Plant CellPhysiol. 37(7): 1007-1012 (1996)
JSPP © 1996
A Lectin from Leaves of Neoregelia flandria Recognizes D-Glucose,
D-Mannose and 7V-Acetyl D-glucosamine, Differing from the MannoseSpecific Lectins of Other Monocotyledonous Plants
Fumio Yagi1, Makiko Hidaka, Yuji Minami and Kenjiro Tadera
Biochemistry and Biotechnology, Faculty of Agricuture, Kagoshima University, Kagoshima, 890 Japan
A mannose-specific lectin was isolated from leaves
of Neoregelia flandria, an ornamental plant that belongs
to Bromeliaceae, a family of monocotyledons. The amino
acid composition and molecular mass of the lectin were
similar to those of mannose-specific lectins from other
monocotyledons. However, in a test to examine the inhibition of hemagglutination, it became apparent that the isolated lectin recognized D-glucose and jV-acetyl D-glucosamine in addition to D-mannose, unlike mannose-specific
lectins from the monocotyledons that have been reported
to date.
Key words: Bromeliaceae — Leaf — Lectin — Monocotyledon — Neoregelia flandria.
Mannose-specific lectins and related lectins have been
isolated from monocotyledonous plants (Kaku et al. 1990,
1992, Mo et al. 1993, Saito et al. 1993, Shibuya et al. 1988,
Van Damme et al. 1987a, b, 1988, 1991a, b, 1994, 1995,
Yagi et al. 1993) that belong to Amaryllidaceae, Alliaceae,
Araceae and Orchidaceae. They differ from the the mannose/glucose-specific lectins (Strosberg et al. 1986) from leguminous plants in terms of molecular mass, sugar specificity, primary structure and requirements for metal ions.
These mannose-specific lectins from monocotyledonous
plants recognize either D-mannose or mannooligosaccharides exclusively and they do not recognize D-glucose, glucobioses and N-acetyl D-glucosamine.
We found that a lectin in an extract of leaves of Neoregelia flandria recognized not only D-mannose but also D-glucose, glucobioses and /V-acetyl D-glucosamine. We describe
Abbreviations: AAA, agglutinin from bulbs of shallot
(Allium ascalonicum); AMA, agglutinin from tubers of Arum
maculatum; ASA, agglutinin from bulbs of garlic (Allium sativum); Con A, concanavalin A; GNA, lectin from bulbs of Galanthus nivalis L.; LOA, agglutinin from leaves of Listera ovata;
NFL, lectin from leaves of Neoregelia flandria; Manal,3Man, 3O-a-D-mannopyranosyl-D-mannopyranose; Manal,6Man, 6-O-aD-mannopyranosyl-D-mannopyranose; Manal,6[Manal,3]Man,
6-0-a-D-mannopyranosyl-[3-0-a-D-mannopyranosyl]-D-mannopyranose; PBS, phosphate-buffered saline (pH 7.2) containing
6.7 mM phosphate (K, Na), 132 mM NaCl and 0.04% NaN3;
PNP-, /7-nitrophenyl.
' To whom correspondence should be addressed.
here the purification of the lectin from leaves of N. flandria
and its carbohydrate-binding specificity.
Materials and Methods
Materials—Toyopearl HW 55F was obtained from Tosoh
(Tokyo, Japan), and mannose-agarose was from Sigma Chemical
Co. (St. Louis, MO, U.S.A.). Mannobioses and mannotriose were
purchased from Funakoshi Co. (Tokyo, Japan). Kojibiose,
nigerose and sophorose were gifts from Professor Ken'ichi Takeo,
Kyoto Prefectural University. Other monosaccharides, glycosides
and glycoproteins were from Sigma Chemical Co. Asialoglycoproteins were prepared from fetuin and thyroglobulin by desialylation with 0.1 M H2SO4 at 80°C.
Hemagglutination assay—Hemagglutinating activity was
determined in wells of microtiter plates by the 2-fold dilution
method, in a final volume of 70 /JL\ of phosphate-buffered saline,
pH 7.2, that contained 6.7 mM phosphate (K, Na), 132 mM NaCl
and 0.04% NaN3 (PBS). Each well contained 50/il of lectin solution and 20 fA of a 4% (v/v) suspension of trypsinized rabbit
erythrocytes.
