Isolation and characterization of a new lectin from plasma of fish Channa punctatus

A soluble lectin is purified to apparent homogeneity from plasma of Channa punctatus by affinity chromatography on N-acetyl-D-galactosamine coupled to epoxy-activated cellulose. The lectin has 140 kDa native molecular mass and 68 kDa subunit molecular mass, as determined by native and sodium dodecyl sulphate denaturing polyacrylamide gel electrophoresis, respectively. The lectin agglutinates human A and AB blood groups and rat, mice and guinea pig erythrocytes in the presence of Ca2+ and Mg2+ or Mn2+ ions. These divalent cations, but not thiol group, are obligatory requirements for the lectin activity. Gal(beta 1----3)GalNAc (0.09 mM) is the most potent inhibitor of the lectin.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.     

Identification and characterization of Sarcophaga lectin receptor on the surface of murine macrophages by use of monoclonal antibodies

The structure of Sarcophaga lectin receptor on the surface of murine macrophages was analyzed using monoclonal antibodies. This receptor was found by gel filtration to have a molecular weight of 460 kDa. SDS-polyacrylamide gel electrophoresis showed that this receptor consists of two subunits of 170 kDa and 110 kDa. The results indicated that it is probably a heterotetramer of two molecules of each subunit. Two monoclonal antibodies recognized epitopes in the 110 kDa subunit, and one of them specifically inhibited the binding of Sarcophaga lectin to macrophages and the cytotoxic reaction mediated by this lectin in the presence of macrophages. Therefore, it is likely that the 110 kDa protein in the receptor plays a role in activation of macrophages by this lectin.     

Chemical and physicochemical characterization of the sialic acid-specific lectin from Cepaea hortensis

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.     

Biochemical characterization of a lectin from Delonix regia seeds

A lectin from Delonix regia (DRL) seeds was purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on diethylaminoethyl-Sepharose and reverse-phase high-performance liquid chromatography on a C18 column. Hemagglutinating activity was monitored using rat erythrocytes. DRL showed no specificity for human erythrocytes of ABO blood groups. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single protein in the presence of 0.1 M of dithiothreitol (DTT) and in nonreducing conditions. Native-PAGE showed that DRL is a monomer with a molecular mass of about 12 kDa, as determined by denaturing gel electrophoresis and gel filtration chromatography. An amino acid composition revealed the absence of cysteine residues, the presence of 1 mol methionine/mol protein and a high proportion of acidic amino acids and glycine. The N-terminal sequence of DRL was determined by Edman degradation, and up to 16 amino acid residues showed more than 90% homology with other lectins from the Leguminosae family. The optimal pH range for lectin activity was between pH 8.0 and 9.0, and the lectin was active up to 60°C. The lectin required Mn²⁺ for hemagglutinating activity and remained active after reduction with 0.1 M of DTT, but lost activity in the presence of 8 M of urea. Sodium metaperiodate had no effect on the activity of DRL.     

A blood group A specific lectin from the seeds of Crotalaria striata

A lectin, monospecific for human blood group A red blood cells was extracted from seeds of Crotalaria striata and purified by molecular sieving on Sephadex G-100 and ion-exchange on DEAE-cellulose. A molecular mass of 30 kDa was determined by SDS-polyacrylamide gel electrophoresis under non-reducing and reducing conditions. Molecular sieving on a Superose 12 column indicated a molecular mass of 110 kDa, suggesting the tetrameric nature of the native protein. Amino-acid composition showed the presence of aminated carbohydrate residues on the lectin. N-terminal amino-acid sequencing showed a striking similarity with the N-terminal sequence of the lectin from Crotalaria juncea, which is blood-group non-specific. The potency order of agglutination inhibition with galactose containing monosaccharides was N-acetyl-D-galactosamine greater than D-galactose greater than D-galactosamine as found for blood-group-A-specific lectins from other species.     

Isolation and characterization of a lectin from Annona muricata seeds

A lectin with a high affinity for glucose/mannose was isolated from Annona muricata seeds (Annonaceae) by gel filtration chromatography on Sephacryl S-200, ion exchange chromatography on a DEAE SP-5 PW column, and molecular exclusion on a Protein Pak Glass 300 SW column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) yielded two protein bands of approximately 14 kDa and 22 kDa. However, only one band was seen in native PAGE. The Mr of the lectin estimated by fast-performance liquid chromatography-gel filtration on Superdex 75 was 22 kDa. The lectin was a glycoprotein with 8% carbohydrate (neutral sugar) and required divalent metal cations (Ca2+, Mg2+, and Mn2+) for full activity. Amino acid analysis revealed a large content of Glx, Gly, Phe, and Lys. The lectin agglutinated dog, chicken, horse, goose, and human erythrocytes and inhibited the growth of the fungi Fusarium oxysporum, Fusarium solani, and Colletotrichum musae.     

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.     

