Purification and characterization of a sialic acid specific lectin from
the hemolymph of the freshwater crab
Paratelphusa jacquemontii
Maghil Denis, P. D. Mercy Palatty, N. Renuka Bai and S. Jeya Suriya
Department of Zoology, Holy Cross College, Rochnagar, Nagercoil Tamil Nadu, India
A naturally occurring hemagglutinin was detected in the
serum of the freshwater crab, Paratelphusa jacquemontii
(Rathbun). Hemagglutination activity with different mam-
malian erythrocytes suggested a strong affinity of the serum
agglutinin for horse and rabbit erythrocytes. The most
potent inhibitor of hemagglutination proved to be bovine
submaxillary mucin. The lectin was purified by affinity
chromatography using bovine submaxillary mucin-coupled
agarose. The molecular mass of the purified lectin was
34 kDa as determined by SDS/PAGE. The hemagglutina-
tion of purified lectin was inhibited by N-acetylneuraminic
acid but not by N-glycolylneuraminic acid, even at a
concentration of 100 m
M
. Bovine submaxillary mucin,
which contains mainly 9-O-acetyl- and 8,9 di-O-acety-N-
acetyl neuraminic acid was the most potent inhibitor of the
lectin. Sialidase treatment and de-O-acetylation of bovine
submaxillary mucin abolished its inhibitory capacity com-
pletely. Also, asialo-rabbit erythrocytes lost there binding
specificity towards the lectin. The findings indicated an
O-acetyl neuraminic acid specificity of the lectin.
Keywords: Paratelphusa jacquemontii; hemolymph; lectin;
sialic acid; O-acetylsialic acid.
Lectins are sugar-specific proteins with multiple combining
sites capable of agglutinating cells or precipitating glyco-

Of the known lectins that have been purified and
characterized few bind sialic acid [20–24]. Sialic acid specific
lectins have been found in several species of crustaceans,
namely Homarus americanus [25], Macrobrachium rosen-
bergii [26], Cancer antennarius [12], Scylla serrata [24] and
Penaeus monodon [27]. The lectin from Scylla serrata
was NeuGc specific [24] and that from Cancer antennarius
was 9-O-NeuAc and 4-O-NeuAc specific [12,28]. Lectins
with defined specificity for different kinds of sialic acids and
their glycosidic linkages could form a library of potential
diagnostic tools for identifying sialyl epitopes in pathogenic
bacteria [29] and malignant tumor cells.
Here we report purification of the lectin from the
hemolymph of P. jacquemontii by affinity chromatography
on bovine submaxillary mucin (BSM) agarose. The binding
specificity of the lectin was also studied.
Experimental procedures
Materials
Polypropylene Econo Columns were purchased from Bio-
Rad. CNBr-activated Sepharose 4B, bovine submaxillary
mucin, porcine stomach mucin, bovine and porcine thyro-
globulin, fetuin, transferrin, N-acetyl mannosamine, gluco-
samine and galactosamine, lactose, glucose-6-phosphate,
sucrose, fucose, glucose, fructose, xylose, raffinose, treha-
lose, melibiose, N-glycolyl- and N-acetylneuraminic acids,
Correspondence to M. Denis, Department of Zoology, Holy Cross
College, Rochnagar, Nagercoil ) 629001, Tamil Nadu, India.
Tel.: +91 98421279184,
2
E-mail: [email protected]

5
The following buffers were used in this study: NaCl/Tris/
CaCl
2
6
(50 m
M
Tris/HCl, pH 7.5, 100 m
M
NaCl, 10 m
M
CaCl
2
), NaCl/Tris/BSA [pH 7.5 containing 0.05% (v/v)
BSA], NaCl/P
i
(10 m
M
sodium phosphate, pH 7.0, 0.15
M
NaCl), high salt buffer (HSB; 50 m
M
Tris/HCl, pH 7.5, 1
M
NaCl, 10 m
M
CaCl
2
), low salt buffer, (LSB; 50 m
M

