From the Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India
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ABSTRACT |
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Two mannose-binding lectins, Allium
sativum agglutinin (ASA) I (25 kDa) and ASAIII (48 kDa), from
garlic bulbs have been purified by affinity chromatography followed by
gel filtration. The subunit structures of these lectins are different,
but they display similar sugar specificities. Both ASAI and ASAIII are
made up of 12.5- and 11.5-kDa subunits. In addition, a complex (136 kDa) comprising a polypeptide chain of 54 ± 4 kDa and the
subunits of ASAI and ASAIII elutes earlier than these lectins on gel
filtration. The 54-kDa subunit is proven to be alliinase, which is
known to form a complex with garlic lectins. Constituent subunits of
ASAI and ASAIII exhibit the same sequence at their amino termini. ASAI and ASAIII recognize monosaccharides in mannosyl configuration. The
potencies of the ligands for ASAs increase in the following order:
mannobiose (Man1-3Man) < mannotriose (Man
1-6Man
1-3Man)
mannopentaose
Man9-oligosaccharide. The addition of
two GlcNAc residues at the reducing end of mannotriose or mannopentaose
enhances their potencies significantly, whereas substitution of both
1-3- and
1-6-mannosyl residues of mannotriose with GlcNAc
at the nonreducing end increases their activity only marginally.
The best manno-oligosaccharide ligand is
Man9GlcNAc2Asn, which bears several
1-2-linked mannose residues. Interaction with glycoproteins
suggests that these lectins recognize internal mannose as well as bind
to the core pentasaccharide of N-linked glycans even when
it is sialylated. The strongest inhibitors are the high
mannose-containing glycoproteins, which carry larger glycan chains.
Indeed, invertase, which contains 85% of its mannose residues in
species larger than Man20GlcNAc, exhibited the highest
binding affinity. No other mannose- or mannose/glucose-binding lectin
has been shown to display such a specificity.
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INTRODUCTION |
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The majority of the well characterized plant lectins have been
isolated from the seeds of dicotyledonous species. But lectins of
non-seed origin from other species are also emerging as promising tools
chiefly because of two reasons: (i) a good number of them might contain
novel sugar-binding sites; and (ii) they can provide valuable
information regarding the biological roles of plant lectins, which to a
large extent still remain elusive. In the recent past, there have been
several reports of non-seed lectins from monocotyledonous families
(1-3), especially Amaryllidaceae. The most remarkable property of
these lectins is that they show strict specificity for mannose (2, 4,
5), unlike other mannose/glucose-binding plant lectins. Hence, they are
being used extensively as affinity ligands for the purification of
glycoproteins, viz. IgM, 2-macroglobulin, haptoglobin, and
-lipoprotein (3, 6).
Van Damme et al. (3) examined a number of species (including Allium sativum) from the family Alliaceae (which is taxonomically close to the family Amaryllidaceae) and found them to accumulate mannose-binding lectins. They observed that lectins from both families share many common properties like their state of oligomerization, sugar specificity, amino acid composition, and serological interaction. We note that the bulbs of the species A. sativum contain an additional lectin (other than the one(s) described by them) that differs in its quarternary structure.
We found that the garlic lectins bind most avidly to invertase (which
contains high mannose residues) among the glycoproteins tested. By
exploiting this property, we developed a simple method to study their
sugar specificities. This study reveals that the binding sites of
these lectins accommodate a number of 1-2-linked mannose residues.
None of the other mannose-binding lectins have been shown to exhibit
this kind of specificity.
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MATERIALS AND METHODS |
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Sugars and Glycoproteins--
Methyl--mannose, mannose,
N-acetylmannosamine,
- and
-methylumbelliferyl
mannose, glucose, methyl-
-glucose, glucosamine, N-acetylglucosamine, galactose,
N-acetylgalactosamine, lactose, melibiose, maltose, altrose,
talose, and Man
1-4Man were purchased from Sigma. Man
1-2Man and
Man
1-3Man were obtained from Carbohydrate International.
