Lectinochemical studies on the glyco-recognition factors of a Tn (GalNAcα1→Ser/Thr) specific lectin isolated from the seeds of Salvia sclarea

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Abstract
The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc α1→Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAcα1→ 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 α1→3GalNAc β1→3Gal α1→4Gal β1→4Glc (Fp), GalNAc α1→3Gal β1→4Glc (A L ), GalNAc α1→3GalNAc β1→Me (F β), GalNAc α1→3GalNAc α1→Me (F α) and GalNAc α1→ Ser/Thr (Tn) were the most active ligands, being 2.5–5.0× 103 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 CH3CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc α1→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 α, F β and GalNAc β1→3Gal α1→ (P α) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.

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