M
HCl,
(pH 8.3)
8
, was added and gently mixed for an additional
hour. The adsorbent was washed thoroughly by three cycles
of alternating pH. Each cycle consists of a wash at pH 4.0
(0.1
M
acetate buffer containing 0.5
M
NaCl) followed by a
wash at pH 8.0 (0.1
M
Tris containing 0.5
M
NaCl)
9
.The
BSM–agarose was stored in cold NaCl/Tris, pH 7.5,
containing 0.02% (w/v) sodium azide. Approximately
70–80% of the BSM was coupled.
Purification of lectin from the hemolymph
of
P. jacquemontii
Clarified serum (10 mL) was applied to 3 mL of BSM–
agarose in an Econo Column (Bio-Rad) previously
equilibrated with NaCl/Tris at 4 °C. The eluant was
collected at a rate of 0.6 mLÆmin
)1

in the fractions. The fractions that contained significant
amount of lectin were pooled and dialysed against 1 m
M
CaCl
2
,at4°C for 18 h, and then for 3 h with fresh 1 m
M
CaCl
2
. The dialysate was then aliquoted, lyophilized
andstoredat)20 °C. The elution profile (Fig. 1) and a
summary of purification (Table 1) give an overall view on
the lectin purification.
Erythrocyte preparation
Blood for HA assay was prepared as described by
Ravindranath et al. [12].
Hemagglutination assay
Hemagglutination assays were performed in microtiter
plates (Falcon) as recommended for a crab hemolymph
lectin [28].
Fig. 1. BSM-affinity elution profile. The affinity column was prepared
using a polypropylene Econo Column (0.8 · 4 cm). BSM–agarose was
equilibrated with NaCl/Tris containing 10 mm CaCl
2
at 4 °C. The
clarified serum (10 mL) was applied and washed with NaCl/Tris
containing 1
M
NaCl and 10 mm CaCl
2

Preparation of asialo-erythrocytes. The procedure fol-
lowed for preparing asialo-erythrocytes was that of Mercy
and Ravindranath [24].
Sialidase treatment of sialoglycoprotein. Asialo-glycopro-
teins were prepared by incubating 2 mg of glycoprotein with
0.1 unit of C. perfringens sialidase (Type X) in 400 lLof
5m
M
acetate buffer pH 5.5 for 3 h at 37 °C. As a control,
glycoproteins were treated similarly without sialidase. The
HAI assay was performed with purified lectins for treated
and untreated glycoproteins against 1.5% of horse erythro-
cytes.
De-O-acetylated preparation of glycoproteins. De-O-
acetylation of glycoproteins was performed following the
procedures of Sarris and Palade [30] and Schauer [6]. A
solution of 750 lL of glycoprotein (5 mgÆmL
)1
) was added
to 250 lLof0.04
M
of NaOH, vortexed, incubated on ice
for 45 min and neutralized with 1 mL of 0.01
M
HCl,
respectively.
Polyacrylamide gel electrophoresis. SDS/PAGE (12.5%
slab gel) was performed according to Laemmli [31]. Samples
were heated for 3 min at 100 °C in sample buffer [25% (v/v)
1

erythrocytes revealed a striking correlation between the
presence O-acetylsialic acid and agglutination ability of the
Table 1. Purification of Paratelphusa jacquemontii lectin. The purification shown is from native hemolymph. Horse erythrocytes (1.5% NaCl/Tris,
0.05% BSA) were used for the hemagglutination assays. One unit of activity is defined as the amount of protein required to give one well of
hemagglutination.
SI No. Sample Volume (ml) Protein (mg) Total activity (HA units) Specific activity (HA unitÆmg
)1
) Purification
1 Serum 20 1040 2 · 10
5
196 1
2 Clarified serum 10 25 1 · 10
5
4096 20
3 Purified 20 1.0 4 · 10
5
409600 2000
Fig. 2. SDS/PAGE of purified lectin from the hemolymph of P. jac-
quemontii. A sample containing about 10 mg of protein from serum
(A) and 5 mg of BSM–agarose-purified lectin (B) was prepared for
electrophoresis as described in the Experimental procedures section.
The lectin was homogenous with the molecular mass of 34 kDa when
compared with standards (C) of known molecular mass (MW): bovine
serum albumin (66 kDa), glutamic dehydrogenase (55 kDa), ovalbu-
min (45 kDa) glyceraldehyde-3-phosphate dehydrogenase (36 kDa),
carbonic anhydrase (29 kDa), trypsinogen (24 kDa), trypsin inhibitor
(20 kDa), a-lactalbumin (14.2 kDa).
4350 M. Denis et al.(Eur. J. Biochem. 270) Ó FEBS 2003
lectin. Horse and rabbit erythrocytes, which have a high
contentof4-O-Ac-NeuAc and 9-O-Ac-NeuAc, respectively,