Man
1-6Man, Man
1-3(Man
1-6)Man, mannopentaose, Man
1-4GlcNAc, GlcNAc2Man3, and
N-acetyllactosamine were purchased from Dextra Laboratories
(London). Man2GlcNAc, Man5GlcNAc, and Man3GlcNAc2 were obtained from Biocarb (Lund,
Sweden). Man9GlcNAc2Asn was a gift from Dr.
M. I. Khan (National Chemical Laboratory, Pune, India).
Man8GlcNAc2Asn and
Man7GlcNAc2Asn prepared from quail ovalbumin
were available in the laboratory from a previous study (7). Invertase,
fetuin, transferrin, IgGs, fibrinogen,
1-acid
glycoprotein, and ovalbumin were products of Sigma. Soybean agglutinin
and jacalin (Artocarpus integrifolia agglutinin) were prepared in the laboratory according to Swamy et al. (8) and Sureshkumar et al. (9), respectively. Glucose oxidase was
purchased from Boehringer Mannheim. All other chemicals used were of
the highest purity available.
Preparation of Mannose-Sepharose Affinity Matrix-- Mannose was coupled to epichlorohydrin-activated Sepharose 6B following the procedure of Sundberg and Porath (10).
Purification of A. sativum Agglutinins (ASAs)1-- Healthy dry bulbs of A. sativum were purchased from the local market. The bulbs were homogenized with a blender using 20 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS). The extract was filtered and centrifuged at 10,000 rpm. The supernatant was subjected to (NH4)2SO4 cut (70%), and centrifuged, and the protein pellet was resuspended in PBS and dialyzed extensively against PBS. The crude protein was loaded on the mannose-Sepharose column at 4 °C. The column was then washed extensively at the same temperature with PBS until A280 nm was below 0.005. The bound protein was eluted with 0.2 M mannose in PBS at room temperature.
The affinity-purified protein was dialyzed extensively initially against 20 mM PBS and finally against 50 mM PBS and concentrated using a Centricon filtration unit. The concentrated protein was then applied to a Bio-Gel P-200 column (1.8 × 110 cm) equilibrated and eluted with 50 mM PBS.Electrophoretic Procedures--
SDS-polyacrylamide gel
electrophoresis under reducing conditions was carried out as described
by Laemmli (11). Molecular masses of the lectins were calculated
according to the method of Weber and Osborn (12) using lysozyme (14.2 kDa), -lactoglobulin (18.4 kDa), trypsinogen (24 kDa), hen egg
ovalbumin (45 kDa), and bovine serum albumin (68 kDa) as the standards.
The proteins were visualized on the gel by Coomassie staining. A P/ACE
system 2100 (Beckman Instruments) was used for capillary
electrophoresis with P/ACE system software controlled by an IBM PS/2
Model 50-Hz computer. Post-run data analysis was performed on System
Gold software (Beckman Instruments). A standard capillary of 27 cm length (20 cm to the detector window) × 20 µm inner diameter, designed for P/ACE cartridges, was obtained from Beckman Instruments. On-line detection was set at 280 nm with a 50 × 200-µm aperture in the P/ACE cartridge. Temperature of the capillary during
electrophoresis was maintained at 25 °C. Samples were introduced by
pressure injection for 5 s. Electrophoresis was performed at a
constant voltage of 8 kV.
Determination of the Amino-terminal Sequence-- Proteins after separation by 15% SDS-PAGE were electroblotted onto polyvinylidene difluoride membrane following the procedure of Matsudaira (13) using a Multiphor II electrophoresis system (Pharmacia Biotech Inc.). N-terminal analysis of the native protein was carried out on a Shimadzu automated gas-phase sequencer (Model PSQ-1) equipped with an on-line C-R4A120A Chromatopac Shimadzu phenylthiohydantoin analyzer.
