Purification and properties of a lectin from lonchocarpus capassa (apple-leaf) seed

A lectin has been purified from L. capassa seed by ammonium sulphate fractionation and affinity chromatography on a column of D-galactose-derivatized Sepharose. The lectin is a glycoprotein which contains 3.8% neutral carbohydrates comprised of mannose, N-acetylglucosamine, xylose and fucose. The subunit M, of the lectin is 29 000, it has only alanine as N-terminal amino acid and contains 240 amino acids with a high content of acidic and hydroxy amino acids, single residues of methionine and histidine and the absence ofcystine. The lectin of L. capassa seed is a metalloprotein in that it contains 0.8 mol Ca²⁺ and 0.4 mol Mn²⁺ per mol. It agglutinates untreated human A, O and B type erythrocytes and rabbit erythrocytes. N-Acetyl-D-galactosamine was the best inhibitor. D-Galactose and various carbohydrates containing this sugar inhibit the hemagglutinating activity of the lectin. The lectin is also inhibited by D-glucose. The amino-terminal sequence of the lectin from L. capassa seed shows a significant degree of homology with many lectins from leguminous plants and is related to concanavalin A by a circularly permuted sequence homology.     

A lactose specific lectin from the sponge Cinachyrella apion: Purification,characterization, N-terminal sequences alignment and agglutinating activity on Leishmania promastigotes

Crude extract from the sponge Cinachyrella apion showed cross-reactivity with the polyclonal antibody IgG anti-CvL (Cliona varians lectin) and also a strong haemagglutinating activity towards human erythrocytes of all ABO groups. Thus, it was submitted to acetone fractionation, IgG anti-deglycosylated CvL Sepharose affinity chromatography, and Fast Protein Liquid Chromatography (FPLC-AKTA Purifier) gel filtration on a Superose 6 10/300 column to purify a novel lectin. C. apion lectin (CaL) agglutinated all types of human erythrocytes with preference for papainized type A erythrocytes. The haemagglutinating activity is independent of Ca²⁺, Mg²⁺ and Mn²⁺ ions, and it was strongly inhibited by the disaccharide lactose, up to a minimum concentration of 6.25 mM. CaL molecular mass, determined by FPLC-gel filtration on a Superose 12 10/300 column and SDS gel electrophoresis, was approximately 124 kDa, consisting of eight subunits of 15.5 kDa, assembled by hydrophobic interactions. The lectin was heat-stable between 0 and 60 °C and pH-stable. The N-terminal amino acid sequence of CaL was also determined and a blast search on amino acid sequences revealed that the protein showed similarity only with a silicatein. Leishmania chagasi promastigotes were agglutinated by CaL and this activity was abolished by lactose, indicating that lactose receptors could be presented in this parasite stage. These findings are indicative of the potential biotechnological application of CaL as diagnostic of pathogenic protozoa.     

Isolation and characterization of an aluminum-sensitive mutant of Pseudomonas fluorescens

An aluminum-sensitive mutant was isolated by chemical mutagenesis using 1-methyl-3-nitro-1-nitrosoguanidine. Although this mutant of Pseudomonas fluorescens was susceptible to aluminum, it did respond in a manner analogous to the wild-type when challenged with either Ca²⁺, or Ga³⁺ or Zn²⁺. However, the aluminum-sensitive mutant did not grow in media with iron. Calcium detoxification appeared to be attained by the elaboration of calcite, a detoxification strategy observed in the wild-type Pseudomonas fluorescens. There was a 40% diminution in biomass when the mutant was cultured in a control medium as compared to the wild-type. The aluminum-sensitive mutant had a characteristic yellow coloration. Electrophoretic analyses of the soluble cellular extract revealed numerous variations between the aluminum-sensitive mutant and wild-type Pseudomonas fluorescens. Bands corresponding to approximate molecular masses of 158 kDa, 116 kDa, 97 kDa, 55 kDa, and 19 kDa were absent in the aluminum-sensitive mutant. Furthermore, bands attributable to molecular masses of 90 kDa and 15 kDa were only evident in the aluminum-sensitive Pseudomonas. It appears that the chemical mutagenesis may have affected only aluminum and iron(III) metabolism in this organism.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.

