Tài liệu Báo cáo khoa học: Purification and characterization of a sialic acid specific lectin from the hemolymph of the freshwater crab Paratelphusa jacquemontii pdf

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-

conjugates [1]. Lectins may recognize specifically the whole
sugar [2], a specific site in a sugar [3], a sequence of sugars [4]
or their glycosidic linkages [5] on cell-surface glycocon-
jugates, namely glycoproteins and glycolipids, or in bacter-
ial polysaccharides. Sialic acids are a family of sugars,
N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic
acid (NeuGc), with more than 20 derivatives, which differs
only in the acyl substitution of the C-5 amino group [6].
Most of the other sialic acids contain one or more O-acetyl
substitutions of the hydroxyl groups at C-4, C-7, or C-9.
The derivatives of sialic acid are very important constit-
uents of the cell surface. They are found at the outermost
ends of the sugar chain of animal glycoconjugate. They act
as an important component of the ligands recognized by the
lectins. Recognition can be affected by specific structural
variations and modifications of sialic acids and their linkage
to the underlying sugar [7]. The most common sialic acid
found in sialoproteins and gangliosides of human tissues is
NeuAc. Modified sialic acids are found in transformed or
neoplastic cells [8–10]. The type of sialic acid and the
glycosidic linkages with the adjacent sugars contribute to a
remarkable diversity of sialyl epitopes in the sialoconjugates
on the cell surface of neoplastic cells [11]. Human malignant
melanoma contains 9-O-acetyl-NeuAc [8,12] and in human
colon carcinoma tissues N-glycolylneuraminic acid [10,13]
as well as a2,6-linked sialic acids [11,14] were detected. Thus,
sialic acid-recognizing lectins would be of immense value in
identifying and discriminating sialic acids on the surface of
cancer cells. The sialic acid-specific lectins are also useful in
distinguishing highly pathogenic strains of bacteria [15–19].

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:

Abbreviations: HA, hemagglutination; HAI, hemagglutination
inhibition; NeuAc, N-acetylneuraminic acid; NeuGc, N-glycolyl-
neuraminic acid.
(Received 15 July 2003, revised 30 August 2003,
accepted 10 September 2003)
Eur. J. Biochem. 270, 4348–4355 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03828.x
Clostridium perfringens sialidasetypeX,protease
enzymes and molecular mass standards were purchased
from Sigma.
Preparation of crab sera
Freshwater field crabs, Paratelphusa jacquemontii were
collected from the local wetlands of Kanyakumari district,
India. The crabs used for experimental purpose were of
either sex, uninjured, in intermolt stage and 30–55 g in
weight. Prior to collecting hemolymph, the first two legs
were cleaned with a cotton swab dipped in water and
70% (v/v) ethanol. After wiping dry, the dactyls
4
were cut
with a pair of scissors and the dripping hemolymph was
collected in a beaker kept on ice. On average about
4–5 mL of hemolymph was collected from each crab. The
clot and cellular elements were removed by centrifugation
at 2000 g at 4 °C. The sera can be stored in the freezer
()20 °C) for 3 months without any change in its hemag-
glutination (HA) activity. Prior to lectin purification, the
sera was centrifuged at 150 000 g for 5 h at 4 °C
(Beckman T65 rotor) to sediment the major portion of
hemocyanin [15].
Buffers

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

Tris/
HCl, pH 7.5, 0.3
M
NaCl, 10 m
M
CaCl
2
, elution buffer (EB;
50 m
M
Tris/HCl, pH 7.5, 0.3
M
NaCl, 10 m
M
EDTA),
coupling buffer (0.05
M
sodium pyrophosphate, pH 8.0).
Preparation of BSM–agarose affinity gel
The CNBr-activated Sepharose gel was prepared as instruc-
ted by the manufacturer. The gel was then transferred to a
solution of BSM (4 mgÆmL
–1
) and the suspension was
mixed gently at room temperature for 3–4 h. The degree of
coupling was checked by the reduction of BSM in the
coupling medium. The protein concentration in the medium
before and after coupling was estimated by Folin–Ciocal-
teau method. Finally, to quench the excess of activated
group (if present), 5 mL of ethanolamine in 1

