Nematotoxicity of Marasmius oreades agglutinin (MOA) depends on glycolipid binding and cysteine protease activity

Fruiting body lectins have been proposed to act as effector proteins in the defense of fungi against parasites and predators. The Marasmius oreades agglutinin (MOA) is a Galα1,3Gal/GalNAc-specific lectin from the fairy ring mushroom that consists of an N-terminal ricin B-type lectin domain and a C-terminal dimerization domain. The latter domain shows structural similarity to catalytically active proteins, suggesting that, in addition to its carbohydrate-binding activity, MOA has an enzymatic function. Here, we demonstrate toxicity of MOA toward the model nematode Caenorhabditis elegans. This toxicity depends on binding of MOA to glycosphingolipids of the worm via its lectin domain. We show further that MOA has cysteine protease activity and demonstrate a critical role of this catalytic function in MOA-mediated nematotoxicity. The proteolytic activity of MOA was dependent on high Ca(2+) concentrations and favored by slightly alkaline pH, suggesting that these conditions trigger activation of the toxin at the target location. Our results suggest that MOA is a fungal toxin with intriguing similarities to bacterial binary toxins and has a protective function against fungivorous soil nematodes.     

Mannose-binding plant lectins: different structural scaffolds for a common sugar-recognition process

Mannose-specific lectins are widely distributed in higher plants and are believed to play a role in recognition of high-mannose type glycans of foreign micro-organisms or plant predators. Structural studies have demonstrated that the mannose-binding specificity of lectins is mediated by distinct structural scaffolds. The mannose/glucose-specific legume (e.g., Con A, pea lectin) exhibit the canonical twelve-stranded beta-sandwich structure. In contrast to legume lectins that interact with both mannose and glucose, the monocot mannose-binding lectins (e.g., the Galanthus nivalis agglutinin or GNA from bulbs) react exclusively with mannose and mannose-containing N-glycans. These lectins possess a beta-prism structure. More recently, an increasing number of mannose-specific lectins structurally related to jacalin (e.g., the lectins from the Jerusalem artichoke, banana or rice), which also exhibit a beta-prism organization, were characterized. Jacalin itself was re-defined as a polyspecific lectin which, in addition to galactose, also interacts with mannose and mannose-containing glycans. Finally the B-chain of the type II RIP of iris, which has the same beta-prism structure as all other members of the ricin-B family, interacts specifically with mannose and galactose. This structural diversity associated with the specific recognition of high-mannose type glycans highlights the importance of mannose-specific lectins as recognition molecules in higher plants.     

Cloning and characterization of a monocot mannose-binding lectin from Crocus vernus (family Iridaceae).

The molecular structure and carbohydrate-binding activity of the lectin from bulbs of spring crocus (Crocus vernus) has been determined unambiguously using a combination of protein analysis and cDNA cloning. Molecular cloning revealed that the lectin called C. vernus agglutinin (CVA) is encoded by a precursor consisting of two tandemly arrayed lectin domains with a reasonable sequence similarity to the monocot mannose-binding lectins. Post-translational cleavage of the precursor yields two equally sized polypeptides. Mature CVA consists of two pairs of polypeptides and hence is a heterotetrameric protein. Surface plasmon resonance studies of the interaction of the crocus lectin with high mannose-type glycans showed that the lectin interacts specifically with exposed alpha-1,3-dimannosyl motifs. Molecular modelling studies confirmed further the close relationships in overall fold and three-dimensional structure of the mannose-binding sites of the crocus lectin and other monocot mannose-binding lectins. However, docking experiments indicate that only one of the six putative mannose-binding sites of the CVA protomer is active. These results can explain the weak carbohydrate-binding activity and low specific agglutination activity of the lectin. As the cloning and characterization of the spring crocus lectin demonstrate that the monocot mannose-binding lectins occur also within the family Iridaceae a refined model of the molecular evolution of this lectin family is proposed.     

Photolabelling the urotensin II receptor reveals distinct agonist- and partial-agonist-binding sites

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.     