A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin.

     

An acid-tolerant lectin coupled with high Hg2+ potentiated hemagglutination enhancing property purified from Amanita hemibapha var. ochracea

A 37.4 kDa acid tolerant lectin was isolated and purified from dried fruiting bodies of Amanita hemibapha var. ochracea designated as AHL. The lectin was not adsorbed on DEAE-cellulose, but rather adsorbed on S-Sepharose and subjected to gel filtration by fast protein liquid chromatography on Superdex 75. The purified lectin was immune from inhibition activities of metal ions. More over, AHL exhibited high agglutination activity on rabbit erythrocytes with accelerating Hg²⁺ ions concentration. Partial peptide sequence analysis (VSNNLLTGPKVVR) of this lectin showed relative similarity to phosphoenolpyruvate carboxykinase [ATP]-like protein as predicted from Fragaria vesca subsp. Vesca. Interestingly, AHL displayed a strong affinity toward α-Lactose, making our study the first report associating Amanita species’ lectin specificity for α-Lactose to the best of our knowledge.     

Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

Studies on lectins: XLIII. Isolation and characterization of the lectin from restharrow boots (Ononis hircina Jacq.)

From 1 kg of dried Ononis hircina Jacq. roots 36 mg of a lectin were isolated by affinity chromatography on O-β-lactosyl polyacrylamide gel. The lectin is homogeneous as judged by ultracentrifugal analysis (s_20,w = 6.2 S), polyacrylamide disc electrophoresis at pH 8.9 or 4.5, gel filtration on thin layers of Sephadex G-200 (M_r = 110 000) and dodecyl sulfate electrophoresis (M_r of sub-units 31 000, both in presence and absence of mercaptoethanol) and disc dodecyl sulfate electrophoresis (pH 9.5). The lectin contains much aspartic and glutamic acids, serine and threonine and also 7.2% of neutral sugar. It is relatively specific for human type O erythrocytes that are agglutinated at a minimal lectin concentration 0.3 μg/ml. The erythroagglutinating activity is not stimulated by Ca²⁺, Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, or Ni²⁺ salts; it is inhibited most effectively by N-acetyl-D-galactosamineandanumberofD-galactosederivatives. Dissociation constants of several lectin · sugar complexes were estimated by affinity electrophoresis. The lectin is not mitogenic in rabbit lymph nodes lymphocytes.     

Lectins (haemagglutinins) in the haemolymph of Glossina fuscipes fuscipes: Isolation,partial characterization,selected physico-chemical properties and carbohydrate-binding specificities

Glossina fuscipes fuscipes haemolymph contained agglutinins (lectins), titre range 2⁻¹¹–2⁻¹⁸, against red blood cells (RBC) of human ABO(H) blood group with highest values detected against “AB” RBC. The use of protease- and neuraminidase-treated RBC in many cases increased titres whilst treatment with galactosidases or glucosidases caused decreased levels. Haemolymph adsorption with “O” RBC reduced titres against “O” and “AB” but to a lesser extent anti-A or -B activity indicating lectin heterogeneity. The carbohydrate-binding specificities for human RBC were directed towards N-acetylated and deoxy derivatives of glucose and/or galactose. In addition the haemagglutinins were reactive against some oligosaccharides, ribose, deoxymannose, deoxygalactose, xylose and xylan with certain of the RBC types. The agglutinins were glycoprotein in nature, thermo-labile, affected by storage, freezing and thawing treatments and exposure to a high dosage of γ-radiation, possessed limited disulphide and hydrogen bonds, and depended upon slightly acid to neutral conditions for optimum agglutination. The haemag-glutinins did not require the presence of divalent cations (Ca²⁺, Mn²⁺ or Cu²⁺ ions) for activity although an elevated concentration of Mg²⁺ ions resulted in increased endpoint titres. However heavy metal ions (Pb²⁺ and Fe²⁺) in the buffer lowered agglutinin levels. The intact lectin molecule had an isoelectric point of 6.2, a relative molecular weight of 710 kDa and comprised approx. 70 kDa subunits.     

Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ? 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ? 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.     

Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon tetrachloride-induced hepatic fibrosis.

The lectin from Euonymus europeus seeds was purified by adsorption onto insoluble polyleucyl hog A + H blood group substance and subsequent elution with lactose. The isolated lectin formed three lines in immunoelectrophoresis against rabbit antisera to the crude seed extract and showed three components on electrophoresis in acrylamide gel at pH 9.4. In analytical isoelectric focusing the purified lectin had six closely spaced bands with pI from 4.3 to 4.7. It sedimented as two peaks: a big symmetrical peak with s_20,w⁰ of 7.8 and another small, diffuse moving peak. The intrinsic viscosity was 0.057 dl/g and the M_r calculated from the sedimentation coefficients, intrinsic viscosity, and V? of 0.71 was about 166,000. In sodium dodecyl sulfate, it gives subunits of M_r 17,000 and 35,000; 20% of the 35,000 subunit resists reduction by dithiothreitol in 7 m guanidine-HCl. The Euonymus lectin is a glycoprotein containing 4.8% d-galactose, 2.9% d-glucose, and 2.8% N-acetyl-d-glucosamine. The purified lectin precipitated well with B and H blood group substances and with the P1 fraction of blood group B substance but not with A₁ substances. It precipitated poorly with Le^a and Le^b and precursor I blood group substances. Inhibition of precipitation with milk and blood group oligosaccharides showed the lectin to be most specific for blood group B oligosaccharides having the structure: dGalα1 → 3[lFucα1 → 2]dGalβ1 → 3 or 4dGlcNAcβ→. It is also inhibited by blood group H oligosaccharides but to a lesser degree. For 50% inhibition of precipitation, 3.5, 850, and 290,000 nmol of B and H oligosaccharides and lactose, respectively are required. The B and H specificities are an intrinsic property of a single lectin site since absorption and elution from an H immunoadsorbent gave material with B as well as H specificity. Millipore-filtered crude extracts of Euonymus europeus preserved with 0.02% sodium azide are stable in the refrigerator for many months and can be used for quantitative precipitin and for quantitative inhibition assays, results being the same as with purified lectin.     

Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed

The thermophilic Bacillus licheniformis strain JS was isolated from a bed of mushrooms, Pleurotus sajor-caju. The organism could produce a novel, single-component, thermostable chitinase that was purified by ion-exchange chromatography using DEAE-cellulose in 7.64% yield and in an 8.1-fold enhancement in purity. Its molecular weight is 22 kDa. The enzyme is a chitobiosidase, since the chitin hydrolysate is N^I,N^II-diacetylchitobiose. The optimum temperature for enzyme activity is 55 °C, and the optimum pH is 8.0. It was completely inhibited by Hg²⁺ ions whereas Co²⁺ ions served as an activator. The thermostability of this enzyme is important in the bioconversion of chitinous waste and for the production of chitooligosaccharides.     

Isolation of a seed coagulant Moringa oleifera lectin

In this work hemagglutinating activity (HA) was investigated in distinct Moringa oleifera tissue extracts. A new lectin from seeds (cMoL) was purified and characterized; hemagglutinating and coagulating activities were evaluated. HA was detected in 0.15 M NaCl extracts from flowers and rachis inflorescence (5%, w/v), seeds, leaves, fundamental tissue of stem and steam bark (10%, w/v). cMoL isolated after saline extraction and guar gel column chromatography was active at pH range 4.0–9.0 agglutinating erythrocytes from rabbit and human blood types. Extracts of tissues and cMoL activities were carbohydrate inhibited; azocasein and asialofetuin abolished cMoL HA. The lectin was thermostable at 100 °C during 7 h. Polyacrylamide gel electrophoresis under reduced conditions revealed a main polypeptide band of 26.5 kDa; native basic cMoL was detected as a unique band. Seed lectin preparations and cMoL showed coagulant activity, similar to aluminium sulphate, the coagulant most widely used in water treatment.     

