Abstract
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.
Highlights
From the §Departmentof Gynecology, Roswell Park Memorial Institute, NewYork State Department of Health, Buffalo, NewYork 14263 and TChembiomed, Limited, Edmonton, AlbertaT6H 4N9,Canada
Hapten inhibition and glycoconanthus caudatus has been isolated by fractionation on jugateprecipitationassayswerealsoconductedfor
Andean countries of South America where it was once used as an importafnotod source [1]. today the Amaranth plant is used principally for ornamental purposes inthe United Statesand Europe, a renewed interest in several Amaranthus species has been expressed due to their nutritional value [1,2].In 1970,Pardoe and co-workers [3]reported that aqueous extracts of A. caudatus seeds contained a lectin which agglutinated A, B, and 0 red blood cells well
Summary
Andean countries of South America where it was once used as an importafnotod source [1]. today the Amaranth plant is used principally for ornamental purposes inthe United Statesand Europe, a renewed interest in several Amaranthus species has been expressed due to their nutritional value [1,2].In 1970,Pardoe and co-workers [3]reported that aqueous extracts of A. caudatus seeds contained a lectin which agglutinated A, B, and 0 red blood cells well. Hapteninhibition assays were carried out using carbonyl)-L-serinemethyl ester [9],Galj31,3Gala-O-CH3(IO),and the 80 pg of amaranthin, 5 pg of AOSM, and theindicated sugar concendisaccharides GalNAca1,BGaland GalNAcal,6Gal[11,12] were pre- trations. 2) After dialysis into metal free buffer, amaranthin was generated from the 8-allyl derivative by acid hydrolysis in 20% acetic incubated for 1h at room temperature in the presence of the indicated acid at 100 "C and subsequently isolated using a Bio-Gel P-2 column EDTA concentrations. All glassware was acid-washed and Purification of A. caudatus Lectin-A. caudatus seeds (60 g) were buffers were passed over a Chelex 100 column (Bio-Rad) to remove finely ground and delipidated by extraction with methanol These fractions were pooledand bohydrate composition was analyzed as described by Perini and applied to a Synsorb-T column (1.5 X 13 cm) equilibrated with PBS.
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