Studies on Galalpha3-binding proteins: comparison of the glycosphingolipid binding specificities of Marasmius oreades lectin and Euonymus europaeus lectin

The carbohydrate binding preferences of the Galalpha3Galbeta4 GlcNAc-binding lectins from Marasmius oreades and Euonymus europaeus were examined by binding to glycosphingolipids on thin-layer chromatograms and in microtiter wells. The M. oreades lectin bound to Galalpha3-terminated glycosphingolipids with a preference for type 2 chains. The B6 type 2 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) was preferred over the B5 glycosphingolipid (Galalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), suggesting that the alpha2-linked Fuc is accommodated in the carbohydrate binding site, providing additional interactions. The lectin from E. europaeus had broader binding specificity. The B6 type 2 glycosphingolipid was the best ligand also for this lectin, but binding to the B6 type 1 glycosphingolipid (Galalpha3[Fucalpha2]Galbeta3GlcNAcbeta3Galbeta4Glcbeta1Cer) was also obtained. Furthermore, the H5 type 2 glycosphingolipid (Fucalpha2Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer), devoid of a terminal alpha3-linked Gal, was preferred over the the B5 glycosphingolipid, demonstrating a significant contribution to the binding affinity by the alpha2-linked Fuc. The more tolerant nature of the lectin from E. europaeus was also demonstrated by the binding of this lectin, but not the M. oreades lectin, to the x2 glycosphingolipid (GalNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GlcNAcbeta3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer. The A6 type 2 glycosphingolipid (GalNAcalpha3[Fucalpha2]Galbeta4GlcNAcbeta3Galbeta4Glcbeta1Cer) and GalNAcalpha3Galbeta4GlcNAcbeta3Galbeta4Glcbeta1-Cer were not recognized by the lectins despite the interaction with B6 type 2 glycosphingolipid and the B5 glycosphingolipid. These observations are explained by the absolute requirement of a free hydroxyl in the 2-position of Galalpha3 and that the E. europaea lectin can accommodate a GlcNAc acetamido moiety close to this position by reorienting the terminal sugar, whereas the M. oreades lectin cannot.     

Studies on lectins. XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis.

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7--9. For concanavalin A and the L-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized alpha-D-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different lentil varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.     

Crystal structure of the Marasmius oreades mushroom lectin in complex with a xenotransplantation epitope

MOA, a lectin from the mushroom Marasmius oreades, is one of the few reagents that specifically agglutinate blood group B erythrocytes. Further, it is the only lectin known to have exclusive specificity for Galalpha(1,3)Gal-containing sugar epitopes, which are antigens that pose a severe barrier to animal-to-human organ transplantation. We describe here the structure of MOA at 2.4 A resolution, in complex with the linear trisaccharide Galalpha(1,3)Galbeta(1,4)GlcNAc. The structure is dimeric, with two distinct domains per protomer: the N-terminal lectin module adopts a ricinB/beta-trefoil fold and contains three putative carbohydrate-binding sites, while the C-terminal domain serves as a dimerization interface. This latter domain, which has an unknown function, reveals a novel fold with intriguing conservation of an active site cleft. A number of indications suggest that MOA may have an enzymatic function in addition to the sugar-binding properties.     

Studies on lectins. XXXVI. Properties of some lectins prepared by affinity chromatography on O-glycosyl polyacrylamide gels

A number of lectins has been purified by affinity chromatography on O-glycosyl polyacrylamide gels. The lectins isolated (and the particular sugar ligands used in the affinity carriers) are as follows: Anguilla anguilla, serum (alpha-L-fucosyl-), Vicia cracca, seeds; Phaseolus lunatus, seeds; Glycine soja, seeds; Dolichos biflorus, seeds; Maclura pomifera, seeds; Sarothamnus scoparius, seeds; Helix pomatia, ablumin glands; Clitocybe nebularis, fruiting bodies (all N-acetyl-alpha-D-galactosaminyl-); Ricinus communis, seeds (beta-lactosyl-); Ononis spinosa, root; Fomes fomentarius, fruiting bodies; Marasmius oreades, fruiting bodies (all alpha-D-galactosyl-), Canavalia ensiformis, seeds, (i.e., concanavalin A) (alpha-D-glucosyl-). Physicochemical properties of Glycine soja, Dolichos biflorus, Phaseolus lunatus, Helix Pomatia and Ricinus communis lectins corresponded well to properties of the preparations studied earlier by other workers. For the other purified lectins the essential physiochemical data (sedimentation coefficient, molecular weight, subunit composition, electrophoretic patterns, amino acid composition, carbohydrate content, isoelectric point) were established and their precipitating, hemagglutinating and mitogenic activities determined.     

