Preparation of Biotinylated Lectins and Application in Microtiter Plate Assays and Western Blotting

Lisowska, Elwira; Duk, Maria; Wu, Albert M.

doi:10.1007/978-3-0348-7349-9_7

Cited by 31 publications

(44 citation statements)

References 14 publications

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“…Besides employing oligosaccharides as inhibitors of ligand binding, determination of their avidity towards determinants in natural glycans is useful to map the profile of their reactive structures (geometrical parameters). In this study, the carbohydrate-binding activity of domain-I of galectin-4 was examined by using a recently described ELLSA method [12,13] and a wide range of distinct gp and oligosaccharide preparations [19,41,42].…”

Section: Discussionmentioning

confidence: 99%

“…The test was performed according to the procedures described by Duk et al [12] and Lisowska et al [13]. The volume of each…”

Section: The Microtitre Plate Lectin-enzyme Binding Assaymentioning

confidence: 99%

“…Towards these aims, it is of vital importance to delineate the binding properties of this galectin for natural glycans. Consequently, the interaction of CRD-I with glycoproteins (gps) and polysaccharide was studied by the biotin\avidin-mediated microtitre plate lectin-binding assay and by the inhibition of agglutinin-glycan interactions with sugar ligands [12,13]. Separate recombinant expression of these two CRDs enabled us to obtain the individual lectin sites of the tandem-repeat-type galectin.…”

Section: Introductionmentioning

confidence: 99%

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Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)

Tsai

et al. 2002

Biochemical Journal

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Galectins, a family of β-galactoside-specific endogenous lectins, are involved in regulating diverse activities such as proliferation\ apoptosis, cell-cell (matrix) interaction and cell migration. It is presently unclear to what extent the carbohydrate fine specificities of the combining sites of mammalian galectins overlap. To address this issue, we performed an analysis of the carbohydraterecognition domain (CRD-I) near the N-terminus of recombinant rat galectin-4 (G4-N) by the biotin\avidin-mediated microtitre plate lectin-binding assay with natural glycoproteins (gps)\poly-saccharide and by the inhibition of galectin-glycan interactions with a panel of glycosubstances. Among the 35 glycans tested for lectin binding, G4-N reacted best with human blood group ABH precursor gps, and asialo porcine salivary gps, which contain high densities of the blood group Ii determinants Galβ1-3GalNAc (the mucin-type sugar sequence on the human erythrocyte membrane) and\or GalNAcα1-Ser\Thr (Tn), whereas this lectin domain reacted weakly or not at all with most sialylated gps. Among the oligosaccharides tested by the inhibition assay, Galβ

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Section: Discussionmentioning

confidence: 99%

“…The test was performed according to the procedures described by Duk et al [12] and Lisowska et al [13]. The volume of each…”

Section: The Microtitre Plate Lectin-enzyme Binding Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)

Tsai

et al. 2002

Biochemical Journal

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“…It is important to elucidate the detailed carbohydrate specificity of this lectin, because it may function as a signaling adhesion molecule and has potential as a tool in experimental glycobiology, biochemistry, and immunochemistry (4 -9). In the present study, we defined the glycan affinity of this lectin by both enzyme-linked biotin/avidin-mediated microtiter plate lectin assay (ELLSA) and also by examination of the inhibition of AGL-glycan interaction (10,11). The great advantage of this method is that the amount of lectin and glycoform required is about 1/10 to 1/1000 of that required for the quantitative precipitin assay (12,13).…”

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confidence: 99%

Defining the Carbohydrate Specificities of AplysiaGonad Lectin Exhibiting a Peculiar d-Galacturonic Acid Affinity