Identification of a 148-kDa surface lectin from Giardia lamblia with specificity for α-methyl-d-mannoside

Abstract A lectin specific for α-methyl-d-mannoside was purified from the membrane extract of Giardia lamblia by a combination of gel filtration chromatography on Sephadex G-75 and Superose 6-HR 10/30. The homogeneity of the lectin was established by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The molecular mass of the native protein was 148 kDa. The lectin agglutinated rabbit erythrocytes in the presence of Ca²⁺ at 37 °C and pH 7.O. The maximum activity of the lectin was obtained after trypsin treatment. The inhibition study clearly suggests that the binding site of the lectin recognizes α-methyl-d-mannoside as the immunodominant sugar.     

Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

A lectin from the marine red alga Gracilaria ornata (Gracilariaceae, Rodophyta) was purified and characterized. The purification procedure consisted of extracting soluble proteins in 0.025 M Tris-HCl buffer, pH 7.5, followed by ammonium sulfate precipitation (70% saturation), ion exchange chromatography on DEAE-cellulose and affinity chromatography on mucin-Sepharose 4B. The purified G. ornata lectin (GOL) showed a single protein band with an apparent molecular mass of 17 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing conditions. The native molecular mass of GOL determined by gel filtration on a Sephadex G-100 column was 17.4 kDa and its carbohydrate content was estimated to be 2.9%. Therefore, GOL is a monomeric glycoprotein. The purified lectin agglutinated trypsin-treated erythrocytes from rabbit and chicken but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested but by the complex glycoproteins porcine stomach mucin, lactotransferrin, asialofetuin and bovine and porcine thyroglobulins. Isoelectric focusing showed that GOL is an acidic protein with a pI of 5.4 with analysis of its amino acid composition revealing high contents of Asx, Glx, Ser, Glu, Ala and Cys. When incorporated in artificial seeds, GOL significantly affected the development of Callosobruchus maculatus larvae, indicating the possibility of using this lectin in a biotechnological strategy for insect management of stored cowpea seeds.     

Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc specific lectin in tuberous roots of Trichosanthes japonica.

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a lectin from peanut roots.

A glucose-specific lectin has been purified to apparent homogeneity from 7-day-old peanut (Arachis hypogaea) roots by affinity chromatography on a Sephadex G-50. The lectin has a 66 kDa native molecular mass and a 33 kDa subunit molecular mass as revealed by native and denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively. The purified lectin, gives a single precipitin line with the antiserum produced against 7-day-old root extract and shows 5 bands in the pH range of 4.4-5.4 in the isoelectric focusing gel. The glucose-specific lectin activity in the peanut roots appears from the fourth day onwards. Lipopolysaccharides isolated from the host specific Rhizobium strain are a 68-fold more potent inhibitor of the lectin as compared to glucose.     

Facilitated glucose transporter of human erythrocyte: proteolytic mapping of the [3H]cytochalasin B photoaffinity-labeled transporter polypeptide

The human erythrocyte D-glucose transporter is an integral membrane glycoprotein with an heterogeneous molecular mass spanning a range 45-70 kDa. The protein structure of the transporter was investigated by photoaffinity labeling with [3H]cytochalasin B and fractionating the labeled transporter according to molecular mass by preparative SDS-polyacrylamide gel electrophoresis. Each fraction was digested with either papain or S. aureus V8 proteinase, and the labeled proteolytically derived peptide fragments were compared by SDS polyacrylamide gel electrophoresis. Papain digestion yielded two major peptide fragments, of approx. molecular mass 39 +/- 2 and 22 +/- 2 kDa; treatment with V8 proteinase resulted in two fragments, with mass of 24 +/- 2 and 15 +/- 2. Proteolysis of each transporter fraction produced the same pattern of labeled peptide fragments, irrespective of the molecular mass of the original fractions. The binding characteristics of [3H]cytochalasin-B-labeled transporter to Ricinis communis agglutinin lectin was examined for each transporter molecular mass fraction. It was found that higher-molecular-mass fractions of intact transporter had a 2-fold greater affinity for the lectin than lower-molecular-mass fractions (i.e., 67 kDa greater than 45 kDa fraction). However, proteolytically derived labeled peptide fragments from each fraction had minimal affinity for the lectin. These results suggest that the labeled peptide fragments have been separated from the glycosylated regions of the parent transporter protein. The present findings indicate that, although transporter proteins have an apparently heterogeneous molecular mass, some regions of the protein share a common peptide. Furthermore, the glycosylated regions appear to be located some distance from the [3H]cytochalasin-B-labeled site(s).     

Purification and characterization of a new mannose-specific lectin from Sternbergia lutea bulbs

A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an α(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin (S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed of two identical subunits of 10 kDa which are linked by non-covalent interactions. The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays. It is an α-D-mannose-specific lectin that interacts to form precipitates with various α-mannans, galactomannan and asialo-thyroglobulin, but not with α-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothryroglobulin precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for terminal α(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence SLA showed 7% homology with that of GNA. Abbreviations: AAA, Allium ascalonicum agglutinin (shallot lectin); ASA, Allium sativum agglutinin (garlic lectin); AUA, Allium ursinum agglutinin (ramsons lectin); DAP, 1,3-diaminopropane; GNA, Galanthus nivalis agglutinin (snowdrop lectin); HHA, Hippeastrum hybr. agglutinin (amaryllis lectin); LOA, Listera ovata agglutinin (orchid twayblade lectin); NPA, Narcissus pseudonarcissus agglutinin (daffodil lectin); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, SLA, Sternbergia lutea agglutinin; SDS, sodium dodecyl sulfate; Me, methyl; Bn, benzyl; PNP, p-nitrophenyl. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and characterization of a lectin from the beetle, Allomyrina dichotoma

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.     

