Interaction of Pseudomonas aeruginosa galactophilic lectin PA-IL with pigeon egg white glycoproteins

Pseudomonas aeruginosa produces a galactophilic lectin, PA-IL, that resembles P-fimbrial adhesins of uropathogenic Escherichia coli strains in binding to human P blood group antigens. We examined, in the present study, its interaction with pigeon egg white glycoproteins carrying N-glycans with terminal Galalpha1-4Gal which inhibit the adhesion of P-fimbriae. For comparison, the lectin concanavalin A (Con A) and additional avian egg whites (of hen and quail) were also examined. The results obtained in both hemagglutination inhibition and Western blot analyses showed that PA-IL, unlike Con A, preferentially reacted with the pigeon egg white glycoproteins. These results, which confirmed PA-IL similarity in sugar specificity to E. coli P-fimbriae, demonstrated the advantage of this purified lectin for representing P-type and additional galactophilic microbial adhesins unavailable in purified stable form, in Western blot analyses.     

Xenopus galectin-VIIa binds N-glycans of members of the cortical granule lectin family (xCGL and xCGL2)

We have identified members of the Xenopus cortical granule lectin (xCGL) family as candidate target glycoproteins of Xenopus galectin-VIIa (xgalectin-VIIa) in Xenopus embryos. In addition to the original xCGL, we also identified a novel member of the xCGL family, xCGL2. Expression of the mRNAs of xCGL and xCGL2, as well as that of xgalectin-VIIa, was observed throughout early embryogenesis. Two and three potential N-glycosylation sites were deduced from the amino acid sequences of xCGL and xCGL2, respectively, and xgalectin-VIIa recognizes N-glycans linked to a common site in xCGL and xCGL2 and also recognizes N-glycans linked to a site specific to xCGL2. However, interaction between xgalectin-Ia and xCGLs was not detectable. We also obtained consistent results on surface plasmon resonance analysis involving xCGLs as ligands and xgalectins as analytes. The Kd value of the interaction between xgalectin-VIIa and xCGLs was calculated to be 35.9 nM. The structures of the N-glycans of xCGLs, which were recognized by xgalectin-VIIa, were analyzed by the two-dimensional sugar map method, and three kinds of N-acetyllactosamine type, biantennary N-glycans were identified as the major neutral N-glycans. The binding specificity of oligosaccharides for xgalectin-VIIa was analyzed by frontal affinity chromatography (FAC). The oligosaccharide specificity pattern of xgalectin-VIIa was similar to that of the human homolog galectin-3, and it was also shown that the N-acetyllactosamine type, biantennary N-glycans exhibit high affinity for xgalectin-VIIa (Kd = 11 microM). These results suggest that xgalectin-VIIa interacts with xCGLs through binding to N-acetyllactosamine type N-glycans and that this interaction might make it possible to organize a lectin network involving members of different lectin families.     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

N-glycan structures of pigeon IgG: a major serum glycoprotein containing Galalpha1-4 Gal termini.

We had shown previously that all major glycoproteins of pigeon egg white contain Galalpha1-4Gal epitopes (Suzuki, N., Khoo, K. H., Chen, H. C., Johnson, J. R., and Lee, Y. C. (2001) J. Biol. Chem. 276, 23221-23229). We now report that Galalpha1-4Gal-bearing glycoproteins are also present in pigeon serum, lymphocytes, and liver, as probed by Western blot with Griffonia simplicifolia-I lectin (specific for terminal alpha-Gal) and anti-P1 (specific for Galalpha1-4Galbeta1-4GlcNAcbeta1-) monoclonal antibody. One of the major glycoproteins from pigeon plasma was identified as IgG (also known as IgY), which has Galalpha1-4Gal in its heavy chains. High pressure liquid chromatography, mass spectrometric (MS), and MS/MS analyses revealed that N-glycans of pigeon serum IgG included (i) high mannose-type (33.3%), (ii) disialylated biantennary complex-type (19.2%), and (iii) alpha-galactosylated complex-type N-glycans (47.5%). Bi- and tri-antennary oligosaccharides with bisecting GlcNAc and alpha1-6 Fuc on the Asn-linked GlcNAc were abundant among N-glycans possessing terminal Galalpha1-4Gal sequences. Moreover, MS/MS analysis identified Galalpha1-4Galbeta1-4Galbeta1-4GlcNAc branch terminals, which are not found in pigeon egg white glycoproteins. An additional interesting aspect is that about two-thirds of high mannose-type N-glycans from pigeon IgG were monoglucosylated. Comparison of the N-glycan structures with chicken and quail IgG indicated that the presence of high mannose-type oligosaccharides may be a characteristic of these avian IgG.     