A novel yeast-binding lectin from hemolymph Cyclina sinensis (Gmelin) and its effects on yeast cells

A lectin, Cyclina sinensis (Gmelin) (CSL), was isolated from hemolymph C. sinensis by ion-exchange on Cellulose DE52 and purified by gel filtration on Sephadex G-100 and HPLC on TSK gel G4000PW_XL. SDS-PAGE showed that the CSL protein had a molecular mass of 72 kDa, had consisted of 40 and 18 kDa subunits. The lectin activity of CSL was Ca²⁺-denpendent. The total carbohydrate content of CSL was found to be 16.2%. According to the principle of β-elimination reaction, the oligosaccharide moiety and peptide moiety of CSL might belong to O-glucosidic linkage. CSL was found to agglutinate rabbit erythrocytes and yeast Saccharomyces cerevisiae. The hemagglutination activity was inhibited by GalNAc and Man. CSL was observed to promote the yeast cells growth and ethanol production by yeast cells. The number of yeast and ethanol production increased with increasing concentration of CSL. In addition, the nitric oxide (NO) production increased as CSL concentration increased. The results indicate that CSL can be a potential yeast stimulator in fermentation process.     

Characterization of α-mannosidase from Erythrina indica seeds and influence of endogenous lectin on its activity

α-mannosidase from Erythrina indica seeds is a Zn²⁺ dependent glycoprotein with 8.6% carbohydrate. The enzyme has a temperature optimum of 50 °C and energy of activation calculated from Arrhenius plot was found to be 23 kJ mol^− 1. N-terminal sequence up to five amino acid residues was found to be DTQEN (Asp, Thr, Gln, Glu, and Asn). In chemical modification studies treatment of the enzyme with NBS led to total loss of enzyme activity and modification of a single tryptophan residue led to inactivation. Fluorescence studies over a pH range of 3–8 have shown tryptophan residue to be in highly hydrophobic environment and pH change did not bring about any appreciable change in its environment. Far-UV CD spectrum indicated predominance of α-helical structure in the enzyme. α-Mannosidase from E indica exhibits immunological identity with α-mannosidase from Canavalia ensiformis but not with the same enzyme from Glycine max and Cicer arietinum. Incubation of E. indica seed lectin with α-mannosidase resulted in 35% increase in its activity, while no such activation was observed for acid phosphatase from E. indica. Lectin induced activation of α-mannosidase could be completely abolished in presence of lactose, a sugar specific for lectin.     

Purification and characterization of a lectin from the banana shrimp Fenneropenaeus merguiensis hemolymph

A lectin from the hemolymph of the banana shrimp Fenneropenaeus merguiensis was purified by affinity chromatography on a fetuin-agarose column following by gel filtration on a Superose-12 column. The native molecular mass of purified F. merguiensis lectin (FmL) determined by gel filtration was 316.2 kDa and its carbohydrate content was estimated to be 4.4%. By SDS-PAGE analysis, purified FmL consisted of 32.3 kDa and 30.9 kDa subunits. These data suggest that this lectin is an oligomer. Two-dimensional electrophoresis showed that it had a pI value of 6.0 and was mainly composed of glycine, serine, histidine, glutamic acids and glutamine, with relatively lower amounts of methionine and tyrosine. Purified FmL expressed higher agglutination activity against rabbit and rat erythrocytes than with those from human, and its activity was Ca²⁺-dependent. The hemagglutinating activity of FmL was stable up to 55 °C and at pH 7.5–8. N-acetylated sugars, such as ManNAc, GlcNAc, GalNAc, and NeuNAc were strong inhibitors of the FmL induced hemagglutinating activity with NeuNAc being most effective. Porcine stomach mucin and fetuin were the most potent inhibitors of FmL. Purified FmL caused selective agglutination of Vibrio harveyi, and Vibrio parahemolyticus both pathogens of this Penaeus species and to a lesser extent Vibrio vulnificus but had no effect on the non-pathogenic strains; Vibrio cholerae, Salmonella typhi and Escherichia coli. Its bacterial agglutination was also completely inhibited by NeuNAc, mucin, fetuin and also anti-FmL antibody. This observation indicates that FmL may contribute to the defense response of this species of penaeid shrimps to potentially pathogenic bacteria.     