Determination of Native Molecular Mass-- The native molecular masses of ASAs were determined by gel filtration on a Bio-Gel P-200 column (1.8 × 110 cm) calibrated with rabbit IgG, Vicia villosa agglutinin, soybean agglutinin, bovine serum albumin, hen egg ovalbumin, and pepsin. Void and inner volumes of the column were determined with blue dextran and myoglobin, respectively. Molecular masses of ASAI and ASAIII were also determined on a Bio-Gel P-60 column (8 × 90 cm) calibrated with hen egg ovalbumin, chymotrypsinogen, myoglobin, and bovine pancreas ribonuclease A.
Protein Estimation-- Protein concentration was determined following the method of Lowry et al. (14) using bovine serum albumin as the standard.
Sugar Assay-- Total neutral sugar content was determined by the phenol-sulfuric acid method of Dubois et al. (15) using mannose as the standard.
Hemagglutination Assay-- Hemagglutination was carried out at room temperature using rabbit and human erythrocytes (16). Hemagglutination inhibition tests were done by preincubating lectin (10 hemagglutinating units) with serially diluted sugars or glycoproteins in microtiter plates. Rabbit erythrocyte suspension (25 µl of 4% (v/v)) was then added to the solution, and the results were noted after 1 h.
Estimation of the Activity of Invertase Bound to ASAs-- An appropriate amount of lectin was coated on an enzyme-linked immunosorbent assay plate and left overnight at 4 °C. The plate was washed with 20 mM PBS and then blocked with 3% bovine serum albumin in PBS. After washing with the blocking buffer, invertase was added to the wells and incubated for 1 h. The plate was then washed with PBS, and 100 µl of 50 mM sucrose solution was added to each well. After incubation for 1 h, the solutions were transferred to separate test tubes. The extent of hydrolysis of sucrose was assayed by estimating the free glucose according to the method of Nelson (17).
Enzyme-linked Lectin Absorbent Assay-- The sugar binding properties of ASAs were studied in detail using a modified enzyme-linked immunosorbent assay technique (Scheme 1). An optimum concentration of the lectin was coated on enzyme-linked immunosorbent assay plates and left overnight at 4 °C. After washing three times with 20 mM PBS, the wells were blocked with 3% bovine serum albumin for 1 h at room temperature. Subsequent to washing thrice with the blocking buffer, different sugars were added at varying concentrations and allowed to interact with the lectin for 1 h. A fixed concentration of invertase was added to each well and incubated for 30 min. The wells were then washed thrice with blocking buffer and once with PBS. An equal volume of 50 mM sucrose solution in 100 mM acetate buffer (pH 5.0) was added to each well. After incubation (for 1 h), glucose oxidase in the same buffer was then added to the solution to produce H2O2 from the liberated glucose. H2O2 thus generated was assayed using horseradish peroxidase and o-phenylenediamine (0.05 mg/well in 100 mM citrate buffer). The reaction was stopped by 2 N HCl, and the absorbance was recorded on an enzyme-linked immunosorbent assay reader at 490 nm.
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RESULTS |
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Bulbs of A. sativum Contain a Group of Mannose-binding Lectins-- Earlier reports (3) have shown that dimeric proteins of 25 kDa occur in the bulbs of A. sativum. By using a modified purification procedure, we have identified an additional lectin designated as ASAIII. The affinity-purified preparation revealed three peaks upon gel filtration on a Bio-Gel P-200 column with molecular masses of 136, 48, and 25 kDa, respectively (Fig. 1). Taken together, the data from SDS-PAGE (Fig. 2) and gel filtration show that peak 3 is a heterodimer of 12.5- and 11.5-kDa subunits, whereas peak 2 is most likely a heterotetramer made up of two pairs of 12.5- and 11.5-kDa polypeptide chains, although occurrence of these subunits in other proportions cannot be ruled out.
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Hemagglutination Properties-- The agglutinins ASAI and ASAIII interact strongly with rabbit erythrocytes, but considerably weakly with human erythrocytes, irrespective of their blood groupings.
