The hemagglutina-tion of purified lectin was inhibited by N-acetylneuraminic acid but not by N-glycolylneuraminic acid, even at a concentration of 100 mM.. Keywords: Paratelphusa jacquemo
Trang 1Purification and characterization of a sialic acid specific lectin from
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 mM 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 Sbacter-ialic 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 galactogluco-samine, 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: maghilthilak@yahoo.com
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)
Trang 2Clostridium perfringens sialidase typ e X, p rotease
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
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 4C 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 4C
(Beckman T65 rotor) to sediment the major portion of
hemocyanin [15]
Buffers
5The following buffers were used in this study: NaCl/Tris/
CaCl2
6 (50 mM Tris/HCl, pH 7.5, 100 mM NaCl, 10 mM
CaCl2), NaCl/Tris/BSA [pH 7.5 containing 0.05% (v/v)
BSA], NaCl/Pi(10 mMsodium phosphate, pH 7.0, 0.15M
NaCl), high salt buffer (HSB; 50 mMTris/HCl, pH 7.5, 1M
NaCl, 10 mMCaCl2), low salt buffer, (LSB; 50 mM Tris/
HCl, pH 7.5, 0.3MNaCl, 10 mMCaCl2, elution buffer (EB;
50 mM Tris/HCl, pH 7.5, 0.3M NaCl, 10 mM EDTA),
coupling buffer (0.05Msodium 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 1M 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
(0.1Macetate buffer containing 0.5MNaCl) followed by a
wash at pH 8.0 (0.1M Tris containing 0.5M NaCl)
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
ofP 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 4C The eluant was
collected at a rate of 0.6 mLÆmin)1 The column was washed with HSB until the A280of the effluent was < 0.002 The column was further washed with LSB at 4C until the
A280of the effluent was < 0.002, and it was then transferred from 4C to 32 C and was washed again with warm (32C) LSB until the A280of the effluent was < 0.002 This stepeluted 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 mM EDTA, and 1 mL fractions collected on ice in polypropy-lene tubes containing 100 lL of 100 mMCaCl2at 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 4C
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 mM CaCl2, at 4C for 18 h, and then for 3 h with fresh 1 mM CaCl2 The dialysate was then aliquoted, lyophilized and stored at )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 C with 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.
Trang 3Hemagglutination 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 (30–35C) 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)1in NaCl/Tris, pH 7.5) with washed
erythro-cytes were incubated at 37C 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 lL of
5 mMacetate buffer pH 5.5 for 3 h at 37C 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 lL of 0.04Mof NaOH, vortexed, incubated on ice
for 45 min and neutralized with 1 mL of 0.01M HCl,
respectively
Polyacrylamide gel electrophoresis SDS/PAGE (12.5%
slab gel) was performed according to Laemmli [31] Samples
were heated for 3 min at 100C in sample buffer [25% (v/v)
1M 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
ofP 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 ofP 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
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).
Trang 4lectin Horse and rabbit erythrocytes, which have a high
content of 4-O-Ac-NeuAc and 9-O-Ac-NeuAc, respectively,
showed the highest titers with the lectin Human A and O,
sheepand 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 mM 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>fetuin¼ 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 lL of a 1.5%
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 groupa
O-acetyl sialic acid content percentage totala HA titer
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
Sialidase (C perfringes Type X) NeuAc- D -Gal NeuAc- D -GalNAc 256 0
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 (%)
NeuGc 0 < 100 > 6.25
Trang 5out 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-acetylneuN-glycolylneu-raminic acid and, 8,9-di-O-acetylneuraminic acid [6] and p roved to be a 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 sheephave 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-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
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
Bovine thyroglobulin + sialidase 2
BSM + 0.04 M NaOH 4 C 45 min 32
Bovine thyroglobulin + 0.01 M NaOH 4 C 45 min 0
Trang 6isolated 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 jacquemontiilectin 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 developan 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 jacquemontiilectin 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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