Semi‐permeable membrane retention of synovial fluid lubricants hyaluronan and proteoglycan 4 for a biomimetic bioreactor

Synovial fluid (SF) contains lubricant macromolecules, hyaluronan (HA), and proteoglycan 4 (PRG4). The synovium not only contributes lubricants to SF through secretion by synoviocyte lining cells, but also concentrates lubricants in SF due to its semi‐permeable nature. A membrane that recapitulates these synovium functions may be useful in a bioreactor system for generating a bioengineered fluid (BF) similar to native SF. The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 µm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes ± IL‐1β + TGF‐β1 + TNF‐α, a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes ± adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 µg/mL) was added to the BF compartment, and HA and PRG4 loss into the NF compartment after 2, 8, and 24 h was determined. Lubricant loss kinetics were analyzed to estimate membrane permeability. Experiment 1: Cytokine‐regulated HA and PRG4 secretion rates on membranes were comparable to those on TC plastic. Experiment 2: Transport of HA and PRG4 across membranes was lowest with 50 nm membranes and highest with 3 µm membranes, and transport of high MW HA was decreased by adherent synoviocytes (for 50 and 90 nm membranes). The permeability to HA mixtures for 50 nm membranes was ～20 × 10^?8 cm/s (? cells) and ～5 × 10^?8 cm/s (+ cells), for 90 nm membranes was ～35 × 10^?8 cm/s (? cells) and ～19 × 10^?8 cm/s (+ cells), for 170 nm membranes was ～74 × 10^?8 cm/s (± cells), and for 3 µm membranes was ～139 × 10^?8 cm/s (± cells). The permeability of 450 kDa HA was ～40× lower than that of 30 kDa HA for 50 nm membranes, but only ～2.5× lower for 3 µm membranes. The permeability of 4,000 kDa HA was ～250× lower than that of 30 kDa HA for 50 nm membranes, but only ～4× lower for 3 µm membranes. The permeability for PRG4 was ～4 × 10^?8 cm/s for 50 nm membranes, ～48 × 10^?8 cm/s for 90 nm membranes, ～144 × 10^?8 cm/s for 170 nm membranes, and ～336 × 10^?8 cm/s for 3 µm membranes. The associated loss across membranes after 24 h ranged from 3% to 92% for HA, and from 3% to 93% for PRG4. These results suggest that semi‐permeable membranes may be used in a bioreactor system to modulate lubricant retention in a bioengineered SF, and that synoviocytes adherent on the membranes may serve as both a lubricant source and a barrier for lubricant transport. Biotechnol. Bioeng. 2010; 106: 149–160. © 2009 Wiley Periodicals, Inc.     

Isolation and characterization of a lectin from Grifola frondosa fruiting bodies

An N-acetylgalactosamine-specific lectin (GFL) was isolated from Grifola frondosa fruiting bodies by affinity chromatographies on acid-treated Sepharose CL-4B and then GalNAc-Toyopearl. The isolated lectin agglutinated all types of erythrocytes equally. Molecular masses estimated by gel filtration under various buffers and matrices varied from 30 to 52 kDa. On the other hand, SDS-PAGE in the presence or absence of 2-mercaptoethanol showed three major bands of 33, 66 and 100 kDa and a faint band of 65 kDa. This lectin exhibited GalNAc-specificity. The protein was a glycoprotein containing 3.3% total sugar, and the amino acid analysis revealed a high content of acidic and hydroxy amino acids and a low content of methionine and histidine. GFL was cytotoxic against HeLa cells. The toxicity did not appear after preincubating the lectin with the haptenic sugar N-acetylgalactosamine.