Analysis of sugar chain-binding specificity of tomato lectin using lectin blot: recognition of high mannose-type N-glycans produced by plants and yeast

The sugar chain-binding specificity of tomato lectin (LEA) against glycoproteins was investigated qualitatively using lectin blot analysis. Glycoproteins containing tri- and tetra-antennary complex-type N-glycans were stained with LEA. Unexpectedly, glycoproteins containing high mannose-type N-glycans and a horseradish peroxidase were stained with LEA. LEA blot analysis of the glycoproteins accompanied by treatment with exoglycosidase revealed that the binding site of LEA for the complex-type N-glycans was the N-acetyllactosaminyl side chains, whereas the proximal chitobiose core appeared to be the binding site of LEA for high mannose-type N-glycans. Despite these results, the glycoproteins did not inhibit the hemagglutinating activity of LEA. Among the chitin-binding lectins compared, potato tuber lectin showed specificity similar to LEA on lectin blot analysis, while Datura stramonium lectin and wheat germ agglutinin (WGA) did not interact with glycoproteins containing high mannose-type N-glycans, except that RNase B was stained by WGA. Based on these observations, LEA blot analysis was applied to sugar chain analysis of tomato glycoproteins. The most abundant LEA-reactive glycoprotein was purified from the exocarp of ripe tomato fruits, and was identified as the tomato anionic peroxidase1 (TAP1). These results suggest that LEA interacts with glycoproteins produced by tomatoes, which participate in biological activities in tomato plants.     

Comparative analysis by frontal affinity chromatography of oligosaccharide specificity of GlcNAc-binding lectins, Griffonia simplicifolia lectin-II (GSL-II) and Boletopsis leucomelas lectin (BLL)

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans.     

Isolation and characterization of a new mannose-binding lectin gene from<Emphasis Type="Italic">Taxus media</Emphasis>

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species,Taxus media. The full-length cDNA ofT. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and thattma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed thattma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.     

A novel mannose-binding tuber lectin from Typhonium divaricatum (L.) Decne (family Araceae) with antiviral activity against HSV-II and anti-proliferative effect on human cancer cell lines

A novel mannose-binding tuber lectin with in vitro antiproliferative activity towards human cancer cell lines and antiviral activity against HSV-II was isolated from fresh tubers of a traditional Chinese medicinal herb, Typhonium divaricatum (L.) Decne by a combined procedure involving extraction, ammonium sulfate precipitation, ion exchange chromatography on DEAE-SEPHAROSE, CM-SEPHAROSE and gel-filtration on sephacryl S-200. The apparent molecular mass of the purified Typhonium divaricatum lectin (TDL) was 48 kDa. TDL exhibits hemagglutinating activity toward rabbit erythrocytes at 0.95 microg/ml, and its activity could be strongly inhibited by mannan, ovomucoid, asialofetuin and thyroglobulin. TDL showed antiproliferative activity towards some well established human cancer cell lines, e.g. Pro-01 (56.7 +/- 6.8), Bre-04 (41.5 +/- 4.8), and Lu-04 (11.4 +/- 0.3). The anti-HSV-II activity of TDL was elucidated by testing its HSV-II infection inhibitory activity in Vero cells with TC(50) and EC(50) of 5.176 mg/ml and 3.054 microg/ml respectively. The full-length cDNA sequence of TDL was 1145 bp and contained an 813-bp open reading frame (ORF) encoding a 271 amino acid precursor of 29-kDa. Homology analysis showed that TDL had high homology with many other mannose-binding lectins. Secondary and three-dimensional structures analyses showed that TDL is heterotetramer and similar with lectins from mannose-binding lectin superfamily, especially those from family Araceae.     

Cloning and characterization of the Atlantic salmon serum lectin,a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.     

