A novel lectin having hemagglutinating and mitogenic activities was isolated from leaf skin of A. arborescens (Koike etal, 1995a, b). The lectin with a molecular mass of 35 kDa consists of subunits containing 109 amino acid residues. The N-terminal amino acid sequence of the intact subunit showed a homology with that of snowdrop lectin.
The homogenates of the leaf skin of A. arborescens were filtered and the filtrates were mixed with two-fold cold acetone. The precipitate was collected by centrifugation and lyophilized. The dried powder was dissolved in phosphate-buffered saline and fractionated on a Sephadex G-25 column. Fractions with the lectin activity were precipitated with 80% saturation of ammonium sulfate. The precipitate was collected by centrifugation and dissolved in 5mM potassium phosphate buffer, pH 8.0. After dialysis the solution was applied to a DEAE 52 column. The active fractions were dialyzed and then lyophilized. The solution of the dried material was applied to a Superdex 75 HR 10/30 column and the fractions with both hemagglutinating and mitogenic activities were combined and used for the experiments.
The lectin showed a molecular mass of about 35 kDa by gel filtration chromatography and the complete amino acid sequence of the subunit was determined. The subunit consisted of 109 amino acid residues having a molecular mass 12.2 kDa and contained an intrachain disulfide bridge. However, it is unclear whether it consists of three or four subunits, because the molecular mass of the intact lectin was estimated to be roughly 35 kDa. Lectins consisting of three subunits are not general but a leek (Allium porrum) lectin being a trimer rather than tetramer of polypeptides with a molecular mass 12.5—13 kDa was reported (Van Damme etal, 1993).
The sequence of the lectin in this study is highly homologous to that of a mannose-binding lectin (105 residues) from snowdrop bulb, belonging to the family Liliaceae as well as A. arborescens (Van Damme etal, 1991).
The aloe lectin exhibited high agglutinating activity toward trypsinized and glutaral-dehyde-fixed rabbit erythrocytes, whereas it exhibited no activity toward human (type A, B and O) or sheep erythrocytes.
Effects of fifteen sugars (D-mannose, methyl-a-D-mannopyranoside, mannan, mannosamine, D-glucose, L-fucose, lactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N-acetyl-D-neuraminic acid, maltose, mannitol, fructose, a-methyl-D-glucoside) on the hemagglutinating activity of the aloe lectin were investigated. D-mannose and methyl-a-D-mannopyranoside showed high activity.
The mitogenic activity of the aloe lectin was examined using BALB/c strain mouse spleen lymphocytes. Cell proliferation was determined by a colorimetric assay which detects the conversion of 3(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazonium bromide (MTT, Sigma) into the formazan product (blue color) by mitochondrial succinate-dehydrogenase (12, 13).
The aloe lectin showed a mitogenic activity toward mouse lymphocytes. The activity was co-eluted with the lectin activity both on the final two steps of DEAE ion exchange and the subsequent gel filtration chromatographies.
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