ASAs Bind Most Avidly to Invertase--
Hemagglutination
inhibition studies of garlic agglutinins were carried out using a
series of simple sugars and several glycoproteins. Among the
monosaccharides tested, only methyl--D-mannopyranoside was found to be inhibitory besides mannose, although the latter was
less potent as an inhibitor (Table I).
Glucose, a C-2 epimer of mannose, was inactive.
Methyl-
-D-glucopyranoside also did not interact.
Replacement of the C-2 hydroxyl group of glucose with other groups did
not alter its inhibitory property as both N-acetyl-D-glucosamine and glucosamine were
inactive. Of all the glycoproteins used in this assay, invertase was
the strongest inhibitor, and the minimum amount of this enzyme needed
for complete inhibition was 0.7 nM (Table I). The invertase
bound to both ASAI and ASAIII was found to retain its catalytic
activity.
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Inhibition of ASA-Invertase Binding by Mono- and
Disaccharides--
Sugar inhibition assays were carried out in
triplicate, and each value is an average of three experiments. The
amount of sugar required for 50% inhibition was calculated from
complete inhibition curves (Fig. 5), and
values are listed in Tables I-III.
Relative affinities of the lectins for different sugars were
determined from the concentration of
sugars required for 50% inhibition of the binding of invertase.
Consistent with hemagglutination inhibition studies,
methyl--D-mannopyranoside was better as an inhibitor than mannose (Tables I and II). The presence of nonpolar
p-nitrophenyl aglycon in D-mannose did not
improve the binding affinity, but the introduction of a nonpolar
4-methylumbelliferyl aglycon at the anomeric position in
-linkage
slightly enhanced its inhibitory potency. The lectins did not bind to
4-methylumbelliferyl-
-mannopyranoside, indicating that
-linked
mannose was not conducive for binding. N-Acetyl-D-mannosamine was inactive, suggesting
that the axially oriented hydroxyl group of mannose cannot be
substituted with a bulky acetamido group. The monosaccharide binding
propensities of ASAI and ASAIII are broadly similar to other well
studied mannose-binding lectins from snowdrop (4) and daffodil and
amaryllis (21). Of all the mannobioses tested, Man
1-3Man was the
most potent inhibitor. Its potency was 12 times greater than that of
mannose. Man
1-2Man and Man
1-6Man were almost eight and six
times more active, respectively, over mannose. But the other
mannobioses, including Man
1-4Man, were poor ligands. Among the
other mannose-binding lectins, GNA recognizes only terminal
Man
1-3Man, whereas NPA (daffodil) and HHA (amaryllis) prefer
1-6-linked mannose. On the other hand, ConA is known to exhibit
greater affinity for Man
1-2Man (22). Artocarpin (A. integrifolia mannose-binding lectin) shows higher specificity for
Man
1-3Man (7). The
-linked disaccharides like Man
1-4GlcNAc
and GlcNAc
1-2Man were poor inhibitors, whereas Man
1-6GlcNAc and
GlcNAc
1-6Man were inactive toward ASAs. The lectins did not
interact with disaccharides like lactose (Gal
1-4Glc), N-acetyllactosamine (Gal
1-4GlcNAc), and melibiose
(Gal
1-6Glc).
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Binding Specificities of ASAI and ASAIII for
Manno-oligosaccharides--
To understand the carbohydrate
specificities of ASAI and ASAIII in greater detail, their binding to a
carefully chosen panel of manno-oligosaccharides was then undertaken
(Table II). The core structure of N-linked oligosaccharides,
Man1-3(Man
1-6)Man, was 30 times stronger an inhibitor than
mannose and showed four, two, and five times more potency than
Man
1-2Man, Man
1-3Man, and Man
1-6Man, respectively. The
relative inhibitory potencies of mannopentaose and mannotriose were
identical. Man5GlcNAc and Man
1-3Man
1-4GlcNAc were
marginally better ligands than mannopentaose and Man
1-3Man,
respectively, unlike artocarpin, in which the addition of GlcNAc at the
reducing end of Man
1-3Man caused a dramatic enhancement of its
binding ability (7).
