Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen.
A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.
Rinderle
S J
SJ
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Goldstein
I J
IJ
Matta
K L
KL
Ratcliffe
R M
RM
D000951:Antigens, Neoplasm / Q000032:analysis; D015295:Antigens, Tumor-Associated, Carbohydrate; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002241:Carbohydrates; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004187:Disaccharides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D006241:Haptens; D006386:Hemagglutination Tests; D037102:Lectins / Q000302:isolation & purification*; D008958:Models, Molecular; D008970:Molecular Weight; D037121:Plant Lectins; D011233:Precipitin Tests; D054788:Ribosome Inactivating Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D012639:Seeds / Q000032:analysis*
D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D002241:Carbohydrates; D004187:Disaccharides; D006241:Haptens; D037102:Lectins; D037121:Plant Lectins; D054789:Ribosome Inactivating Proteins, Type 1; C061152:amaranthin protein, Amaranthus; C016467:Thomsen-Friedenreich antigen; D054788:Ribosome Inactivating Proteins