sialoglycoproteins differ in the composition of neuraminic
acid and its derivatives. Hence free sialic acids NeuAc and
NeuGc were tested as inhibitors of HA. NeuGc did not
inhibit HA whereas NeuAc inhibited HA (Table 4). To
further define the possible role of sialic acid as potent
inhibitor of lectin, the sialoglycoproteins were enzymatically
or chemically modified and their derivatives were examined
for HAI. Sialidase treatment of BSM abolished its inhi-
bitory properties completely (Table 6). This clearly points
Table 2. Correlation between the presence of O-acetyl groups on
erythrocytes and hemagglutination by P. jacquemontii lectin. Purified
lectins suspended in NaCl/Tris (pH 7.5) containing 0.05% BSA, were
serially diluted in microtiter plates and mixed with 25 lLofa1.5%
suspension of erythrocytes obtained from various mammalian species.
The HA titer was determined as the reciprocal of the highest dilution of
serum giving complete agglutination after 60 min at room temperature
(30–35 °C).
Erythrocyte
types
Position of major
O-acetyl group
a
O-acetyl sialic acid
content percentage
total
a
HA titer
Horse C-4 40 256
Rabbit C-9 < 20 128
Pig – – 16

The sugars selected for the study were reconstituted in NaCl/Tris to
100 m
M
concentration and 25 lL of each sugar was diluted serially in
microtiter plates and mixed with 25 lL of lectin previously adjusted to
2 HA units. After 60 min of incubation at room temperature
(30–35 °C), 25 lL of 1.5% suspension of horse erythrocytes were
added to each microtiter well and mixed. The values of the HAI titer
were determined after 60 min of incubation and expressed as the
highest dilution of sugars that inhibited the agglutination of erythro-
cytes. Mannose, Fructose, Xylose, Raffinose, Trehalose, and Melibiose
failed to inhibit hemagglutination of purified lectin.
Sugars HAI titer
Minimum concentration
required for
inhibition in m
M
Relative
inhibitory
potency (%)
ManNAc 32 3.125 100
GalNAc 16 6.25 50
Lactose 16 6.25 50
Glc6P 8 12.5 25
GlcNAc 8 12.5 25
Sucrose 4 25 12.5
Fucose 2 50 6.25
Glucose 2 50 6.25
NeuAc 2 50 6.25
NeuGc 0 < 100 > 6.25

