Lectinochemical studies on the glyco-recognition factors of a Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Salvia sclarea.

Abstract

The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc alpha1-->Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAcalpha1--> related oligosaccharides and glycopeptides. Thus, the requirements of functional groups in monosaccharide and high-density polyvalent carbohydrate structural units for SSL binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested for interaction, a high density of exposed Tn-containing glycoproteins such as in the armadillo salivary Tn glycoprotein and asialo ovine salivary glycoprotein reacted best with SSL. When the gps were tested for inhibition of SSL binding, which was expressed as 50% nanogram inhibition, the high density polyvalent Tn present in macromolecules was the most potent inhibitor. Among the monosaccharide and carbohydrate structural units studied, which were expressed as nanomole inhibition, GalNAc alpha1-->3GalNAc beta1-->3Gal alpha1-->4Gal beta1-->4Glc (Fp), GalNAc alpha1-->3Gal beta1-->4Glc (A(L)), GalNAc alpha1-->3GalNAc beta1-->Me (F beta), GalNAc alpha1-->3GalNAc alpha1-->Me (F alpha) and GalNAc alpha1--> Ser/Thr (Tn) were the most active ligands, being 2.5-5.0 x 10(3) and 1.25-2.5 times more active than Gal and GalNAc, respectively. From the results, it is suggested that the combining site of SSL is a shallow groove type, recognizing the monosaccharide of GalNAc as the major binding site or Tn up to the Forssman pentasaccharide (Fp). It can be concluded that the three critical factors for SSL binding are the -NH CH(3)CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc alpha1-->Ser/Thr) present in macromolecules. These results should assist in understanding the glyco-recognition factors involved in carbohydrate-lectin interactions in biological processes. The effect of the polyvalent F alpha, F beta and GalNAc beta1-->3Gal alpha1--> (P alpha) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.