Purification and Characterization of a Mannose-Specific Lectin from Shallot (Allium ascalonicum) Bulbs

Abstract

A new mannose-binding lectin was isolated from shallot (Allium ascalonicum) bulbs by affinity chromatography on an immobilized D-mannose column. The lectin (A. ascojonicum agglutinin, AAA) appeared homogeneous by polyacrylamide gel electrophoresis at pH 4.3 and gave a single protein band with an apparent Mr of 11 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a single symmetrical peak of 11 kDa by gel filtration oa a Sephacryl S-200 HR column, indicating that AAA exists as a monomeric protein at neutral pH under the gel filtration condition employed. However, chemical cross-linking studies revealed that some degree of self-association of the lectin molecules occurs and that the lectin exists in solution as a mixture of monomers and oligomers. Scatchard analysis of equilibrium dialysis data showed the presence of one carbohydrate binding site for Man ([alpha]1-3) Man-[alpha]-O-Me per monomer, with Ka = 1.62 x 104 M-1. The carbohydrate-binding properties of the purified AAA were investigated by quantitative precipitation and hapten inhibition assays. Purified AAA precipitated asialofetuin, asialotransferrin, asialothyroglobulin, asialoorosomucoid, as well as their agalacto derivatives, but did not precipitate either sialylated glycoproteins or mucins. AAA also reacted strongly with the highly branched yeast mannan obtained from Saccharomyces cerevisiae. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the AAA-asialofetuin precipitation system, whereas D-glucose, D-altrose, D-talose, N-acetyl-D-mannosamine, and derivatives of D-mannose, including 2-deoxy-, 2-deoxy-2-fluoro-, 3-deoxy-, and 6-deoxy-D-mannose were noninhibitors. These results suggest that the presence of equatorial hydroxyl groups at the C-3 and C-4 positions, an axial hydroxyl group at the C-2 position, and a free hydroxyl group at the C-6 position of the pyranose ring are the most important loci for the binding of D-mannose to AAA. Of the oligosaccharides tested, the best inhibitors were oligosaccharides containing terminal Man([alpha]1-6)[Man([alpha]1-3)]Man groups. Oligosaccharides containing either Man([alpha]1-3)Man or Man([alpha]1-6)Man units were also moderately good inhibitors of the AAA-asialofetuin precipitation system. These results indicate that AAA has an extended carbohydrate-binding site, which is most complementary to a branched mannotriosyl residue, i.e., Man([alpha]1-6)[Man([alpha]1-3)]Man. A comparison is presented of the detailed carbohydrate-binding properties and molecular structure of the A. ascalonicum lectin and several other mannose-binding lectins from different species of the plant family Amaryllidaceae, i.e., snowdrop lectin (Galanthus nivalis), daffodil lectin (Narcissus pseudonarcissus), and amaryllis lectin (Hippeastrum hybr.).