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

.Thecolumnwas
washed with HSB until the A
280
of the effluent was < 0.002.
The column was further washed with LSB at 4 °C until the
A
280
of the effluent was < 0.002, and it was then transferred
from 4 °Cto32°C and was washed again with warm
(32 °C) LSB until the A
280
of the effluent was < 0.002. This
step eluted additional inert proteins and was necessary for
obtaining homogenous lectin. In all these steps the buffers
contained the calcium required for binding of lectin to
BSM–agarose. Lectin was eluted with EB containing 10 m
M
EDTA, and 1 mL fractions collected on ice in polypropy-
lene tubes containing 100 lLof100m
M
CaCl
2
at a rate of
0.3 mLÆmin
)1
. The presence of calcium chloride was
required in the collected fractions because the lectin was
unstable in the presence of EDTA. The fractions were
vortexed immediately after collection and stored at 4 °C.
HA assay was carried out to determine the presence of lectin

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

at 4 °C, until the A
280
of the
effluent was 0.002. The column was transferred to a water bath at
32 °C and washed with NaCl/Tris containing 300 mm NaCl until the
A
280
of the effluent was 0.002. Elution was carried out at 32 °Cwith
NaCl/Tris containing 10 mm EDTA. One millilitre fractions were
collected and tested for hemagglutination with a 1.5% suspension of
horse erythrocytes in NaCl/Tris containing 0.5% BSA at pH 7.5.
Ó FEBS 2003 A sialic acid specific lectin from P. jacquemontii (Eur. J. Biochem. 270) 4349
Hemagglutination inhibition (HAI) assay
The hemagglutination inhibition (HAI) was performed
following the procedure of Ravindranath et al. [12].
Enzyme treatment of the erythrocytes
Protease treatment. Following the procedure of Pereira
et al. [28], horse and rabbit erythrocytes were washed five
times with NaCl/Tris (pH 7.5) by centrifugation at 400 g for
5 min at room temperature (30–35 °C) and resuspended in
the same buffer. Equal volumes each of trypsin, chymo-
trypsin (1 mgÆmL
)1
in NaCl/Tris, pH 7.5) and pronase
(0.25 mgÆmL
)1
in NaCl/Tris, pH 7.5) with washed erythro-
cytes were incubated at 37 °C for 1 h. The erythrocytes were
washed in NaCl/Tris five times and used for the hemagglu-
tination assay.

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

M
Tris/HCl, pH 6.8, 4% (w/v) SDS, 2% (v/v) 2-merca-
ptoethanol and 5% (v/v) glycerol]. Gels were fixed and
stained with a solution containing 0.05% (w/v) Coomassie
Blue R-250, 10% (v/v) acetic acid, and 25% (v/v) isopropyl
alcohol, and destained with a solution containing 5% (v/v)
methanol and 7% (v/v) acetic acid at room temperature.
Estimation of protein. The protein concentration was
determined following the procedure of Lowry et al. [32].
Results
Purification of lectin from the hemolymph
of
P. jacquemontii
A column profile depicting purification of lectin from the
hemolymph of P. jacquemontii by affinity chromatography
on BSM-coupled Sepharose 4B is shown in Fig. 1. Clarified
serum (20 mL) was applied and on elution with EDTA
yielded 1.0 mg of pure lectin. The specific activity of purified
lectin increased about 2000-fold from 196 (crude hemo-
lymph) to 409 600 of hemagglutinin per mg protein
(Table 1). Analysis of purified lectin on SDS/PAGE in the
presence of 2-mercaptoethanol revealed a major band at
molecular mass of 34 kDa (Fig. 2).
Erythrocyte-binding specificity of
P. jacquemontii
lectin
The Paratelphusa lectin agglutinated only a limited range of
erythrocytes. Out of 12 erythrocyte types tested the lectin
could agglutinate only six erythrocyte types (Table 2). Our
study on the sialoconjugates found on the surface of

