Binding Studies on the Combining Site of a GalNAcα1→‐Specific Lectin with Thomsen‐Friedenreich Activity Prepared from Green Marine Algae Codium fragile Subspecies Tomentosoides

The combining site of a GalNAα1→‐specific lectin (CFT) with Thomsen‐Friedenreich (T, Galβ1→‐3‐GalNAcα1→Ser/Thr) activity, purified from the subspecies tomentosoides of green marine algae Codium fragile was studied by quantitative precipitin and precipitin‐inhibition assays. Of 27 glycoforms tested, Tn (GalNAcα1→Ser/Thr) glycoprotein from armadillo submandibular glands, and asialo porcine submandibular glycoprotein, which contains T, Tn and GalNAcα1→3Gal(A) sequences, completely precipitated the lectin added, and less than 1 μg glycoprotein was required to precipitate 50% 4.7 μg lectin nitrogen. However, CFT precipitated negligibly with Pneumococcus type‐XIV polysaccharide and asialo human α₁‐acid glycoprotein, that contain exclusively the human blood‐type‐II precursor sequence (II, Galβ1→4GlcNAc) at the nonreducing ends. Among the sugar inhibitors tested, the human blood A‐active trisaccharide [Ah, GalNAcα1→3(lFucα1→2)Gal] was the best inhibitor; it was about twice as active as the T disaccharide. Oligosaccharides without GalNAcα1→ as part of their sequences were inactive, indicating that the acetamido group at C2 of galactose is essential for binding and that GalNAc is the main contributor in the T sequence for binding. From the data provided, it is clear that the combining site of CFT requires an α‐anomer of GalNAc and recognizes A_h, internal GalNAcα1→ of T and Tn determinants of glycans, but not the blood group I/II (Galβ1→3/4GlcNAc) sequences. Consequently, CFT is a useful reagent for detecting GalNAcα1→‐containing glycoconjugates.