Ricinus communis agglutinin I (RCA120) is considered a versatile tool for the detection of galactose-containing oligosaccharides. However, possible contamination by the highly toxic isolectin 'ricin' has become a critical issue for RCA120's continued use. From a practical viewpoint, it is necessary to find an effective substitute for RCA120. For this purpose, we examined by means of frontal affinity chromatography over 100 lectins which have similar sugar-binding specificities to that of RCA120. It was found that Erythrina cristagalli lectin (ECL) showed the closest similarity to RCA120. Both lectins prefer Gal beta1-4GlcNAc (type II) to Gal beta1-3GlcNAc (type I) structures, with increased affinity for highly branched N-acetyllactosamine-containing N-glycans. Their binding strength significantly decreased following modification of the 3-OH, 4-OH and 6-OH of the galactose moiety of the disaccharide, as well as the 3-OH of its N-acetylglucosamine residue. Several differences were also observed in the affinity of the two lectins for various other ligands, as well as effects of bisecting GlcNAc and terminal sialylation. Although six other Erythrina-derived lectins have been reported with different amino acid sequences, all showed quite similar profiles to that of ECL, and thus, to RCA120. Erythrina lectins can therefore serve as effective substitutes for RCA120, taking the above differences into consideration.

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A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.