Interaction of ASAI and ASAIII with Glycoproteins-- The ability of several mannose-containing glycoproteins to inhibit the binding of ASAI or ASAIII to invertase was checked to confirm their specificities. The extent of binding of these glycoproteins was qualitatively consistent with the relative affinities exhibited by manno-oligosaccharides.
The soybean lectin and ovalbumin, which bear several terminal Man ![]() |
DISCUSSION |
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The high mannose-binding lectins (ASAI and ASAIII) from garlic bulbs were purified in two steps using affinity chromatography and gel filtration. Van Damme et al. (3) reported a mannose-binding lectin that resembles ASAI in its subunit composition. However, it differs from ASAI reported by them in displaying the same amino-terminal sequence for both of the polypeptide chains. ASAI and ASAIII are the heterodimer and heterotetramer, respectively, of similar polypeptide chains, present in equimolar proportion, that vary slightly in their molecular masses, viz. 12.5 and 11.5 kDa, yet the N-terminal sequences of their large (12.5 kDa) and smaller (11.5 kDa) subunits are identical. Despite the dissimilarities in their subunit compositions, the contours of the carbohydrate-binding sites of ASAI and ASAIII are identical. The biosynthetic and functional significance of the occurrence of lectins that differ in their subunit composition but display similar binding propensities within the same tissue is not presently understood. Our failure to isolate ASAII as a homodimer of 12-kDa subunits is perhaps related to differences in the purification methods used by us and by Van Damme et al. (3). It is also possible that our isolation procedure permits the purification of isolectins that have high propensities for interaction with alliinase (20). To ascertain their biological function and to develop them as potential tools for research, a knowledge of the carbohydrate specificities of these lectins becomes imperative. The sugar specificities of ASAI and ASAIII were elucidated in two steps: hemagglutination inhibition and a coupled enzyme-based assay. The hemagglutination inhibition study confirmed its exclusive specificity for D-mannose, like three other Amaryllidaceae lectins (GNA, NPA, and HHA) that do not recognize D-glucose (4, 17). Our studies also show that these lectins display extraordinary avidity for invertase as compared with other glycoproteins tested. Based on this finding, a sensitive enzyme-based assay system was designed to investigate their detailed binding specificities.
The subunit molecular mass of one of the lectins isolated by Gupta and Sandhu (31) is similar to that of the larger subunit of peak 1 on the gel filtration column. However, unlike the alliinase-ASA complex or ASAI and ASAIII, the former is not retained on a mannose-Sepharose matrix. In the absence of the N-terminal sequence and comprehensive sugar binding properties of the high molecular mass lectin reported in Ref. 31, it is not possible to conclude that it is derived from another isolectin rather than being a glycoprotein contaminant, viz. alliinase. Garlic plant contains at least five different lectins and lectin genes (32). The processing and post-translational modifications of the primary translation products of monocot mannose-binding lectins are rather complex as evident from the report of several lectins with different molecular masses (33).
Compared with other mannose-binding lectins, ASAI and ASAIII bind to
mannose very weakly. Methyl--D-mannopyranoside is
six times better an inhibitor than mannose. The relative potencies of
D-mannose and its epimers suggest that the equatorial
orientation of the hydroxyl groups at C-3, C-4, and C-6 and an axial
hydroxyl group at C-2 as in mannose are necessary for interaction with these lectins. Compared with other mannose-binding lectins, some of the
mannobioses show much higher potency than mannose. Of all the
mannobioses tested, Man
1-3Man exhibited the highest affinity as
found by Kaku et al. (34), but unlike the present result, they recorded a lower potency of trimannoside than Man
1-3Man. The
difference might be attributed to the different techniques used.