Agglutination was assessed after incubation for 1 h at room
temperature, and hemagglutinating activity was expressed as the
titer, namely, the reciprocal of the highest dilution that gave a positive result. The specific hemagglutinating activity was defined as
the titer (mg lectin)"1.
Quantitation of protein and carbohydrate—Protein was quantitated by the method of Lowry et al. (1951) with bovine serum
albumin as the standard, and carbohydrate was quantitated by the
phenol-sulfuric acid method of Dubois et al. (1956) with D-mannose as the standard.
Electrophoresis—PAGE was carried out by the method of
Davis (1964) at pH 8.9. Proteins on gels were stained with
Coomassie brilliant blue R-250. SDS-PAGE was performed using
a discontinuous system, as described by Laemmli (1970). Proteins
on gels were stained with Coomassie brilliant blue R-250.
Estimation of molecular mass—The molecular mass of the
purified lectin was estimated by gel filtration on a column of
Toyopearl HW 55F (2.5 cm i.d. x 140 cm), by SDS-PAGE, and by
HPLC. HPLC was performed with a pump (model 880 PU;
JASCO, Tokyo, Japan), a UV detector (model 875; JASCO) and
a Fine Pak SIL AF-102 column (JASCO). The column was eluted
with 10 mM sodium phosphate, pH 6.8, that contained 0.2 M
sodium sulfate, at a flow rate of 0.5 ml min" 1 , and the absorption
at 280 nm of the effluent was monitored.
Amino acid analysis and determination of N-terminal amino
acid sequence—The purified lectin (50 fig) was hydrolyzed with
constant-boiling HC1 in a sealed evacuated glass tube for 48 h at
110°C. The hydrolysate was analyzed with an amino acid analyzer
(model 835; Hitachi, Tokyo, Japan). Amino-terminal sequencing
was performed with an automated protein sequencer (model 492;
Applied Biosystems-Perkin Elmer Japan, Tokyo) fitted with a
1007
A lectin from leaves of Neoregelia flandria
1008
PTH analyzer (model HOC; Applied Biosystems-Perkin Elmer
Japan).
Purification of the lectin—Hemagglutinating activity was
measured throughout the entire purification procedure with trypsinized rabbit erythrocytes. A total of 40 g of leaves of N. flandria,
purchased at a local market in Kagoshima, was homogenized with
400 ml of PBS at 4°C, and the homogenate was filtered through
two layers of gauze and centrifuged at 8,000 xg. Ammonium
sulfate was added to the supernatant to 90% saturation. The precipitate was collected by centrifugation and dissolved in 20 ml of
PBS. The solution was centrifuged to remove insoluble material.
The supernatant was loaded onto a mannose-agarose affinity column (0.8 cm i.d. x 7 cm) that had been equilibrated with PBS. The
column was washed with 120 ml of PBS, and then the lectin was
eluted with 0.2 M D-mannose in PBS and dialyzed against PBS.
Elution was monitored at 280 nm and by examining the hemagglutinating activity.
B
Results
Purification of a novel lectin from leaves of Neoregelia
flandria—A lectin was isolated by one-step chromatographic purification on an affinity column of mannose-agarose (Fig. 1). When we investigated the hemagglutinating activity in extracts of leaves, we found that trypsinized rabbit
erythrocytes were agglutinated but human erythrocytes
were not. From 40 g of leaves, we obtained 0.5 mg of protein and the specific activity of the lectin was 30,000 titer
(mg protein)" 1 , with 120-fold purification from the crude
extract. The yield was 30% on the basis of hemagglutinating activity.
Fig. 2 PAGE and SDS-PAGE of the purified lectin from leaves
of Neoregelia flandria. A, PAGE (7.5% gel) at pH 8.9; B, SDSPAGE (15% gel), a and b, the lectin from Neoregelia and molecular-mass markers (Oriental Yeast, Tokyo, Japan), respectively.
Native PAGE and SDS-PAGE (Fig. 2) confirmed the
homogeneity of the purified preparation of the lectin.
Table 1 Amino acid compositions of the lectin from
Neoregelia and GNA
Amino acid
- 1,000
10 15 20 25 30 35 40 45
Fraction no.