Characterization and cloning of GNA-like lectin from the mushroom Marasmius oreades

A new mannose-recognizing lectin (MOL) was purified on an asialofetuin-column from fruiting bodies of Marasmius oreades grown in Japan. The lectin (MOA) from the fruiting bodies of the same fungi is well known to be a ribosome-inactivating type lectin that recognizes blood-group B sugar. However, in our preliminary investigation, MOA was not found in Japanese fruiting bodies of M. oreades, and instead, MOL was isolated. Gel filtration showed MOL is a homodimer noncovalently associated with two subunits of 13 kDa. The N-terminal sequence of MOL was blocked. The sequence of MOL was determined by cloning from cDNA and by protein sequencing of enzyme-digested peptides. The sequence shows mannose-binding motifs of bulb-type mannose-binding lectins from plants, and similarity to the sequences. Analyses of sugar-binding specificity by hemagglutination inhibition revealed the preference of MOL toward mannose and thyroglobulin, but asialofetuin was the strongest inhibitor of glycoproteins tested. Furthermore, glycan-array analysis showed that the specificity pattern of MOL was different from those of typical mannose-specific lectins. MOL preferred complex-type N-glycans rather than high-mannose N-glycans.     

High mannose-binding lectin with preference for the cluster of alpha1-2-mannose from the green alga Boodlea coacta is a potent entry inhibitor of HIV-1 and influenza viruses

The complete amino acid sequence of a lectin from the green alga Boodlea coacta (BCA), which was determined by a combination of Edman degradation of its peptide fragments and cDNA cloning, revealed the following: 1) B. coacta used a noncanonical genetic code (where TAA and TAG codons encode glutamine rather than a translation termination), and 2) BCA consisted of three internal tandem-repeated domains, each of which contains the sequence motif similar to the carbohydrate-binding site of Galanthus nivalis agglutinin-related lectins. Carbohydrate binding specificity of BCA was examined by a centrifugal ultrafiltration-HPLC assay using 42 pyridylaminated oligosaccharides. BCA bound to high mannose-type N-glycans but not to the complex-type, hybrid-type core structure of N-glycans or oligosaccharides from glycolipids. This lectin had exclusive specificity for α1-2-linked mannose at the nonreducing terminus. The binding activity was enhanced as the number of terminal α1-2-linked mannose substitutions increased. Mannobiose, mannotriose, and mannopentaose were incapable of binding to BCA. Thus, BCA preferentially recognized the nonreducing terminal α1-2-mannose cluster as a primary target. As predicted from carbohydrate-binding propensity, this lectin inhibited the HIV-1 entry into the host cells at a half-maximal effective concentration of 8.2 nm. A high association constant (3.71 × 10(8) M(-1)) of BCA with the HIV envelope glycoprotein gp120 was demonstrated by surface plasmon resonance analysis. Moreover, BCA showed the potent anti-influenza activity by directly binding to viral envelope hemagglutinin against various strains, including a clinical isolate of pandemic H1N1-2009 virus, revealing its potential as an antiviral reagent.     

Isolation of a novel plant lectin with an unusual specificity from Calystegia sepium

A novel plant lectin has been isolated from the rhizomes of Calystegia sepium (hedge bindweed) and partially characterized. The lectin is a dimeric protein composed of two identical non-covalently linked subunits of 16kDa. Hapten inhibition studies indicate that the novel lectin is best inhibited by maltose and mannose and hence exhibits a sugar binding specificity that differs in some respects from that of all previously isolated plant lectins. Mitogenicity tests have shown that the Calystegia lectin is a powerful T-cell mitogen. Affinity purification of human, plant and fungal glycoproteins on immobilized C. sepium lectin demonstrates that this novel lectin can be used for the isolation of glycoconjugates from various sources. Moreover, it can be expected that by virtue of its distinct specificity, the new lectin will become an important tool in glycobiology. Abbreviations: Calsepa, lectin isolated from Calystegia sepium; ConA, concanavalin A; LPS, lipopolysaccharide; PBS, phosphate buffered saline (1.5 mMKH2PO4, 10 mM Na2HPO4, 3 mM KCl, 140 mM NaCl, pH 7.4) This revised version was published online in November 2006 with corrections to the Cover Date.     

Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin

The carbohydrate-binding specificity of Aleuria aurantia lectin was investigated by analyzing the behavior of a variety of fucose-containing oligosaccharides on an A. aurantia lectin-Sepharose column. Studies with complex-type oligosaccharides obtained from various glycoproteins by hydrazinolysis and their partial degradation fragments indicated that the presence of the alpha-fucosyl residue linked at the C-6 position of the proximal N-acetylglucosamine moiety is indispensable for binding to the lectin column. Binding was not affected by the structures of the outer chain moieties nor by the presence of the bisecting N-acetylglucosamine residue. These results indicated that A. aurantia lectin-Sepharose is useful for the group separation of mixtures of complex-type asparagine-linked sugar chains. Studies of glycosylated Bence Jones proteins indicated that this procedure is also applicable to intact glycoproteins. The behavior of oligosaccharides isolated from human milk and the urine of patients with fucosidosis indicated that the oligosaccharides with Fuc alpha 1----2Gal beta 1----4GlcNAc and Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups interact with the lectin, but less strongly than complex-type sugar chains with a fucosylated core. Lacto-N-fucopentaitol II, which has a Gal beta 1----3(Fuc alpha 1----4)GlcNAc group, interacts less strongly than the above two groups with the matrix. Oligosaccharides with Fuc alpha 1----2Gal beta 1----3GlcNAc and Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4(Fuc alpha 1----3)GlcNAc groups showed almost no interaction with the matrix.     

Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography

During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependent since lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.     

Sugar-binding properties of VIP36, an intracellular animal lectin operating as a cargo receptor

The vesicular integral protein of 36 kDa (VIP36) is an intracellular animal lectin that acts as a putative cargo receptor, which recycles between the Golgi and the endoplasmic reticulum. Although it is known that VIP36 interacts with glycoproteins carrying high mannose-type oligosaccharides, detailed analyses of the sugar-binding specificity that discriminates isomeric oligosaccharide structures have not yet been performed. In the present study, we have analyzed, using the frontal affinity chromatography (FAC) method, the sugar-binding properties of a recombinant carbohydrate recognition domain of VIP36 (VIP36-CRD). For this purpose, a pyridylaminated sugar library, consisting of 21 kinds of oligosaccharides, including isomeric structures, was prepared and subjected to FAC analyses. The FAC data have shown that glucosylation and trimming of the D1 mannosyl branch interfere with the binding of VIP36-CRD. VIP36-CRD exhibits a bell-shaped pH dependence of sugar binding with an optimal pH value of approximately 6.5. By inspection of the specificity and optimal pH value of the sugar binding of VIP36 and its subcellular localization, together with the organellar pH, we suggest that VIP36 binds glycoproteins that retain the intact D1 mannosyl branch in the cis-Golgi network and recycles to the endoplasmic reticulum where, due to higher pH, it releases its cargos, thereby contributing to the quality control of glycoproteins.     

Purification and characterization of lectin from fruiting body of Ganoderma lucidum: lectin from Ganoderma lucidum

A novel 114 kDa hexameric lectin was purified from the fruiting bodies of the mushroom Ganoderma lucidum. Biochemical characterization revealed it to be a glycoprotein having 9.3% neutral sugar and it showed hemagglutinating activity on pronase treated human erythrocytes. The lectin was stable in the pH range of 5-9 and temperature up to 50 degrees C. The hemagglutinating activity was inhibited by glycoproteins that possessed N-as well as O-linked glycans. Chemical modification of the G. lucidum lectin revealed contribution of tryptophan and lysine to binding activity. The thermodynamics of binding of bi- and triantennary N-glycans to G. lucidum lectin was studied by spectrofluorimetry. The lectin showed very high affinity for asialo N-linked triantennary glycan and a preference for asialo glycans over sialylated glycans. The binding was accompanied with a large negative change in enthalpy as well as entropy, indicating primarily involvement of polar hydrogen, van der Waals and hydrophobic interactions in the binding.     

Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.     