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,

showed the highest titers with the lectin. Human A and O,
sheep and goat that contain largely NeuAc were not
agglutinated. The specific binding was based on O-acetyl
linkages. Horse and rabbit erythrocytes treated with pro-
tease enzymes did not change the binding affinity of the
erythrocytes to the lectin (Table 3). However, sialidase
treated rabbit erythrocytes lost the capacity to hemagglu-
tinate the lectin. Horse erythrocytes that contain 4-O-
Ac-NeuAc and are resistant to C. perfringens sialidase
showed no change in HA (Table 3). This suggested that the
major binding site of the lectin was sialic acid on the surface
of erythrocytes.
The binding specificity of crab lectin
Inhibition studies with various sugars was helpful in
deducing the binding specificity of the lectin. Fucose and
lactose inhibited HA activity of purified lectin. Sucrose,
glucose and glucose-6-phosphate inhibit at concentrations
less than 25 m
M
. However, N-acetyl derivatives, namely
N-acetylglucosamine (GlcNAc), N-acetylgalactosamine
(GalNAc) and N-acetyl mannosamine (ManNAc) were
better inhibitors than their respective sugars (Table 4). This
clearly suggested that the lectin recognizes the acetyl group.
The hemagglutination inhibition (HAI) assays with a
variety of sialoglycoproteins (Table 5) showed BSM as the
most potent inhibitor of the purified lectin with an HAI of
524 288. The HAI of the sialoglycoproteins can be graded
as follows: BSM>transferrin>fetuin ¼ porcine thyroglob-
ulin>porcine stomach mucin>bovine thyroglobulin. The

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

Guinea pig – – 16
Dog – – 4
Donkey – – 4
Human A – 0 0
Human B – 0 2
Human O – 0 0
Cow – 0 0
Goat – 0 0
Sheep – 0 0
a
Data from [30] and [63].
Table 3. The effect of enzyme treatment of rabbit and horse erythrocytes on hemagglutination assay of P. jacquemontii. NaCl/Tris-washed rabbit and
horse erythrocytes are incubated with specific concentration of enzymes for a period of 45–60 min at 37 °C, washed and reconstituted in NaCl/Tris
(pH 7.5) as a 1.5% suspension and used for HA assay.
Enzymes used Site of enzyme activity
HA titer
Horse erythrocytes Rabbit erythrocytes
None – 256 128
Sialidase (C. perfringes Type X) NeuAc-
D
-Gal NeuAc-
D
-GalNAc 256 0
Trypsin (1 mgÆmL
)1
) Arg; Lys- 256 128
Pronase (0.25 mgÆmL
)1
) Tyr-, Trp-, Phe-, Leu 256 128
Table 4. Inhibition of P. jacquemoutii lectin hemagglutination by sugars.

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

Ó FEBS 2003 A sialic acid specific lectin from P. jacquemontii (Eur. J. Biochem. 270) 4351
out that the sialic acid present in BSM was an important
binding determinant. Asialofetuin and asialotransferrin
showed weak inhibition due to nonspecific binding of acetyl
groups conjugated to proteins or other sugars. Base
treatment specific for hydrolysis of the O-acetyl groups of
sialic acids without cleavage of peptide bonds [6] consider-
ably reduced the ability of BSM and bovine thyroglobulin
to inhibit HA (Table 6). The bovine mucin was estimated to
contain 65% O-acetylneuraminic acid [12]. Taking together
these observations, it was suggested that the inhibitory
potency of BSM and other sialoglycoproteins was due to
O-acetyl sialic acid.
The HAI results when summarized suggest that the crab
lectin was sialic acid-specific with a high affinity for
O-acetylated NeuAc.
Discussion
The presence of naturally occurring agglutinins in the
hemolymph of several crustaceans has been well known
since the beginning of the 20th century [34]. An evaluation
of the literature revealed that purification of lectin from the
hemolymph of crustaceans was most successful by affinity
chromatography as it gave a higher fold of purification and
percentage of recovery [20,24,27,35–39]. The lectin was
purified from the hemolymph of P. jacquemontii by affinity
chromatography. It yielded a 2000-fold increase in specific
activity. Analysis of the lectin on SDS/PAGE gave a single
band at apparent molecular mass of 34 kDa.
The binding affinity of the lectin in the hemolymph of
the freshwater crab, Paratelphusa jacquemontii, expressed