Fig. 1 Purification by affinity chromatography of the lectin
from Neoregelia. The ammonium sulfate precipitate of the crude
extract was dissolved in PBS, and the clear supernatant after centrifugation was applied onto a column of mannose-agarose (0.8
cm i.d. x 7 cm) that had been equilibrated with PBS. The column
was washed with PBS and then protein was eluted with 0.2 M Dmannose in PBS. The arrow indicates the start of elution with 0.2
M D-mannose. Dotted line, y^sol solid line, hemagglutinating activity (HA) with trypsinized rabbit erythrocytes. The volume of
each fraction was 5 ml.
Asx
Thr
Ser
Glx
Pro
Gly
Ala
l/2Cys
Val
Met
He
Leu
Tyr
Phe
Lys
His
Arg
Trp
Neoregelia
GNA"
(residue/subunit)
9.5
7.8
8.0
10.0
6.5
17.7
6.6
1.0
5.9
1.0
6.4
7.0
2.9
3.6
2.2
0.7
5.0
—
17.0
9.0
7.4
6.8
3.7
11.2
3.4
2.9
5.6
1.7
5.5
8.8
5.8
3.0
2.8
0.9
2.9
1.7
The values for the lectin from Neoregelia are based on an assumed
molecular mass of 12 kDa.
" Van Damme et al. (1987a).
A lectin from leaves of Neoregelia flandria
1009
1
10
20
30
Neoregelia (present work) DNILYTNDKL SGRQSLTLGN YRLTMQTDCN LVLYDGGS- -GNA (snowdrop)1)
EHILXSGETL £TGEFLNY£S FVFIMQEEQJ LVLYDVDK-- AAA (shallot)2)
RUVLVNUEGL YAGQSLVEEQ YTFIMQDEQI LVLYEYST
LOA (twayblade)3)
SHTLGGGERL NSGESLIESA CVFIMQEEQI LSLXESSR--AMA (arum)4)
VGSHYLLSSETL NTDGRLING.D FTLIMQGECH LVLYNGGW-- Fig. 3 Comparison of N-terminal amino acid sequences of the lectin from Neoregelia and of four lectins from other monocotyledons.
Bold-faced letters show conserved residues in the five sequences; underlined letters show those residues that are identical to those in
NFL. 1,3,4: Van Damme et al. (1991a, 1994, 1995). 2: Mo et al. (1993). 3 and 4 (AMA1) are sequences deduced from the corresponding
cDNAs.
From the results of gel-filtration chromatography and
HPLC, the molecular mass of the lectin was estimated to
be about 22 kDa (data not shown). Only a single band of
12-kDa protein was detected after SDS-PAGE (Fig.2B).
Table 2 Inhibition of the hemagglutinating activity of the lectin from Neoregelia by saccharides
innioitor
D-Fructose
D-Glucose
D-Mannose
D-Galactose
TV-Acetyl D-glucosamine
JV-Acetyl D-galactosamine
D-Glucosamine
Me-a-D-glucoside
Me-/?-D-glucoside
Me-a-D-mannoside
Me-yS-D-mannoside
2-Deoxy-D-glucose
3-O-Me-D-glucose
D-Xylose
D-Lyxose
Lactose
Sucrose
Trehalose (a,a)
Kojibiose (a 1,2)
Sophorose 091,2)
Nigerose (a 1,3)
Laminaribiose 091,3)
Maltose (a 1,4)
Cellobiose 051,4)
Isomaltose (a 1,6)
Gentiobiose 031,6)
Af-Acetylchitobiose
Manal,3Man
Manal,6Man
Manal ,6[Manal ,3]Man
Minimum cone.
(mM)
3.12
3.12
0.78
N (200)
1.56
N (100)
6.25
3.12
12.5
1.56
6.25
6.25
1.56
N (100)
100
N (200)
0.39
0.39
N (100)
6.25
1.56
1.56
12.5
25
1.56
3.12
12.5
0.19
0.39
0.78
Relative inhibitory potency "
Con A*
Neoregelia lectin
0.25
0.25
1.0
< 0.004
0.50
< 0.008
0.125
0.25
0.0625
0.50
0.125
0.125
0.50
< 0.008
0.008
< 0.004
2.0
2.0
< 0.008
0.125
0.50
0.50
0.063
0.031
0.50
0.25
0.063
4.0
2.0
1.0
0.50
0.22
1.0
<0.04
1.76
0.07
7.33
0.19
0.63
<0.04
1.02
<0.04
2.00
0.04
16.1 e
13.9 C
950 c
Minimum cone, necessary for complete inhibition of hemagglutinating activity with a titer of 4 are shown.
