Crystallographic characterization of a principal non-toxic lectin from seeds of Ricinus communis

Wei, Chin Hsuan; Koh, C.

doi:10.1016/0022-2836(78)90216-4

Cited by 15 publications

(4 citation statements)

References 23 publications

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“…Ricin [ricin D according to the nomenclature of Wei and Koh (27)] was from Inland Laboratories (Austin, TX). 2-(4′-Maleimidylanilino)naphthalene-6-sulfonic acid, sodium salt (MIANS) 1 was purchased from Molecular Probes, Inc. (Eugene, OR).…”

Section: Methodsmentioning

confidence: 99%

Identification of Three Oligosaccharide Binding Sites in Ricin

Steeves¹,

Denton²,

Barnard³

et al. 1999

Biochemistry

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The galactoside-binding sites of ricin B chain can be blocked by affinity-directed chemical modification using a reactive ligand derived from asialoglycopeptides containing triantennary N-linked oligosaccharides. The terminal galactosyl residue of one branch of the triantennary oligosaccharide is modified to contain a reactive dichlorotriazine moiety. Two separate galactoside-binding sites have been clearly established in the ricin B chain by X-ray crystallography [Rutenber, E., and Robertus, J. D. (1991) Proteins 10, 260-269], and it is necessary to covalently attach two such reactive ligands to the B chain to block its binding to galactoside affinity matrixes. A method was developed using thiol-specific labeling of the ligand combined with subsequent immunoaffinity chromatography which allowed the isolation of ricin B chain peptides covalently linked to the ligand from proteolytic digests of purified blocked ricin. The sites of covalent attachment of the two ligands in blocked ricin were inferred from sequence analysis to be Lys 62 in domain 1 of the B chain and Tyr 148 in domain 2. A minor species of blocked ricin contains a third covalently attached ligand. From the analysis of peptides derived from blocked ricin enriched in this species, it is inferred that Tyr 67 in domain 1 is the specific site on the ricin B chain where a third reactive ligand becomes covalently linked to the protein. These results are interpreted as providing support for the notion that the ricin B chain has three oligosaccharide binding sites.

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Section: Methodsmentioning

confidence: 99%

Identification of Three Oligosaccharide Binding Sites in Ricin

Steeves¹,

Denton²,

Barnard³

et al. 1999

Biochemistry

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“…Ricin, which is ricin D according to the nomenclature of Wei and Koh (1978), was purchased from Sigma Chemical Co. (St. Louis, MO). Aminoethylpolyacrylamide P-150 was supplied by Bio-Rad (Richmond, CA).…”

Section: Methodsmentioning

confidence: 99%

Modification of the binding site(s) of lectins by an affinity column carrying an activated galactose-terminated ligand

Moroney¹,

D'Alarcao²,

Goldmacher³

et al. 1987

Biochemistry

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An affinity column approach is described, aimed at the modification of the galactose binding site(s) of ricin in an effort to block the binding of ricin to cells. The affinity column was prepared by linking N-(2'-mercaptoethyl)lactamine to pyridyldithio-activated polyacrylamide heads. The linker between the ligand and the solid support thus contained a disulfide bond and an unmodified terminal galactose moiety. The amino group of the ligand was allowed to react with the bifunctional cross-linking reagent 2,4-dichloro-6-methoxytriazine. The lectin was then allowed to bind to the galactose functions on the activated column at pH 7.0, prior to raising the pH to 8.6 to initiate the cross-linking reaction between the ligand and the lectin. Lectin that was not covalently linked to the functionalized galactose residues on the column was eluted with galactose or lactose. Finally, the covalent ligand-lectin complexes were released from the solid support by reducing the disulfide bond between the ligand and the support. The affinity column was used in this way to modify the galactose binding site(s) of ricin. Upon release from the affinity column, blocked ricin was purified from unmodified ricin by affinity chromatography on columns of immobilized asialofetuin (a ligand to which ricin binds very tightly). The sulfhydryl group formed by cleavage of the ligand-ricin complex from the column was labeled with [3H]-N-ethylmaleimide to provide evidence that one blocking ligand was linked per ricin molecule. The blocked ricin and a conjugate of the blocked ricin with the monoclonal antibody J5 were toxic for cultures of Namalwa cells in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…For example, lectins from mushroom species including Agaricus bisporus, Boletus satanus, Flamulina velutipes , and Ganoderma lucidm have antitumor activities (Wang et al 1998). Lectins from Arisaema tortuosum as well as alga Dasa villosa and Ricinus communis were shown to have antitumor and/or antiproliferative activities (Wei et al 1978; Dhuna et al 2005). …”

Section: Introductionmentioning

confidence: 99%

Purification and Characterization of a New D-Galactose-Specific Lectin from the Housefly,Musca domestica, and Its Antiproliferative Effect on Human K562 and MCF-7 Tumor Cells

Cao

Sun

Wang

et al. 2010

Journal of Insect Science

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In the present work, a D-galactose-specific lectin with novel N-terminal sequence was purified from Musca domestica L. (Diptera: Muscidae) pupae. The purification was performed using affinity chromatography, ultra-filtration, and HPLC. The haemagglutinating activity of M. domestica lectin was specifically inhibited by D-galactose. The haemagglutinating activity of this lectin was stable at temperatures up to 65° C and in pH ranging from 4 to 8. Salts including FeCl3 and MnCl2 inhibited the haemagglutinating process, whereas NaCl, KCl, CaCl2, MgCl2, ZnCl2, and AlCl3 did not. By SDS-PAGE, purified M. domestica pupae lectin yielded a single band with a molecular weight of 40 kDa, with or without reduction of β-mercaptoethanol, and it could be stained with Alcian Blue 8 GX. The morphology of purified lectin was observed by atomic force microscopy, which indicated that M. domestica lectin was an 8.27 nm high, globular shaped glycoprotein with a 1.41 nm high polysaccharide chain. In addition, antiproliferative activity of this lectin against tumor cells K562 and MCF-7 was determined with a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, which showed that the antiproliferative process was time- and dose-dependent with an IC50 of 5.7 and 6.7 at 24 h, 5.5 and 6.4 at 36 h, 5.2 and 6.5 µM at 48 h, respectively.

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Crystallographic characterization of a principal non-toxic lectin from seeds of Ricinus communis

Cited by 15 publications

References 23 publications

Identification of Three Oligosaccharide Binding Sites in Ricin

Identification of Three Oligosaccharide Binding Sites in Ricin

Modification of the binding site(s) of lectins by an affinity column carrying an activated galactose-terminated ligand

Purification and Characterization of a New D-Galactose-Specific Lectin from the Housefly,Musca domestica, and Its Antiproliferative Effect on Human K562 and MCF-7 Tumor Cells

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