Purification and characterization of a Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc and HSO3(-)-->6Gal beta 1-->GlcNAc 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 Fuc alpha 1-->2Gal beta/GalNAc beta 1-->groups, and the other one, which passed through a porcine stomach mucin-Sepharose 4B column, was purified by sequential chromatography on a human alpha 1-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-I has four equal binding sites per molecule, and the association constant toward tritium-labeled Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcOT is Ka = 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 Neu5Ac alpha 2-->6Gal beta 1-->4GlcNAc or HSO3(-)-->6Gal beta 1-->4GlcNAc group showed tremendously stronger binding ability than oligosaccharides with a Gal beta 1-->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.(ABSTRACT TRUNCATED AT 250 WORDS)
Yamashita
K
K
Department of Biochemistry, Sasaki Institute, Tokyo, Japan.
Umetsu
K
K
Suzuki
T
T
Ohkura
T
T
D000595:Amino Acid Sequence; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D037102:Lectins / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000302:isolation & purification* / Q000378:metabolism; D037121:Plant Lectins; D010944:Plants / Q000737:chemistry*; D013379:Substrate Specificity
D006023:Glycoproteins; D037102:Lectins; D009844:Oligosaccharides; D037121:Plant Lectins