Lectinochemical characterization of a GalNAc and multi-Galbeta1-->4GlcNAc reactive lectin from Wistaria sinensis seeds.
An agglutinin that has high affinity for GalNAcbeta1-->, was isolated from seeds of Wistaria sinensis by adsorption to immobilized mild acid-treated hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 M lactose. The binding property of this lectin was characterized by quantitative precipitin assay (QPA) and by inhibition of biotinylated lectin-glycan interaction. Of the 37 glycoforms tested by QPA, this agglutinin reacted best with a GalNAcbeta1-->4 containing glycoprotein (GP) [Tamm-Horsfall Sd(a+) GP]; a Galbeta1-->4GlcNAc containing GP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human alpha1-acid GP) and a GalNAcalpha1-->3GalNAc containing GP (asialo bird nest GP), but poorly or not at all with most sialic acid containing glycoproteins. Among the oligosaccharides tested, GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Fp) was the most active ligand. It was as active as GalNAc and two to 11 times more active than Tn cluster mixtures, Galbeta1--> 3/4GlcNAc (I/II), GalNAcalpha1-->3(L-Fucalpha1-->2)Gal (Ah), Galbeta1-->4Glc (L), Galbeta1-->3GalNAc (T) and Galalpha1--> 3Galalpha-->methyl (B). Of the monosaccharides and their glycosides tested, p-nitrophenyl betaGalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding alpha anomer and GalNAc (or Fp), respectively. GalNAc was 53.3 times more active than Gal. From the present observations, it can be concluded that the Wistaria agglutinin (WSA) binds to the C-3, C-4 and C-6 positions of GalNAc and Gal residues; the N-acetyl group at C-2 enhances its binding dramatically. The combining site of WSA for GalNAc related ligands is most likely of a shallow type, able to recognize both alpha and beta anomers of GalNAc. Gal ligands must be Galbeta1-->3/4GlcNAc related, in which subterminal beta1-->3/4 GlcNAc contributes significantly to binding; hydrophobicity is important for binding of the beta anomer of Gal. The decreasing order of the affinity of WSA for mammalian structural carbohydrate units is Fp >/= multi-II > monomeric II >/= Tn, I and Ah >/= E and L > T > Gal.
Song
S S
SS
Glyco-Immunochemistry Research Lab, Institute of Molecular and Cellular Biology, Chang-Gung Medical College, Kwei-san, Tao-yuan, Taiwan.
Chen
Y Y
YY
Herp
A
A
Tsai
M S
MS
Wu
A M
AM
D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006386:Hemagglutination Tests; D006801:Humans; D066298:In Vitro Techniques; D037102:Lectins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D019684:Magnoliopsida / Q000737:chemistry*; D008969:Molecular Sequence Data; D037121:Plant Lectins; D012639:Seeds / Q000737:chemistry
D006023:Glycoproteins; D037102:Lectins; D037121:Plant Lectins