Intracellular Trafficking and Glycobiology of TbPDI2, a Stage-Specific Protein Disulfide Isomerase in Trypanosoma brucei

Trypanosoma brucei protein disulfide isomerase 2 (TbPDI2) is a bloodstream stage-specific lumenal endoplasmic reticulum (ER) glycoprotein. ER localization is dependent on the TbPDI2 C-terminal tetrapeptide (KQDL) and is mediated by TbERD2, an orthologue of the yeast ER retrieval receptor. Consistent with this function, TbERD2 localizes prominently to ER exit sites, and RNA interference (RNAi) knockdown results in specific secretion of a surrogate ER retention reporter, BiPN:KQDL. TbPDI2 is highly N-glycosylated and is reactive with tomato lectin, suggesting the presence of poly-N-acetyllactosamine modifications, which are common on lyso/endosomal proteins in trypanosomes but are inconsistent with ER localization. However, TbPDI2 is reactive with tomato lectin immediately following biosynthesis—far too rapidly for transport to the Golgi compartment, the site of poly-N-acetyllactosamine addition. TbPDI2 also fails to react with Erythrina cristagalli lectin, confirming the absence of terminal N-acetyllactosamine units. We propose that tomato lectin binds the Manβ1-4GlcNAcβ1-4GlcNAc trisaccharide core of paucimannose glycans on both newly synthesized and mature TbPDI2. Consistent with this proposal, α-mannosidase treatment renders oligomannose N-glycans on the T. brucei cathepsin L orthologue TbCatL reactive with tomato lectin. These findings resolve contradictory evidence on the location and glycobiology of TbPDI2 and provide a cautionary note on the use of tomato lectin as a poly-N-acetyllactosamine-specific reagent.     

Further characterization of the sialic acid-binding lectin from the horseshoe crab Carcinoscorpius rotunda cauda

A sialic acid-binding lectin, named carcinoscorpin, has been isolated from the horseshoe crab Carcinoscorpius rotunda cauda. It is a glycoprotein of molecular-weight 420,000, having two subunits of molecular weight 27,000 and 28,000, both subunits responding to glycoprotein stain. Leucine was detected as the only NH₂-terminal amino acid. The sedimentation constant of the native lectin was found to be 12.7 s. On digestion with trypsin, the lectin gave 18 soluble tryptic peptides. This lectin was found to be antigenically unrelated to another sialic acid-binding lectin, limulin, isolated from the horseshoe crab Limulus polyphemus. A lectin-specific disaccharide alcohol namely O-(N-acetylneuraminyl) (2 → 6)2-acetamido-2-deoxy-d-galactitol was found to quench the typical tryptophan fluorescence of the native lectin at 332 nm. The association constant for this interaction was determined spectrofluorimetrically and found to be 1.82 × 10³m^?1.     

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.     

Purification,characterization of mosquito larvicidal lectin from Annona muricata and its eco-toxic effect on non-target organism

The present study investigates the purification and characterization of mosquito larvicidal lectin from the seed kernel extract of Annona muricata and toxic effects on non-target organism Chironomus costatus. Soursop lectin was purified by anion-exchange column chromatography using DEAE-cellulose with approximately molecular mass of 260 kDa and with seven distinct subunits (16, 18, 21, 22, 24, 73 and 95 kDa). Soursop lectin highest Hemagglutination (HA) titer value of 128 was recorded against hen indicator RBC type with the influence of different divalent cations such as Ca²⁺, Ba²⁺ and Mn²⁺. The lectin mediated HA activity was highly inhibited by monosaccharides of glucose and mannose and disaccharides such as trehalose and melibiose. It was found to be EDTA sensitive (at 30 mM), pH-dependent (between 6–9) and heat-labile (upto 60 °C). A significant homology for soursop lectin was recorded to leguminous seed lectin from Psophocarpus scandens in MALDI-TOF-MS analysis with 66 % of sequence coverage. Finally, the toxicity bioassay of soursop lectin resulted in 100 % larval mortality for A. aegypti at 48 h whereas only 10 % mortality recorded to non-target organism C. costatus. Therefore, we concluded that soursop lectin could be a potent insecticidal agent in integrated pest management for controlling various insect pests.     