Purification and characterisation of a lectin isolated from the Manila clam Ruditapes philippinarum in Korea

The characteristics of a lectin from the marine bivalve Ruditapes philippinarum (Manila clam) were investigated in this study. A method was developed for the isolation of the Manila clam lectin (MCL). Affinity chromatography using mucin-Sepharose, ion-exchange chromatography with DEAE-Toyoperl, and gel filtration with Superose 6 were used for MCL isolation. SDS-PAGE showed that the MCL protein had a molecular mass of 138 kDa, and consisted of 74-, 34-, and 30-kDa subunits. The native lectin in solution behaved as a 274-kDa protein in gel filtration chromatography. The lectin activity of MCL was Ca2+ -dependent, and the optimal Ca2+ concentration for MCL activity was 20 mM. MCL activity was stable between pH 6 and pH 9, and was temperature-dependent; incubation of MCL at 90 degrees C led to irreversible denaturation. The activity of MCL was not inhibited by the presence of monosaccharides, such as Man, Fuc, Gal, Glc, GlcNAc, and NeuNAc. In contrast, the lectin activity of MCL was strongly inhibited by the presence of porcine mucins. MCL activity was also inhibited by N-acetyl-d-galactosamine, human embryonic alpha-1-acid glycoprotein, and highly branched mannans from marine halophilic bacteria. It appears that MCLs have unusual carbohydrate specificities for N-acetyl-d-galactosamine, which contains both mucin-type carbohydrate chains and highly branched mannans. Immunofluorescence staining revealed that MCL was bound to the surfaces of purified hypnospores from Perkinsus sp., which is a protozoan parasite of Manila clams.     

Comparative analysis of galactoside-binding lectins isolated from mammalian spleens

Galactoside-inhibitable lectins have been isolated from rabbit, rat, mouse, pig, lamb, calf, and human spleens. Native molecular mass, subunit structure, pI, and hemagglutinating activity have been compared for these lectins. The yields of lectin varied from 1.8 mg/kg for rabbit spleen to 79 mg/kg for lamb spleen. Pig, lamb, calf, and human spleen lectins yielded single protein peaks when subjected to Superose 12 fast-protein liquid chromatography. The apparent molecular mass for these lectins was 33-34 kDa. In contrast, rat and mouse spleen lectin preparations were separated into three components ranging from 8.4 to 34 kDa. Superose 12 chromatography of rabbit spleen lectin revealed the presence of at least six components. Gradient slab gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of single polypeptides for pig, calf, lamb, and human lectins corresponding to a molecular mass of 14-14.5 kDa. Multiple polypeptides were detected for the mouse, rat, and rabbit lectins. The molecular mass of the major polypeptides were 15, 15, and 17 kDa for rat, mouse, and rabbit, respectively. The presence of isolectins in all preparations was shown by isoelectric focusing. The major isolectins were acidic proteins with pI 4.38-4.80. Hemagglutination and hemagglutination inhibition assays demonstrated similarities as well as differences among the lectin preparations. Hemagglutinating activity could not be demonstrated in rabbit spleen extracts nor for isolated putative lectin. Human buffy coat cells were reversibly agglutinated by calf and human spleen lectins, demonstrating the presence of leucocyte cell surface lectin receptors.     

PURIFICATION OF A SEX‐SPECIFIC LECTIN INVOLVED IN GAMETE BINDING OF AGLAOTHAMNION CALLOPHYLLIDICOLA (RHODOPHYTA)1

Egg and sperm binding and correct recognition is the first stage for successful fertilization. In red algae, spermatial attachment to female trichogynes is mediated by a specific binding between the lectin(s) distributed on the surface of trichogyne and the complementary carbohydrates on the spermatial surface. A female‐specific lectin was isolated from Aglaothamnion callophyllidicola by agarose‐bound fetuin affinity chromatography. Two proteins, 50 and 14 kDa, eluted from the fetuin column were separated using a native‐polyacrylamide gel electrophoresis method and subjected to a gamete binding assay. The 50 kDa protein, which blocked spermatial binding to female trichogynes, was used for further analysis. Internal amino acid sequence of the 50 kDa protein was analyzed using matrix‐assisted laser desorption/ionization‐mass spectrometry and degenerated primers were designed based on the information. A full‐length cDNA encoding the lectin was obtained using rapid amplification of cDNA ends polymerase chain reaction (PCR). The cDNA was 1552 bp in length and coded for a protein of 450 amino acids with a deduced molecular mass of 50.7 kDa, which agreed well with the protein data. Real‐time PCR analysis showed that this protein was up‐regulated about 10‐fold in female thalli. As the protein was novel and showed no significant homology to any known proteins, it was designated Rhodobindin.