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: 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) 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]. 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