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

O-acetyl NeuAc was reflected in preferential binding to
erythrocytes which predominantly express O-acetyl sialic
acid on their cell surface. Horse erythrocytes, which contain
4-O-Ac-NeuAc [43], and rabbit erythrocytes, which contain
9-O-Ac-NeuAc [41], showed maximum haemagglutination.
On the other hand, human blood cells A, B and O [44,45],
and sheep have surface glycoconjugates rich in NeuAc [44]
and thereby the lectin showed poor binding affinity towards
these erythrocytes.
The O-acetyl NeuAc specificity of P. jacquemontii lectin
is sufficiently evident from its inhibition and hemagglutin-
atin study. The lectin is unique from that of other sialic acid-
specific lectins. O-Acetyl sialic acid-specific lectin was
Table 5. Inhibition of P. jacquemontii lectin hemagglutination by sialoglycoproteins. The glycoproteins (25 lL), reconstituted in NaCl/Tris
(5 mgÆmL
)1
) were serially diluted in microtiter plates and mixed with 25 lL of lectin/NaCl/Tris-BSA, 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 HAI titer were determined after 60 min of incubation and expressed as the highest dilution of glycoprotein that inhibited the
agglutination of erythrocytes.
Sialoglycoproteins HAI titer
Minimal concentration
required lgÆmL
)1
Relative inhibitory
potency percentage
BSM 524288 0.0096 100
PSM 16 312.5 0.003
Bovine thyroglobulin 8 625 0.0015
Porcine thyroglobulin 32 156.25 0.006

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

brachyuran crabs [12,24,36]. The other crustaceans such as
the barnacles [47,48], the freshwater prawn [26], marine
prawn [50] and the lobsters [34,37], are distinctly marked by
the presence of multiple lectins in its hemolymph. At
present, the most popular belief is that lectins function
primarily as recognition molecules [51]. The single lectin in
brachuryan crabs suggests a specialization towards recog-
nition of nonself. The sialic acids are widely distributed in
nature, generally as components of oligosaccharide units in
mucins, glycoproteins, gangliosides, milk oligosaccharides
and certain microbial polymers [52–55]. It is highly likely
that sialic acid-binding lectins in crab may recognize and
bind to such organisms that contain sialic acid [12]. Lectins
have the ability to agglutinate bacteria [56,57], interact with
microorganisms [58] and enhance phagocytosis of bacteria
by hemocytes [59]. Also it is known that the hepatopancreas
sequesters both extraneous proteins and the bacteria
invading the body cavity [60,61]. Hence sialic acid of
exogenous origins does occur in the hepatopancreas [62].
The freshwater crab, Paratelphusa jacquemontii, found in
the ponds, lakes and paddy fields is adapted to an
environment rich in organic decomposing matter, contain-
ing sialic acid in microbial polymers with which it would
have interacted to develop an innate immunity.
Invasion of parasite, repeated injury, and exposure to
drugs such as phenylhydrazine induce the production of
O-acetyl sialic acid [6,63]. Moreover, O-acetylation of sialic
acid may change with transformation or other alteration in
the environment of the cell [63]. The normal human tissues
contain NeuAc, while malignant tumour cells contain

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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