PURIFICATION AND CHARACTERIZATION OF A NEU5AC-ALPHA-2-]6GAL-BETA-1-]4GLCNAC AND HSO3--]6GAL-BETA-1-]4GLCNAC SPECIFIC LECTIN IN TUBEROUS ROOTS OF TRICHOSANTHES-JAPONICA

Two lectins were purified from tuberous roots of Trichosanthes japonica. The major lectin, which was named TJA-II, interacted with Fucalpha1-->2Galbeta/GalNAcbeta1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha1-antitrypsin-Sepharose 4B column and named TJA-I. The molecular mass of TJA-I was determined to be 70 kDa by sodium dodecyl sulfate gel electrophoresis. TJA-I is a heterodimer of 38-kDa (36-kDa) and 32-kDa (30-kDa) subunits with disulfide linkage(s), and the difference between 38 and 36 kDa, and between 32 and 30 kDa, is due to secondary degradation of the carboxyl-terminal side. It was determined by equilibrium dialysis that TJA-1 has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Acalpha2-->6Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->4Glc(OT) is K(a) = 8.0 x 10(5) M-1. The precise carbohydrate binding specificity was studied using hemagglutinating inhibition assay and immobilized TJA-I. A series of oligosaccharides possessing a Neu5Acalpha2-->6Galbeta1-->4GlcNAc or HSO3--->6Galbeta1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Galbeta1-->4GlcNAc group, indicating that TJA-I basically recognizes an N-acetyllactosamine residue and that the binding strength increases on substitution of the beta-galactosyl residue at the C-6 position with a sialic acid or sulfate. A TJA-I column is useful for separating oligosaccharides and glycoproteins with a Neu5Acalpha2-->6 (or HSO3--->6) Galbeta1-->4GlcNAc group from those with a Neu5Acalpha2-->3 (or HSO3--->3) Galbeta1-->4GlcNAc group.