The Lectin Pathway of Complement Activation Is a Critical Component of the Innate Immune Response to Pneumococcal Infection

The complement system plays a key role in host defense against pneumococcal infection. Three different pathways, the classical, alternative and lectin pathways, mediate complement activation. While there is limited information available on the roles of the classical and the alternative activation pathways of complement in fighting streptococcal infection, little is known about the role of the lectin pathway, mainly due to the lack of appropriate experimental models of lectin pathway deficiency. We have recently established a mouse strain deficient of the lectin pathway effector enzyme mannan-binding lectin associated serine protease-2 (MASP-2) and shown that this mouse strain is unable to form the lectin pathway specific C3 and C5 convertases. Here we report that MASP-2 deficient mice (which can still activate complement via the classical pathway and the alternative pathway) are highly susceptible to pneumococcal infection and fail to opsonize Streptococcus pneumoniae in the none-immune host. This defect in complement opsonisation severely compromises pathogen clearance in the lectin pathway deficient host. Using sera from mice and humans with defined complement deficiencies, we demonstrate that mouse ficolin A, human L-ficolin, and collectin 11 in both species, but not mannan-binding lectin (MBL), are the pattern recognition molecules that drive lectin pathway activation on the surface of S. pneumoniae. We further show that pneumococcal opsonisation via the lectin pathway can proceed in the absence of C4. This study corroborates the essential function of MASP-2 in the lectin pathway and highlights the importance of MBL-independent lectin pathway activation in the host defense against pneumococci.     

Biosynthesis of the Snowdrop (Galanthus nivalis) Lectin in Ripening Ovaries

The biosynthesis and processing of the Galanthus nivalis agglutinin were studied in vivo in ripening snowdrop ovaries. Using labeling and pulse chase labeling experiments it could be demonstrated that the snowdrop lectin is synthesized as a precursor of relative molecular weight (M_r) 15,000 which is posttranslationally converted into the authentic lectin polypeptide of M_r 13,000 with a half-life of about 6 hours. Gel filtration of an extract of [³H]leucine labeled ovaries on Sepharose 4B showed that a significant portion of the newly synthesized lectin is associated with the particulate fraction. When the organellar fraction was fractionated on isopycnic sucrose gradients this lectin banded in the same density region as the endoplasmic reticulum (ER) marker enzyme NADH cytochrome c reductase. Both radioactivity in lectin and in enzyme activity shifted towards a higher density in the presence of 2 millimolar Mg-acetate indicating that the labeled lectin was associated with the rough ER. Labeled lectin could be chased from the ER with a half-life of 4 hours and then accumulated in the soluble fraction. Whereas the ER-associated lectin contains exclusively polypeptides of M_r 15,000 the soluble fraction contains both precursor molecules and mature lectin polypeptides. The snowdrop lectin in the ER is fully capable of binding immobilized mannose. It is associated into tetramers with an appropriate molecular weight of 60,000. These results indicate that newly synthesized snowdrop lectin is transiently associated with the ER before transport and processing.     

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.     

Binding-site specificity of lectins from Bauhinia purpurea alba,Sophora japonica,and Wistaria floribunda

The binding-site specificities of lectins isolated from the seeds of Baihinia purpurea alba, Sophora japonica, and Wistaria floribunda were studied by hemagglutination-inhibition assays utilizing a variety of saccharides as inhibitors. For Bauhinia lectin, 2-acetamido-2-deoxy-d-galactose was found to be the best monosaccharide inhibitor and the free monosaccharide inhibitor was as active as its glycosides. d-Galactose was a weak inhibitor and so were some of its glycosides. Some of the oligosaccharides having a d-galactose nonreducing terminus were good inhibitors, but substitution on the d-galactose or 2-acetamido-2-deoxy-d-galactose residues with other saccharides abolished the inhibitory activity. No specificity for anomeric configuration or linkage position could be demonstrated. The presence of aromatic aglycon groups did not enhance inhibitory activity of the saccharides tested and, in some cases, the inhibitory activity was decreased. In contrast to the results for the Bauhinia lectin, compounds having aromatic aglycon groups were markedly better inhibitors for Sophora and Wistaria lectins than the corresponding compounds without aromatic aglycons. d-Galactose was a weak inhibitor for Sophora and Wistaria lectins, whereas 2-acetamido-d-galactose was a poor inhibitor of Sophora lectin but a good inhibitor of Wistaria lectin. Sophora and Wistaria lectins were somewhat similar in their activity as some of the saccharides having a d-galactose in penultimate position to an l-fucose residue were weak inhibitors. However, Sophora lectin has a binding preference for β anomers, whereas Wistaria lectin did not demonstrate a clear preference for α or β anomers. For some pairs of compounds, the α was a better inhibitor than, the β anomer; in other cases, the reverse was true.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Sucrose-binding lectin in regenerating cockroach (Periplaneta americana) legs: Its purification from adult hemolymph