Peanut (Arachis hypogaea) lectin recognizes α-linked galactose, but not N-acetyl lactosamine in N-linked oligosaccharide terminals

Peanut (Arachis hypogaea) agglutinin (PNA) is extensively used as tumour marker as it strongly recognises the cancer specific T antigen (Galβ1→3GalNAc-), but not its sialylated version. However, an additional specificity towards Galβ1→4GlcNAc (LacNAc), which is not tumour specific, had been attributed to PNA. For correct interpretation of lectin histochemical results we examined PNA sugar specificity using naturally occurring or semi-synthetic glycoproteins, matrix-immobilised galactosides and lectin-binding tissue glycoproteins, rather than mono- or disaccharides as ligands. Dot-blots, transfer blots or polystyrene plate coatings of the soluble glycoconjugates were probed with horse-radish peroxidase (HRP) conjugates of PNA and other lectins of known specificity. Modifications of PNA-binding glycoproteins, including selective removal of O-linked oligosaccharides and treatment with glycosidases revealed that Galβ1→4GlcNAc (LacNAc) was ineffective while terminal α-linked galactose (TAG) as well as exposed T antigen (Galβ1→3 GalNAc-) was excellent as sugar moiety in glycoproteins for their recognition by PNA. When immobilised, melibiose was superior to lactose in PNA binding. Results were confirmed using TAG-specific human serum anti-α-galactoside antibody.     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Hevein,an allergenic lectin from rubber latex,activates human neutrophils' oxidative burst

Hevein is an N-acetyl-D-glucosamine (GlcNAc) specific lectin that has been hypothesized to participate in the IgE-mediated allergic reactions in patients with latex allergy. In this work we assessed the specificity and biological effect of hevein purified from rubber latex on human leukocytes, using epifluorescence microscopy and flow cytometry. Purified human granulocytes were stimulated in vitro with hevein, and production of oxidative radicals was measured by reduction of nitroblue tetrazolium formazan. Histochemical staining and flow cytometry showed that hevein recognizes specifically monocytes (CD14+) and neutrophils (CD16+), but not lymphoid cells. Hevein induced oxidative response in purified granulocytes; this effect was 1.3–1.5-fold higher than the effect observed with the lectin WGA (wheat germ agglutinin), or other lectins with different sugar specificity. The induced reactions and cellular recognition by hevein were inhibited with GlcNAc and its oligomers; as well as by glycoproteins containing tri-and tetra-antennary N-glycosydically linked glycans. Our findings suggest that neutrophils are the main target for latex hevein; this lectin induces production of oxidative radicals, which seem to play an important role in tissue damage during latex allergy.     

Interaction with lectin of kappa opioid binding sites solubilized from human placenta

Kappa opioid binding sites from human placenta, prelabeled with 3H-etorphine and solubilized, were retained on wheat germ agglutinin (WGA) agarose and specifically eluted with N-acetylglucosamine. No significant retention was found with other immobilized lectins, including Concanavalin A (Con A), soybean seed lectin (SBA), Pisum sativum lectin (PsA), Lens culinaris Medik. lectin (LcA), and Lathyrus tingitanus lectin(LtA). About 23% of applied kappa sites were specifically eluted from WGA agarose, less than half of the proportion of rat brain opioid binding sites eluted from the same lectin (55%). Receptors from placental extracts were compared with those from other tissues enriched in either kappa or mu sites. The proportion of applied kappa sites from guinea pig cerebellum eluted specifically from WGA agarose was 36%, whereas elution of binding sites from rat thalamus and rabbit cerebellum (enriched in mu sites) was at a level of 55%. This difference in the level of retention on and elution from WGA may reflect differences in the sugar composition of the glycoproteins of the two types of receptors. Succinylation of WGA abolished its ability to retain opioid binding sites, consistent with involvement of sialic acid. However, currently available evidence suggests that differences in retention on WGA between kappa and mu sites may be due to differences in either sialic acid or N-acetylglucosamine content or both.     

Carbohydrate-binding specificity of Tetracarpidium conophorum lectin.

The carbohydrate-binding specificity of a novel plant lectin isolated from the seeds of Tetracarpidium conophorum (Nigerian walnut) has been studied by quantitative hapten inhibition assays and by determining the behavior of a number of oligosaccharides and glycopeptides on lectin-Sepharose affinity columns. The Tetracarpidium lectin shows preference for simple, unbranched oligosaccharides containing a terminal Gal beta 1----4GlNAc sequence over a Gal beta 1----3GlcNAc sequence and substitution by sialic acid or fucose of the terminal galactose residue, the subterminal N-acetylglucosamine or more distally located sugar residues of oligosaccharides reduce binding activity. Branched complex-type glycans containing either Gal beta 1----4GlcNAc or Gal beta 1----3GlcNAc termini bind with higher affinity than simpler oligosaccharides. The lectin shows highest affinity for a tri-antennary glycan carrying Gal beta 1----4GlcNAc substituents on C-2 and C-4 of Man alpha 1----3 and C-2 of Man alpha 1----6 core residues. Bi- and tri-glycans lacking this branching pattern bind more weakly. Tetra-antennary glycans and mono- and di-branched hybrid-type glycans also bind weakly to the immobilized lectin. Therefore, Tetracarpidium lectin complements the binding specificities of well-known lectins such as Datura stramonium agglutinin, Phaseolus vulgaris agglutinin, and lentil lectin and will be a useful additional tool for the identification and separation of complex-type glycans.     

