Recognition factors of Ricinus communis agglutinin 1 (RCA₁)

Ricinus communis agglutinin (RCA₁) is one of the most important applied lectins that has been widely used as a tool to study cell surfaces and to purify glycans. Although the carbohydrate specificity of RCA₁ has been described, the information obtained was mainly focused on inhibition of simple Galβ1-related oligosaccharides and simple clusters. Here, all possible recognition factors of RCA₁ of glycan binding were examined by enzyme-linked lectinosorbent (ELLSA) and inhibition assays, using known mammalian Gal/GalNAc carbohydrate structural units and natural polyvalent glycans. Among the glycoproteins (gps) tested and expressed as 50% nanogram inhibition, the high-density polyvalent Galβ1-4GlcNAc (II) glycotopes occurring in natural gps, such as Pneumococcus type 14 capsular polysaccharide which is composed of repeating poly II residues, resulted in 9.0 × 10 ⁴, 1.5 × 10⁵, 2.3 × 10⁴ and 2.1 × 10⁴-fold higher affinities to RCA₁ than the monomeric Gal, linear I/II and Tri-antennary-II (Tri-II). Of the ligands tested and expressed as nanomoles of 50% inhibition, Tri-II was the best, being about 2, 4, 25.6 and 33.3 times better inhibitor than Di-II, II, I (Galβ1-3GlcNAc) and Gal, respectively. From the results of this study, it is concluded that: (a) Galβ1-4GlcNAc and other Galβ1-related oligosaccharides are essential for lectin binding and their polyvalent form in macromolecules should be the most important recognition factor for RCA ₁; (b) the combining site of RCA₁ may be a groove type, recognizing Galβ1-4GlcNAc (II) as the major binding site; (c) its combining size may be large enough to accommodate a tetrasaccharide of β-anomeric Gal at the non-reducing end and most complementary to human blood group I Ma active trisaccharide (Galβ1-4GlcNAcβ1-6Gal) and lacto-N-neotetraose (Galβ1-4GlcNAcβ1-3Galβ1-4Glc); (d) RCA₁ has a preference for the β-anomer of Gal oligosaccharides with a Galβ1-4 linkage > Galβ1-6 ≥ Galβ1-3; (e) configuration of carbon-2, -3 -4 and -6 in Gal are essential for binding; (f) hydrophobic interaction in the vicinity of the binding site useful for sugar accommodation increases affinity. These results should be helpful for understanding the functional role of RCA₁ and for characterizing glycotopes of mammalian complex carbohydrates.