(Although assay-dependent differences in binding affinities are not common, they have, however, been seen in some instances. For
example, GNA shows altered affinities for murine IgM under different
assay conditions such as in a precipitation assay and in immobilized
form (34).)
The observation that ASAs are complementary to 1-3-mannosyl units
merits reconsideration as (i) extension of
1-3Man as in Man
1-3Man
1-3Man
1-3Man
1-2Man-ol diminishes its binding
ability (34); (ii) the affinity increases in the order of
Man
1-3Man < mannotriose
mannopentaose
Man9-oligosaccharides; and (iii) it may not be an
1-3-linked mannopyranosyl residue, but the number of residues in
1-2-linkage at the nonreducing end as discussed subsequently that
determine higher affinity of manno-oligosaccharides. This is evident by
the dramatic increase in the potency of oligosaccharides that carry
increasing numbers of
1-2-linked mannose residues. Unlike ASAI and
ASAIII, mannotriose is the most complementary inhibitor for ConA,
whereas artocarpin displays only slightly higher affinity for
mannopentaose over mannotriose. When the reducing end of Man
1-3Man
and mannopentaose was substituted with a GlcNAc residue, no appreciable
change in activity was observed. For artocarpin, the same substitution
led to an enhancement in potency by severalfold (7). The reducing end
GlcNAc in ASAs is probably accommodated adjacent to the primary binding
site without involving any significant interactions with the lectin.
When the terminal mannose residues of mannotriose are substituted with
two GlcNAc residues as in GlcNAc2Man3, it leads
to some improvement in activity, suggesting that the combining site of
ASAs can access the masked/internal mannose residues of the
oligosaccharides. On the other hand, the addition of two GlcNAc
residues in
1-4-linkage at the reducing end of mannotriose
(i.e. the core pentasaccharide of N-linked glycans) improves the affinity by five to six times. These observations are in accordance with the speculation, made by Barre et al.
(33), that the mannose-binding site of the monocot lectins is part of a
more extended site, which explains the stronger binding of ASAI and
ASAIII to complex glycans. Interestingly enough, the extension of the
core trimannosidic structure by
1-2-linked mannosyl residues as in
the glycopeptides of quail ovalbumin and soybean lectin leads to
increased potencies. This enhancement also highlights the preference of
garlic lectins for a cluster of
1-2-linked mannose residues.
The mode of interaction of ASAs with the glycoproteins studied
substantiates the above findings. The ability to recognize internal
mannose and the affinity for core pentasaccharide were, once again,
proved through the interaction of ASAs with some glycoproteins. It
appears that the binding site can withstand the terminal sialic acids
as well as the penultimate galactose residues, although the removal of
sialic acids enhances potency. Sheep IgG and jacalin displayed moderate
binding potencies compared with other glycoproteins. Since the
substitution of reducing end GlcNAc with 1-6-linked fucose as in
Man3GlcNAc2Fuc is impervious to binding, the
relatively weak interaction of jacalin, horseradish peroxidase, and its
oligosaccharide as well as goat IgG appears to be due to the
substitution of the
-linked 3,6-disubstituted mannose with xylose in
1-2-linkage (as in jacalin and horseradish peroxidase) and its
substitution with GlcNAc in
1-4-linkage (as in sheep IgG). Using a
different protocol (surface plasmon resonance analysis), Barre et
al. (33) found that immobilized fetuin and asialofetuin do not
bind to garlic lectins (viz. ASAI and ASAII). But
hemagglutination and the enzyme-based assay reported here show that
garlic lectins (ASAI and ASAIII) can bind to both fetuin and
asialofetuin. Surface plasmon resonance analysis in the concentration
range used appears to have failed to detect this interaction because of
their moderate affinities. Notwithstanding the identity of the
polypeptide chain in the lectin preparation reported by Gupta and
Sandhu (31), they had purified the garlic lectin(s) using an
asialofetuin-silica affinity column, confirming that they indeed are
able to bind glycoproteins such as asialofetuin.