* Potency relative to that of D-mannose.
* Goldstein and Poretz (1986).
c
Values from Bhattacharyya et al. (1987) were multiplied by 7.33, the relative potency of Me-a-D-mannose/D-mannose.
N: No inhibition at the concentration shown in parentheses.
1010
A lectin from leaves of Neoregelia flandria
Therefore, the lectin appeared to be a homodimer.
The amino acid composition and N-terminal amino
acid sequence of the lectin from Neoregelia (NFL)—The
amino acid composition of NFL is shown in Table 1, in
comparison with that of an agglutinin from bulbs of
snowdrop, Galanthus nivalis (GNA; Van Damme et al.
1987a). No carbohydrate was detected by the phenolsulfuric acid method. Levels of Asx and Tyr in NFL were
lower than those in GNA, whereas those of Glx, Pro, Gly,
Ala and Arg in NFL were higher than those in GNA.
The sequence of 38 residues from N-terminus of NFL
was determined and is shown in Fig. 3, together with the
corresponding sequences of GNA, shallot lectin (AAA), an
agglutinin from Listera ovata (LOA) and an agglutinin
from tubers of Arum maculatum (AMA). These plants
belong to different families of monocotyledons, namely,
Bromeliacea, Amaryllidaceae, Alliaceae, Orchidaceae and
Araceae, respectively. When any two sequences were compared, the extent of homology was found to be around
60%. Only 13 residues were conserved among the five N-terminal sequences.
The inhibition of hemagglutination by saccharides—
Table 2 shows the inhibition of hemagglutination of NFL
by saccharides in comparison with that of a typical mannose-specific lectin from a legume, concanavalin A (Con A;
Goldstein and Poretz 1986, Bhattacharyya et al. 1987).
Among the monosaccharides tested, D-mannose was the
most potent inhibitor of hemagglutination by NFL. However, D-glucose, //-acetyl D-glucosamine, D-glucosamine
and D-fructose also had the inhibitory effects. The inhibition by methyl a-D-glucoside and methyl a-D-mannoside
was not stronger than that by the corresponding free sugars.
These results for NFL were very different from those for
Con A.
Sucrose and trehalose were more potent inhibitors
than D-mannose. Glucobioses, were the exception of kojibiose, had inhibitory effects on the hemagglutination.
However, the inhibition by maltose and isomaltose of
hemagglutination by Con A was as strong as that by D-mannose, whereas these disaccharides were weak inhibitors of
NFL-mediated hemagglutination, as compared with D-mannose. Of the mannooligosaccharides tested, 3-O-a-D-mannopyranosyl-D-mannopyranose (Manal,3Man) was the
most potent inhibitor, but its inhibitory potency was 4.0
(relative to that of D-mannose, which was taken as 1.0),
lower than the value of 16.1 for Con A. In particular, the
inhibitory potency of 6-O-a-D-mannopyranosyl-[3-O-a-Dmannopyranosyl]-D-mannopyranose (Manctl,6[Manal,3]Man) was the same as that of D-mannose for hemagglutination by NFL, showing a marked difference from that for
Con A.
Table 3 shows the inhibition of hemagglutination by
various glycoproteins. Hemagglutination was most strongly inhibited by asialothyroglobulin, which has high man-
Table 3 Inhibition of the hemagglutinating activity of
the lectin from Neoregelia by glycoproteins
Minimum cone.
C"gmr')
Inhibitor
Ovalbumin
Ovomucoid
Ovomucoid (quail)
Fetuin
Asialofetuin
Thyroglobulin
Asialothyroglobulin
Horseradish peroxidase
Ascorbate oxidase
(Cucurbita pepo medullosa)
Yeast mannan
(Saccharomyces cerevisiae)
Glycogen (rabbit)
62.5
125
1.95
31.3
31.3
3.90
1.95
N (500)
N (500)
N
(500)
N (1,000)
Minimum cone, necessary for complete inhibition of hemagglutinating activity with a titer of 4 are shown.