Previously, we purified Periplaneta lectin from the hemolymph of adult Periplaneta americana (American cockroach) (Kubo and Natori Eur. J. Biochem. 168, 75–82, 1987). Immunoblotting analysis using antibody against Periplaneta lectin showed that the cockroach hemolymph contains another lectin that cross reacted immunologically with Periplaneta lectin. We have purified this new lectin (regenectin) to homogeneity. Affinity purified antibody against regenectin cross reacted with Periplaneta lectin. Thus, Periplaneta lectin and this new lectin were found to be different lectins sharing common antigenicity. After leg amputation, this new lectin was found to appear transiently at a specific stage of regeneration as revealed by immunoblotting, suggesting that it plays a role in the regeneration process.     

Terminal N-Acetylgalactosamine-Specific Leguminous Lectin from Wisteria japonica as a Probe for Human Lung Squamous Cell Carcinoma

Millettia japonica was recently reclassified into the genus Wisteria japonica based on chloroplast and nuclear DNA sequences. Because the seed of Wisteria floribunda expresses leguminous lectins with unique N-acetylgalactosamine-binding specificity, we purified lectin from Wisteria japonica seeds using ion exchange and gel filtration chromatography. Glycan microarray analysis demonstrated that unlike Wisteria floribunda and Wisteria brachybotrys lectins, which bind to both terminal N-acetylgalactosamine and galactose residues, Wisteria japonica lectin (WJA) specifically bound to both α- and β-linked terminal N-acetylgalactosamine, but not galactose residues on oligosaccharides and glycoproteins. Further, frontal affinity chromatography using more than 100 2-aminopyridine-labeled and p-nitrophenyl-derivatized oligosaccharides demonstrated that the ligands with the highest affinity for Wisteria japonica lectin were GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc, with K _a values of 9.5 × 10⁴ and 1.4 × 10⁵ M^-1, respectively. In addition, when binding was assessed in a variety of cell lines, Wisteria japonica lectin bound specifically to EBC-1 and HEK293 cells while other Wisteria lectins bound equally to all of the cell lines tested. Wisteria japonica lectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence of N-acetylgalactosamine, but not galactose, mannose, or N-acetylglucosamine, and was completely abrogated by β-hexosaminidase-digestion of these cells. These results clearly demonstrate that Wisteria japonica lectin binds to terminal N-acetylgalactosamine but not galactose. In addition, histochemical analysis of human squamous cell carcinoma tissue sections demonstrated that Wisteria japonica lectin specifically bound to differentiated cancer tissues but not normal tissue. This novel binding characteristic of Wisteria japonica lectin has the potential to become a powerful tool for clinical applications.     

Lectin Domains of Polypeptide GalNAc Transferases Exhibit Glycopeptide Binding Specificity

UDP-GalNAc:polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) constitute a family of up to 20 transferases that initiate mucin-type O-glycosylation. The transferases are structurally composed of catalytic and lectin domains. Two modes have been identified for the selection of glycosylation sites by GalNAc-Ts: confined sequence recognition by the catalytic domain alone, and concerted recognition of acceptor sites and adjacent GalNAc-glycosylated sites by the catalytic and lectin domains, respectively. Thus far, only the catalytic domain has been shown to have peptide sequence specificity, whereas the primary function of the lectin domain is to increase affinity to previously glycosylated substrates. Whether the lectin domain also has peptide sequence selectivity has remained unclear. Using a glycopeptide array with a library of synthetic and recombinant glycopeptides based on sequences of mucins MUC1, MUC2, MUC4, MUC5AC, MUC6, and MUC7 as well as a random glycopeptide bead library, we examined the binding properties of four different lectin domains. The lectin domains of GalNAc-T1, -T2, -T3, and -T4 bound different subsets of small glycopeptides. These results indicate an additional level of complexity in the initiation step of O-glycosylation by GalNAc-Ts.     