Cloning and characterization of the Atlantic salmon serum lectin,a long-form C-type lectin expressed in kidney

We report the cloning of four distinct cDNAs and a genomic sequence encoding a multimeric serum lectin found in the blood of Atlantic salmon (Salmo salar). The sequence variation among the cDNAs as well as genomic Southern blotting analysis revealed a multi-gene family. Expression of the salmon serum lectin (SSL) was specific to kidney, as demonstrated by RT-PCR. Analysis of the 173-amino acid sequence of SSL confirmed that it is a member of the C-type lectin superfamily. Sequence alignments and intron/exon structure of the SSL gene showed it to belong to the type VII C-type lectins, which normally bind to galactose or other ligands, whereas the SSL protein sequence contains the EPN motif of mannose-binding C-type lectins, that bind mannose or related carbohydrates.     

Primary structure and carbohydrate binding specificity of a potent anti-HIV lectin isolated from the filamentous cyanobacterium Oscillatoria agardhii

The primary structure of a lectin, designated Oscillatoria agardhii agglutinin (OAA), isolated from the freshwater cyanobacterium O. agardhii NIES-204 was determined by the combination of Edman degradation and electron spray ionization-mass spectrometry. OAA is a polypeptide (Mr 13,925) consisting of two tandem repeats. Interestingly, each repeat sequence of OAA showed a high degree of similarity to those of a myxobacterium, Myxococcus xanthus hemagglutinin, and a marine red alga Eucheuma serra lectin. A systematic binding assay with pyridylaminated oligosaccharides revealed that OAA exclusively binds to high mannose (HM)-type N-glycans but not to other N-glycans, including complex types, hybrid types, and the pentasaccharide core or oligosaccharides from glycolipids. OAA did not interact with any of free mono- and oligomannoses that are constituents of the branched oligomannosides. These results suggest that the core disaccharide, GlcNAc-GlcNAc, is also essential for binding to OAA. The binding activity of OAA to HM type N-glycans was dramatically decreased when alpha1-2 Man was attached to alpha1-3 Man branched from the alpha1-6 Man of the pentasaccharide core. This specificity of OAA for HM-type oligosaccharides is distinct from other HM-binding lectins. Kinetic analysis with an HM heptasaccharide revealed that OAA possesses two carbohydrate binding sites per molecule, with an association constant of 2.41x10(8) m-1. Furthermore, OAA potently inhibits human immunodeficiency virus replication in MT-4 cells (EC50=44.5 nm). Thus, we have found a novel lectin family sharing similar structure and carbohydrate binding specificity among bacteria, cyanobacteria, and marine algae.     

Primary structure of a fucose-specific lectin obtained from a mushroom, Aleuria aurantia

Aleuria aurantia lectin (AAL) is a protein composed of two identical subunits having no carbohydrate chain and shows sugar-binding specificity for L-fucose. Full-length cDNA encoding for the lectin has been isolated from a lambda gt11 library, screened with an antiserum directed against AAL. The cDNA clone contained 1,370 nucleotides and an open reading frame of 939 nucleotides encoding 313 amino acids. The amino-terminal sequence (residues 1-30) of the lectin isolated from the mushroom coincided with the deduced amino acid sequence starting from proline at the 2nd residue, indicating that the mature AAL consists of 312 amino acids. Its molecular weight is calculated to be 33,398. The deduced amino acid sequence shows that AAL includes six internal homologous regions, and has considerable homology with a hemagglutinin from a Gram-negative bacterium, Myxococcus xanthus, which forms a fruiting body. No significant homology was observed with higher plant or animal lectins. The recombinant AAL produced by Escherichia coli JM109 carrying the AAL expression plasmid pKA-1 [Fukumori, F. et al. (1989) FEBS Lett. 250, 153-156] was purified from the cell lysate by affinity chromatography using a fucose-starch column, and hundreds of milligrams of the lectin was obtained. The recombinant lectin showed the same biochemical characteristics and sugar binding specificity as did the natural AAL.     

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 ? resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer.     