Effect of Hypoxia on Endothelial Cell Surface Glycoprotein Expression: Modulation of Glycoprotein IIIa and Other Specific Surface Glycoproteins

Exposure to hypoxia alters many aspects of endothelial cell metabolism and function; however, changes in surface glycoconjugates under these conditions have not been extensively evaluated. In the current studies, we examined surface glycoproteins of cultured bovine aortic (BAEC) and pulmonary arterial (BPAEC) endothelial cells under standard culture conditions (21% oxygen) and following exposure to hypoxia (0% oxygen) for varying time periods (30 min to 18 h) using a system of biotinylation, lectin binding (concanavalin A, Con A; Griffonia simplicifolia , GSA; Arachis hypogaea, PNA; Ricinus communis, RCA; or Triticum vulgaris, WGA), subsequent strep-avidin binding, and staining. Using these methods, we identified differences in lectin binding between the two cell types cultured in 21% oxygen with all lectins except PNA. With exposure to 0% oxygen, there was no change in lectin binding to most surface glycoproteins. Several surface glycoproteins, including glycoprotein IIIa on both cell types, demonstrated a time-dependent decrease in lectin binding; in addition, there was an increase in lectin binding to a few specific surface glycoproteins on each cell type within 30-60 min of exposure to 0% oxygen. These changes in specific surface glycoproteins were confirmed in both cell types by ¹²⁵I labeling. Increased lectin binding was observed for Con A binding BAEC glycoproteins at molecular weight (MW) 116, 130, and 205 kDa, GSA binding BAEC glycoproteins at MW 120 and 205 kDa, and RCA binding BPAEC glycoproteins at MW 140 and 205 kDa. Increased binding of WGA or PNA was not observed during exposure to hypoxia. The specificity of lectin binding was further confirmed by competitive inhibition with the appropriate sugar. These studies demonstrate that there are baseline differences between BAEC and BPAEC cell surface glycoproteins and that exposure to hypoxia is associated with little change in lectin binding to most surface glycoproteins. There is, however, increased surface expression of a few glycoproteins that differ depending of the origin of the endothelial cell. Although the mechanism of this increase in lectin binding is not yet clear, subsequent studies suggested that it is due to increased availability of select carbohydrate moieties. The time course of these alterations suggests a possible role in the endothelial cell response to decreases in ambient oxygen tension.     

Occurrence of highly yielded lectins homologous within the genus Eucheuma

We previously reported that the red alga Eucheuma serra contains large amounts of mitogenic isolectins (ESA-1 and ESA-2), the hemagglutinating activities of which were strongly inhibited by glycoproteins bearing high mannose-type N-glycans. We therefore further examined two other species, E. amakusaensis and E. cottonii. Several lectins were isolated easily by a combination of extraction with aqueous ethanol, precipitation with cold ethanol, gel filtration, and ion exchange chromatography from both species, respectively. The purified lectins were designated as EAA-1, EAA-2, EAA-3, ECA-1 and ECA-2 after the specific names of both algae. The yields of EAAs and ECAs were as high as 2.8 and 2.7 mg g⁻¹ of dry tissue, respectively, indicating that both species would also be good sources for high lectin yields. The five purified lectins shared the same properties in hemagglutinating activity, mitogenic activity, and hemagglutination-inhibition test in which glycoproteins bearing high mannose-type N-glycans were the most inhibitory. They also had almost identical molecular weight and 20 N-terminal amino acid sequence to each other and to those of ESAs, and only differed in the isoelectric point, indicating that they are isolectins to each other. The study thus demonstrated that several species of Eucheuma contain high yields of lectins homologous between species, suggesting that the genus as a whole may be considered as a valuable source of lectin proteins. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.