Multiple molecular forms of peanut lectin: Classification of isolectins and isolectin distribution among genotypes of the genus Arachis

Peanut lectin (or an immunologically indistinguishable material) is present in seeds of 4556 genotypes of the peanut, Arachis hypogaea, and in 65 genotypes of related species of Arachis. Seeds of one line of A. villosa and three lines of unclassified Arachis spp. are devoid of the lectin. Peanut lectin from 116 A. hypogaea genotypes is resolved by isoelectric focusing into three related isolectin profiles, which are designated the V, S, and V₂ types. Each is composed of from six to eight separate isolectins. Peanut lectin from A. monticola, A. pusilla, and one genotype of Arachis spp. is of the V type; isolectin profiles from other wild Arachis genotypes are variable, but comprise several distinct groups. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolves peanut lectin preparations from 37 genotypes into the lectin subunit of M_r 30,000 and a second polypeptide of M_r 60,000. Lectin preparations from five genotypes lack the M_r 60,000 polypeptide band and have a subunit that migrates slightly faster (and therefore probably is of lower molecular weight) than the subunits of all other tested lines. Peanut lectin preparations from 62 lines have specific hemagglutinating activities ranging from 1024 to 4196 with desialyzed human Type O erythrocytes. The lectin from one genotype exhibits substantially less hemagglutinating activity and is hemolytic.     

Purification and Characterization of a Mucin Specific Mycelial Lectin from Aspergillus gorakhpurensis: Application for Mitogenic and Antimicrobial Activity

Background

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis.

Methods

Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay.

Results

Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5–9.5, while optimum temperature for lectin activity was 20–30°C. Lectin was stable within a pH range of 7.0–10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae.

Conclusion

This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this 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.     

Regulation of Adherence and Virulence by the Entamoeba histolytica Lectin Cytoplasmic Domain, Which Contains a β2 Integrin Motif

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the β2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin’s role in virulence.     

Conformation and activity ofPhaseolus coccineus var. rubronanus lectin

The conformation of native and denaturedPhaseolus coccineus var. rubronanus lectin was studied by circular dichroism (CD) and correlated to the hemagglutinating activity. The far-UV CD spectrum at 25°C showed a broad, negative band around 223 nm and a positive one at 196 nm. CD data analysis of the lectin indicated a β-sheet-rich protein. At high temperatures, the spectrum was blue-shifted with increasing magnitude; these changes correlated well with the loss of the activity. The conformation of lectin betweenpH 2 and 10 remained essentially unchanged. AtpH 13 the CD spectrum resembled that of unordered form with a negative band near 200 nm and the activity was completely lost. The denatured lectin in 6 M guanidine hydrochloride would be renatured upon diluting the denaturant to 0.75 M; the changes in CD spectrum again correlated well with the loss of the activity. The effect of sodium dodecyl sulfate on the lectin was drastic; it sharply increased thea-helix at the expense of the β-sheet and reduced the activity; the changes reached a plateau above 20 mM surfactant.     

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.

     

Interaction of pneumococcal S-14 polysaccharide with lectins from Ricinus communis,Triticum vulgaris, and bandeiraea simplicifolia

Two purified lectins, namely, wheat-germ agglutinin (from Triticum vulgaris) and the hemagglutinin from Ricinus communis seeds, readily form a precipitate with pneumococcal S-14 polysaccharide, whereas the Bandeiraea simplicifolia lectin (BS 1) does not. Exhaustive periodate oxidation and borohydride reduction of S 14 modifies terminal β-D-galactopyranosyl residues, as well as chain D-glucopyranosyl residues, and abolishes reactivity with both the R. communis lectin and wheat-germ agglutinin. Controlled periodate oxidation followed by Smith degradation cleaves only terminal β-D-galactopyranosyl residues, giving a linear polymer, the structure of which was determined by methylation analysis. This derived polymer, containing (1→6)-linked 2-acetamido-2-deoxy-β-D-glucosyl residues, readily precipitated wheat-germ agglutinin, but not the R. communis lectin.     

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.