Molecular diversity of skin mucus lectins in fish

Among lectins in the skin mucus of fish, primary structures of four different types of lectin have been determined. Congerin from the conger eel Conger myriaster and AJL-1 from the Japanese eel Anguilla japonica were identified as galectin, characterized by its specific binding to β-galactoside. Eel has additionally a unique lectin, AJL-2, which has a highly conserved sequence of C-type lectins but displays Ca²⁺-independent activity. This is rational because the lectin exerts its function on the cutaneous surface, which is exposed to a Ca²⁺ scarce environment when the eel is in fresh water. The third type lectin is pufflectin, a mannose specific lectin in the skin mucus of pufferfish Takifugu rubripes. This lectin showed no sequence similarity with any known animal lectins but, surprisingly, shares sequence homology with mannose-binding lectins of monocotyledonous plants. The fourth lectin was found in the ponyfish Leiognathus nuchalis and exhibits homology with rhamnose-binding lectins known in eggs of some fish species. These lectins, except ponyfish lectin, showed agglutination of certain bacteria. In addition, pufflectin was found to bind to a parasitic trematode, Heterobothrium okamotoi. Taken together, these results demonstrate that skin mucus lectins in fish have wide molecular diversity.     

First report of a xylose-specific lectin with potent hemagglutinating, antiproliferative and anti-mitogenic activities from a wild ascomycete mushroom

From fresh fruiting bodies of the wild ascomycete mushroom (Xylaria hypoxylon) a lectin with N-terminal sequence resemblance to a part of Aspergillus oryzae genome and only slight similarity to fungal immunomodulatory protein from the mushroom Flammulina velutipes was isolated. The protocol comprised extraction with water, precipitation from the aqueous extract using 80% saturated (NH(4))(2)SO(4), ion exchange chromatography on DEAE-cellulose and CM-cellulose, and then gel filtration by fast protein liquid chromatography on Superdex 75. Lectin activity was adsorbed on DEAE-cellulose and unadsorbed on CM-cellulose. The lectin appeared as a single band with a molecular mass of 14.4 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single 28.8-kDa peak in gel filtration on Superdex 75. The lectin exhibited highly potent antiproliferative activity toward tumor cell lines, and exerted a potent anti-mitogenic action on mouse splenocytes. The hemagglutinating activity of the lectin was inhibited by inulin and xylose. It was stable up to 35 degrees C. At 40 degrees C its hemagglutinating activity was reduced by 50%, and it dwindled to 12.5% of the original activity at 50 degrees C. The hemagglutinating activity was also sensitive to NaOH and HCl solutions. The hemagglutinating activity was unaffected by CaCl(2) and ZnCl(2), and was potentiated substantially in the presence of AlCl(3) and FeCl(3). The distinctive features of this lectin comprise a unique sugar specificity, and highly potent hemagglutinating, antiproliferative and anti-mitogenic activities. X. hypoxylon lectin differs in molecular mass, N-terminal sequence and sugar specificity from previously reported ascomycete mushroom lectins.     

Lectins homologous to those of monocotyledonous plants in the skin mucus and intestine of pufferfish,Fugu rubripes

We have characterized pufflectin, a novel mannose-specific lectin, from the skin mucus of the pufferfish, Fugu rubripes. Molecular mass estimations by gel filtration and matrix-assisted laser desorption ionization time-of-flight mass spectrometry and the SDS-PAGE pattern suggest that pufflectin is a homodimer composed of non-covalently associated subunits of 13 kDa. The full-length pufflectin cDNA consists of 527 bp, with 116 amino acid residues deduced from the open reading frame. The amino acid sequence of pufflectin shows no homology with any known animal lectin. Surprisingly, pufflectin shares sequence homology with mannose-binding lectins of monocotyledonous plants and has conserved two of three carbohydrate recognition domains of these plant lectins. The pufflectin gene is expressed in gills, oral cavity wall, esophagus, and skin. In addition, an isoform occurs exclusively in the intestine. Pufflectin differs from mannose-binding lectins purified from the blood plasma of Fugu. Whereas pufflectin did not agglutinate five bacterial species tested, it was demonstrated to bind to the parasitic trematode, Heterobothrium okamotoi. This finding suggests that pufflectin contributes to the parasite-defense system in Fugu.