Wu¹,

Song²,

Chen³

et al. 2000

Journal of Biological Chemistry

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Aplysia gonad lectin (AGL), which has been shown to stimulate mitogenesis in human peripheral lymphocytes, to suppress tumor cells, and to induce neurite outgrowth and improve cell viability in cultured Aplysia neurons, exhibits a peculiar galacturonic acid/galactose specificity. The carbohydrate binding site of this lectin was characterized by enzyme-linked lectino-sorbent assay and by inhibition of AGL-glycan interactions. Examination of the lectin binding with 34 glycans revealed that it reacted strongly with the following glycoforms: most human blood group precursor (equivalent) glycoproteins (gps), two Gal␣134Gal-containing gps, and two D-galacturonic acid (GalUA)-containing polysaccharides (pectins from apple and citrus fruits), but poorly with most human blood group A and H active and sialylated gps. Among the GalUA and mammalian saccharides tested for inhibition of AGL-glycan binding, GalUA mono-to trisaccharides were the most potent ones. They were 8.5 ؋ 10 4 times more active than Gal and about 1.5 ؋ 10 3 more active than the human blood group P k active disaccharide (E, Gal␣134Gal). This disaccharide was 6, 28, and 120 times more efficient than Gal␤133GlcNAc(I), Gal␤133GalNAc(T), and Gal␤13 4GlcNAc (II), respectively, and 35 and 80 times more active than melibiose (Gal␣136Glc) and human blood group B active disaccharide (Gal␣133Gal), respectively, showing that the decreasing order of the lectin affinity toward ␣-anomers of Gal is ␣134 > ␣136 > ␣133. From the data provided, the carbohydrate specificity of AGL can be defined as GalUA␣134 trisaccharides to mono GalUA > branched or cluster forms of E, I, and II > > monomeric E, I, and II, whereas GalNAc is inactive.

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“…Therefore, it seemed worthwhile to investigate the carbohydrate-binding specificity of Morniga M in more detail using well-characterized mammalian glycan ligands. In order to obtain a more precise binding profile for clustered glycans and to search for the structural requirements of Morniga M with mammalian glycotopes, its binding properties were explored using an enzyme-linked lectinosorbent assay (ELLSA) and our developed inhibition assay [3,6] with our glycan/ligand collection [17,[22][23][24] (see also Appendix). This approach revealed that the lectin chiefly recognizes the di-, tri-, and oligomannosyl structural units of N-glycans.…”

Section: Introductionmentioning

confidence: 99%

A Novel Lectin (Morniga M) from Mulberry <i>(Morus nigra)</i> Bark Recognizes Oligomannosyl Residues in N-Glycans

Wu¹,

Wu²,

Singh³

et al. 2004

J Biomed Sci

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Morniga M is a jacalin-related and mannose-specific lectin isolated from the bark of the mulberry (Morus nigra). In order to understand the function and application of this novel lectin, the binding property of Morniga M was studied in detail using an enzyme-linked lectinosorbent assay and lectin-glycan inhibition assay with extended glycan/ligand collection. From the results, it was found that the di-, tri-, and oligomannosyl structural units of N-glycans such as those of the bovine α₁-acid glycoprotein (gp) and lactoferrin were the most active gps, but not the O-glycans or polysaccharides including mannan from yeast. The binding affinity of Morniga M for ligands can be ranked in decreasing order as follows: gps carrying multiple N-glycans with oligomannosyl residues >> N-glycopeptide with a single trimannosyl core > Tri-Man oligomer [Manα1→6(Man α1→3) Man], Penta-Man oligomer [Manα1→6(Manα1→3)Manα1→6(Manα1→3) Man] ≧ Man α1→2, 3 or 6 Man > Man > GlcNAc, Glc >> L-Fuc, Gal, GalNAc (inactive), demonstrating the unique specificity of this lectin that may not only assist in our understanding of cell surface carbohydrate ligand-lectin recognition, but also provide informative guidelines for the application of this structural probe in biotechnological and clinical regimens, especially in the detection and purification of N-linked glycans.

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Preparation of Biotinylated Lectins and Application in Microtiter Plate Assays and Western Blotting

Cited by 31 publications

References 14 publications

Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)

Fine specificity of domain-I of recombinant tandem-repeat-type galectin-4 from rat gastrointestinal tract (G4-N)

Defining the Carbohydrate Specificities of AplysiaGonad Lectin Exhibiting a Peculiar d-Galacturonic Acid Affinity

A Novel Lectin (Morniga M) from Mulberry <i>(Morus nigra)</i> Bark Recognizes Oligomannosyl Residues in N-Glycans

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