O-acetyl sialic acid specificity. Inhibition studies with
glycoproteins and sugars were helpful in deriving the
binding affinity of the humoral agglutinin. BSM contains
the sialic acids, N-acetylneuraminic acid, N-glycolylneu-
raminic acid, N-acetyl 9-O-acetylneuraminic acid and,
8,9-di-O-acetylneuraminicacid[6]andprovedtobea
potent inhibitor. On the other hand PSM, which contains
90% (v/v) N-glycolylneuraninic acid, 10% (v/v) NeuAc and
traces of N-acetyl-O-acetyl neuraminic acid [39], showed
weak inhibitory potency. Moreover, free NeuAc could
inhibit haemagglutination but NeuGc had no inhibitory
potency. This explains the strong inhibitory potency of the
NeuAc-containing glycoprotein, BSM. Transferrin, PSM,
fetuin, bovine and porcine thyroglobulin, which are rich in
NeuGc are weak inhibitors. Moreover, the NeuAc-linked
glycoprotein oligosaccharides are more inhibitory than free
sialic acids. This can be attributed to the differences in
glycosidic linkages. BSM contains O-acetyl-NeuAc-a(2-
6)GalNAc (1–0) ser/thr-sequence while N-acetylneuraminic
acid occurs in a(2-3)-glycosidic linkage to galactose [41,52].
Evidently strong inhibitory potency of BSM was mainly due
to O-acetyl NeuAc showing a(2–6) linkage.
The sialic acid affinity of the Paratelphusa lectin was
further proved by its inability to inhibit sialidase-treated
BSM and bovine thyroglobulin. Also the lectin failed to
agglutinate desialylated rabbit erythrocytes. BSM, which
contains 85.5% NeuAc in 9-O-acetyl and 8,9di-O-acetyl
forms [41] on de-O-acetylation lost its inhibitory potency
completely, thus suggesting the importance of O-acetyl
NeuAc in the binding affinity of the lectin. The affinity for

Fetuin 32 156.25 0.006
Transferrin 128 39.06 0.0024
Table 6. Hemagglutination inhibition of purified lectin from the hemo-
lymph of P. jacquemontii by sialoglycoproteins before and after
de-O-acetylation and desialylation. Base treated BSM and bovine
thyroglobulin were neutralized before serial dilution. Enzyme buffer
controls were maintained for desialylation experiments.
Desialylated and de-O-acetylated glycoproteins HAI
BSM + sialidase 0
Bovine thyroglobulin + sialidase 2
Fetuin + sialidase 8
Transferrin + sialidase 16
BSM + 0.04
M
NaOH 4 °C 45 min 32
Bovine thyroglobulin + 0.01
M
NaOH 4 °C 45 min 0
4352 M. Denis et al.(Eur. J. Biochem. 270) Ó FEBS 2003
isolated from the hemolymph of the marine crab Cancer
antennarius [28] and Liocarcinus depurator [36]. The horse
shoe crab Limulus polyphemus [22] and slug Limax flavus
[23,40] lectin were inhibited by BSM but base treatment had
no influence on the inhibitory potency of L. polyphemus and
enhanced inhibition in L. flavus. On the other hand, lectin
from C. antennarius was inhibited by BSM and inhibition
was completely abolished on de-O-acetylation [12]. Thus
P. jacquemontii lectin resembled C. antennarius lectin in its
unique sugar specificity.
The presence of a single lectin is a unique feature among

O-acetyl sialic acid [8,64]. An O-acetyl sialic acid-specific
lectin isolated from Cancer antennarius [12] was used to
recognize the human melanoma tumor cells that contain
O-acetyl sialic acid [65]. The sialoglycoproteins on the cell
surface of leukemia erythrocytes show distinct alterations
and the differentiation between several leukemia erythro-
cytes was marked by a 9-O-acetyl sialic acid-specific lectin
purified from the hemolymph of the snail Achatina fulica
[64,66].
Lectins are used for verification of the sugar specificity of
the auto-antibodies found in the individuals reported to
have tumour [65]. Cell surface sialic acids of murine
erythroleukemia cells when transformed to 9-O-acetyl
derivatives can affect a variety of biological recognition
phenomena [66]. Besides this, lectins are valuable probes for
analyzing cell surface carbohydrates by cell agglutination,
and for studying immunofluorescence and staining of tissue
sections [51,67]. Clinical trials to inhibit cancer metastasis
and bacterial infections by blocking specific glycoconjugate
on the target cell surface using lectins are very promising
[68].
Taking the different applications that O-acetyl sialic acid-
specific lectins can be put to, it can be envisioned that
P. jacquemontii lectin may be used as a valuable tool in the
localization and assessment of the functions of glycocon-
jugates containing O-acetyl sialic acid.
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