and SCA was 61%, 59%, 55.1% and
55%, respectively. The three mannose-binding boxes of all the lectins were
strictly conserved and therefore were functional, except the second and third
boxes in PCL (Fig. 3).
The QDNVY in PCL was replaced by HNNVY and PDNVY in the subdomain II and III of
PCL.

Molecular modeling and
docking of PCL

The amino acid sequence
of PCL exhibits higher identity and homology with the snowdrop lectin, GNA. Whereas
the three-dimensional structure of GNA has been resolved by X-ray diffraction
analysis, its coordinates can be used to model the structure of lectins with
homologous amino acid sequences. To determine if the overall folding of PCL and
the structure of the carbohydrate-binding sites also resembled that of GNA
[21], hydrophobic cluster analysis (HCA) [22,23] and molecular modeling were
carried out using GNA as a model protein. Molecular modeling of PCL was carried
out using the Swiss-Model program (Biozentrum) [24].

In addition to these
sequence similarities, the minor insertions or deletions mainly occurred in
loops. Structure similarities suggested that both proteins had very similar
three-dimensional structures, so the localization of the 12 strands of b-sheet, occurring along the HCA plot of GNA, were
readily recognized on the HCA plots (Figs. 4 and 5). The three tandem subdomains were connected by loops to
form a 12-strand b-barrel
containing three
putative mannose-binding sites, which were located in the clefts formed by the
three bundles of b-sheet. However, the three mannose-binding sites in PCL
had undergone some changes from those found in GNA. Gln57 and Asp59 of the binding site of subdomain II of GNA
were replaced by His58 and Asn

Forecast of PCL mRNA
structure and translation ­initiation region analysis

The translation
initiation region (TIR) has an important impact on gene expression, and the
secondary structure of this region will influence gene expression directly. Too
high a concentration of GC in TIR will highly stabilize the secondary
structure, thus the transcription unit can not pass smoothly, thus inhibiting
gene expression [27]. For the calculation of RNA DG and the forecast of secondary structure, refer to http://www.genebee.msu.su/services/rna2_reduced.html
[28]. After analysis, the GC concentration in PCL TIR was 37%, while it was 50%
in PCL cDNA. The DG in TIR was –3.19 KJ/Mol and no typical stem-loop structure was
formed (Fig. 10). The high instability of TIR in PCL mRNA can make
the transcription unit pass smoothly and so benefited high expression of
protein production, which accorded with the role of PCL as the predominant
protein in rhizomes of Polygonatum cyrtonema Hua.

Discussion

In this study, we tested
the HIV inhibitory activity of lectin from the rhizomes of Polygonatum
cyrtonema Hua, in MT-4 and CEM cells and compared this with GNA, LRA, LOA
and SCA, the classic anti-HIV MBLs. All the tested MBLs were found to be active
without significant cytotoxicity. However, PCL exhibited a much lower EC50 value and the most effective anti-HIV activity,
which was 10- to 100-fold more potent than other tested MBLs. Also, the
cytotoxicity was lower than other MBLs from Amaryllidaceae, Liliaceae and
Orchidaceae.

In the monocot MBL
superfamily, GNA was researched early and its anti-HIV activity mechanism was
clearly represented. GNA had high affinity for a-(1-3)-D-mannose oligomers and the crystal
structure of GNA has been resolved [25]. It was potent inhibitor of the
HIV-induced cytopathicity and directly interfered the virus-cell membrane
fusion process by binding to the high-mannose glycans on gp120, the crucial
envelope glycoprotein of HIV-I during the infection and blocking the binding of
HIV to target cells [29]. A number of studies had clearly shown that the
binding of MBL to HIV was dependent on the high-mannose glycans on gp120, and
gp120 was extensively glycosylated with N-linked complex and high mannose
carbohydrates accounting for about half of the molecular weight [30,31].
Research showed that gp120 of HIV-I contains approximately 50% of the
high-mannose-type oligosaccharides. All 24 N-linked glycosylation sites were
used in gp120, and 11 of them contained hybrid and/or high-mannose structures,
while 13 of the sites contained complex-type oligosaccharides. Furthermore, many of the complex oligosaccharides
were sialylated [32–34]. The
anti-HIV potency (EC50) of PCL was approximately 10-fold higher, while the
cytotoxicity (CC50)
was about 10-fold less toxic than GNA in MT-4 and CEM cells. It might be that
unlike the other MBLs binding mannose only, PCL binds not only to mannose
oligomers but also to sialic acid on gp120 because it is a mannose- and sialic ­acid-binding
lectin [10]. Therefore, PCL could effectively block the infection of HIV toward
MT-4 and CEM cells, then inhibit the syncytia formation by binding more
saccharides on gp120 and exhibit more effective anti-HIV activity.

Using degenerate
primers, which were specifically designed according to the N-terminal sequences
of PCL, and the RACE-PCR technique, the full-length cDNA of PCL was obtained.
PCL closely resembles the classic monocot MBLs with respect to its
molecular structure and amino acid sequence. However, in contrast with most
other lectins in this family, it differs from the above-mentioned lectins
chiefly in its higher affinity for both mannose and sialic acid, and also
differs with respect to its biological activities [10]. Analysis of the primary
structure of PCL revealed the presence of three mannose binding sites (QDNVY)
as other MBLs, but the subdomain II and III had undergone some changes. The Gln58 and Asp60 were replaced by His58 and Asn

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