N: No inhibition at the concentration shown in parentheses.
nose N-linked sugar chains. By contrast, plant glycoproteins with complex N-linked sugar chains had no inhibitory
effects.
Discussion
One group of lectins in monocotyledons exclusively recognizes D-mannose or mannooligosaccharides (Van
Damme et al. 1988, 1991b, 1994, 1995). No members of
this group reported to date have been shown to bind glucobioses. In this study, we showed that a lectin from N. flandria bound most of glucobioses tested, in addition to D-glucose and N-acetyl D-glucosamine (Table 2). Allen et al.
(1976) reported the strong inhibitory effects of glucobioses
(trehalose, kojibiose, maltose, isomaltose and sophorose)
on the hemagglutinating activity of four mannose/glucosespecific lectins from legume seeds. Unlike these lectins,
NFL did not bind kojibiose (al ,2) and it bound maltose
(al,4) and sophorose 081,2) only weakly. However, in
assay of the inhibition by mannosaccharides of hemagglutination, NFL appeared to be similar to the agglutinin
from garlic (ASA; Kaku et al. 1992), with strong inhibition
by Manctl,3Man.
A possible role of mannose-specific lectins in monocotyledons was proposed for ASA and GNA, which protected
plants against chewing insects in feeding experiments, and
the toxicity of GNA toward sucking insects was also demonstrated in experiments with transgenic plants (Peumans and
Van Damme 1995). If the specificity of these lectins toward
mannosaccharides is reflected in their effects against insects, NFL might behave in intact plants in a similar man-
A lectin from leaves of Neoregelia flandria
ner to ASA. Moreover, NFL has the strong affinity for
trehalose, which is found in fungi and some insects. From
these results, we can speculate that NFL might disrupt the
metabolism of trehalose in such organisms. The broad
carbohydrate-binding specificity of NFL might be advantageous when NFL functions as a defense protein. Plants
adapted to their environments as a consequence of interactions with many other surrounding organisms. The carbohydrate-binding specificity of NFL might have been
changed from those of other mannose-specific lectins during the adaptation of N. flandria to the environment.
NFL did not recognize horseradish peroxidase and
ascorbate oxidase (Table 3), which have the xylose/fucose- or xylose-containing N-linked chains, respectively
(McManus et al. 1988 and d'Andrea et al. 1988). However,
mannooligosaccharides have also been reported as N-linked carbohydrate chains of plant glycoproteins (Misaki and
Goldstein 1977, Kimura et al. 1988). At present, we do not
know the nature of carbohydrate chains of glycoproteins in
the leaves of N.flandria. Accordingly, it is unknown
whether or not N. flandria has endogenous receptors for
NFL.
Hester et al. (1995) established the three-dimensional
structure of a complex of Me-a-D-mannoside and GNA,
which is a typical example of a mannose-specific lectin
from monocotyledons. The characteristics of the structure
were found to be different from those of Con A and an agglutinin from Pisum sativum L. In the structures of mannose/glucose-specific lectins from legumes, no amino acid
residue is hydrogen-bonded to the proton of the C-2 hydroxyl group of mannose, whereas Asn 91 and Asp 93 are
hydrogen-bonded to the C-2 hydroxyl group in GNA.
These hydrogen bonds were shown to be important for
fixing the axial hydroxyl group at C-2 of the mannose molecule. Our amino acid analysis showed that the level of Asx
in NFL was very low compared with that in other mannosespecific lectins from monocotyledons. We did not determine the sequence beyond 39th residue from the N-terminus in this study, and it is possible that the entire
sequence of NFL might be rather different from those of
other lectins. In most of the cDNA sequences for lectins
from monocotyledons reported by Van Damme et al.
(1991a, 1994, 1995), the residues corresponding to Asn 91
and Asp 93 in GNA are conserved. However, as deduced
from a cDNA of a lectin from Araceae (Van Damme et al.
1995), these residues can be replaced by other amino acid
residues. In this connection, we note that Allen (1995)
reported a lectin from tubers of Arum maculatum that recognized N-acetyllactosamine. The molecular structure of
this lectin from Arum has not been elucidated and the relationship between the lectin and mannose-specific lectins is
unknown. It is likely that new carbohydrate-binding specificities will be found for lectins that are homologous to
the mannose-specific lectins from monocotyledons.
1011
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(Received May 17, 1996; Accepted August 19, 1996)