Lectinochemical characterization of a GalNAc and multi-Gal beta 1 -> 4GlcNAc reactive lectin from Wistaria sinensis seeds

An agglutinin that has high affinity for GalNAc beta 1-->, was isolated fro m seeds of Wistaria sinensis; by adsorption to immobilized mild acid-treate d hog gastric mucin on Sepharose 4B matrix and elution with aqueous 0.2 M l actose. The binding property of this lectin was characterized by quantitati ve precipitin assay (QPA) and by inhibition of biotinylated lectin-glycan i nteraction. Of the 37 glycoforms tested by QPA, this agglutinin reacted bes t with a GalNAc beta 1-->4 containing glycoprotein (GP) [Tamm-Horsfall Sd(a (+)) GP]; a Gal beta 1-->4GlcNAc containing CP (human blood group precursor glycoprotein from ovarian cyst fluid and asialo human alpha(1)-acid GP) an d a GalNAc alpha 1-->3GalNAc containing GP (asialo bird nest GP), but poorl y or not at all with most sialic acid containing glycoproteins. Among the oligosaccharides tested, GalNAc alpha 1-->3GalNAc beta 1-->3Gal a lpha 1-->4Gal beta 1-->4Glc (Fp) was the most active ligand. It was as acti ve as GalNAc and two to 11 times more active than Tn cluster mixtures, Gal beta 1-->, 3/4GlcNAc (I/II), GalNAc alpha 1-->3(L-Fuc alpha 1-->2)Gal (A(h) ), Gal beta 1-->4Glc (L), Gal beta 1-->3GalNAc (T) and Gal alpha 1--> 3Gal alpha-->methyl (B). Of the monosaccharides and their glycosides tested, p-n itrophenyl beta GalNAc was the best inhibitor; it was approximately 1.7 and 2.5 times more potent than its corresponding alpha anomer and GalNAc (or F p), respectively. GalNAc was 53.3 times more active than Gal. From the present observations, it can be concluded that the Wistaria agglut inin (WSA) binds to the C-3, C-4 and C-6 positions of GalNAc and Gal residu es; the N-acetyl group at C-2 enhances its binding dramatically. The combin ing site of WSA for GalNAc related ligands is most likely of a shallow type , able to recognize both ct and beta anomers of GalNAc. Gal ligands must be Gal beta 1-->3/4GlcNAc related, in which subterminal beta 1-->3/4 GlcNAc c ontributes significantly to binding; hydrophobicity is important for bindin g of the beta anomer of Gal. The decreasing order of the affinity of WSA fo r mammalian structural carbohydrate units is Fp greater than or equal to mu lti-II > monomeric II greater than or equal to Tn, I and A(h) greater than or equal to E and L > T > Gal.