Invertase was the strongest glycoprotein ligand. This is attributed to
the presence of several 1-2-linked mannose residues. Invertase
contains nine N-linked high mannose oligosaccharides, seven
of which are accessible. About 85% of the mannose of the accessible
oligosaccharides is in species larger than Man20GlcNAc (30). We believe that the availability of these high mannose oligosaccharides with several
1-2-linked mannose residues at their
nonreducing ends is instrumental for the observed potency of ASAs. This
is also borne out by the inhibitory activities of the glycopeptide from
soybean agglutinin. From molecular modeling studies, it is suggested
(33) that the garlic lectins, like their counterparts in the monocot
mannose-binding lectin family, possess three identical mannose-binding
sites per monomer and that the mannose-binding site is part of a more
extended site. As a result, these lectins are expected to accommodate a
larger number of mannose residues. This explains, at least in part, (i) the comparatively low affinity for the monosaccharide (mannose), (ii)
the increased affinity with increasing numbers of mannose residues, and
(iii) the enhanced avidity for high mannose-containing oligosaccharides/glycoproteins. Although there is no report on the
interaction of high mannose-containing glycoproteins, such as
invertase, with other members of this lectin family, at least one
member (bulb lectin from Allium cepa) demonstrates identical affinity for invertase.3 The
interaction of high mannose-containing oligosaccharides/glycoproteins and other larger glycans with the members of the monocot
mannose-binding lectin family constitutes a novel specificity among
lectins studied to date. If compared with the mannose-binding (GNA,
NPA, and HHA) and mannose/glucose-binding (ConA and artocarpin)
lectins, the topology of the binding site(s) of ASAs would appear quite
distinct. GNA recognizes only terminal
1-3-linked mannose residues.
NPA and HHA interact with both the terminal and internal mannosyl residues, but the best inhibitors of NPA and HHA are
1-6-linked mannotrioses and oligosaccharides with
1-3- or
1-6-mannose
residues, respectively. ConA displays high affinities for
oligosaccharides containing
1-2-linked mannose, but its binding
site is most complementary to mannotriose. Artocarpin does not
recognize
1-2-linked high manno-oligosaccharides and is most
complementary to Man3GlcNAc2Fuc containing
a xylose
1-2-linked to the 3,6-disubstituted core mannose. In
conclusion, our studies illustrate that the exquisite specificity of
lectin-glycoprotein enzyme interaction when coupled with the catalytic
power of an enzyme provides a simple and sensitive method for
elucidating the carbohydrate recognition propensities of lectins. The
ability of ASAs to bind high mannose-containing oligosaccharides and
glycoprotein with enhanced potencies places them in a unique position
among the mannose-binding lectins reported so far. This specificity of
ASAs can be utilized for several biochemical studies, viz.
biosynthesis and functional aspects of high mannose oligosaccharides
and purification of high mannose-containing glycoproteins like
invertase and carboxypeptidase Y.
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FOOTNOTES |
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* This work was supported by a grant from the Departments of Science and Technology and Biotechnology, Government of India.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Post-doctoral Fellow in the Core Support Program in Drug and
Molecular Design supported by a grant from the Department of Biotechnology, Government of India (to the Indian Institute of Science).
§ To whom correspondence should be addressed. Fax: 91-080-3341683.
1 The abbreviations used are: ASAs, A. sativum agglutinins; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; GNA, Galanthus nivalis agglutinin; NPA, Narcissus pseudonarcissus agglutinin; HHA, Hippeastrum hybr. agglutinin; ConA, concanavalin A.
2 Ovalbumin as a glycoprotein is highly heterogeneous, and individual molecules in a population exhibit either high mannose chains that vary in the extent of mannosylation or hybrid-type chains.
3 T. K. Dam and A. Surolia, unpublished observation.
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REFERENCES |
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