The interaction of the Maackia amurensis hemagglutinin (MAH) with various glycopeptides and oligosaccharides was investigated by means of immobilized lectin affinity chromatography.An amino terminal octapeptide obtained from human glycophorin A having three Neu5Aca2+3Gal/I1+3(Neu5Aca2+6)GalNAc tetrasaccharide chains, designated as CB-II, was found to have an extremely strong affinity for MAH. Therefore, it is strongly suggested that hemagglutination by MAH was caused by its interaction with Ser/Thr-linked carbohydrate chains of human glycophorin A on erythrocyte membranes.

Maackia amurensis hemagglutinin (MAH) and leukoagglutinin (MAL) are leguminous lectins which recognize carbohydrate chains containing sialic acid residues linked alpha2,3 to penultimate galactose residues. In the present investigation, cDNA clones encoding MAL were isolated from a cDNA library constructed from germinated Maackia amurensis seeds and sequenced. From the reading frame of the cloned cDNAs, MAL was predicted to be composed of 287 amino acid residues, and showed strong similarity to MAH (86.2% identity). In leguminous lectins, most amino acid residues involved in sugar-binding were previously shown to be conserved. However, in both MAL and MAH lectins, the conserved glycine and asparagine were shown to be substituted by lysine and aspartic acid, respectively. Substitutions were made at position 105 and/or 135 of MAH to examine the roles of amino acid residues postulated to be important in binding to sialic acids. Recombinant MAH bound to the sialic acid-containing CB-II glycopeptide of human glycophorin A. By contrast, mutant lectins with lysine-105 substituted with glycine and/or aspartic acid-135 with asparagine did not bind to sialic acid residues. This indicates that these characteristic substitutions are important in sialic acid binding.

Inflammatory and tumoricidal macrophages express galactose- and N-acetylgalactosamine-specific Ca(2+)-dependent lectins on their surfaces. This lectin is a family member of membrane-bound C-type animal lectins and consists of 304 amino acid residues (molecular weight 34,595). In the present study, expression vectors containing a nucleotide sequence corresponding to the carbohydrate-binding domain of mouse macrophage lectin cDNA have been prepared. The carbohydrate-binding specificity of the recombinant macrophage lectin expressed in Escherichia coli was investigated by comparing elution profiles of various glycopeptides having defined carbohydrate structures on immobilized lectins. When elution profiles of high mannose-type and complex-type Asn-linked carbohydrate chains were compared, the degree of retardation from immobilized macrophage lectin column was in the order tetraantennary complex-type with terminal galactosyl residues > triantennary complex-type with terminal galactosyl residues > biantennary complex-type with terminal galactosyl residues > high mannose-type glycopeptides. N-Terminal octapeptides from human glycophorin A that bore three NeuAc alpha 2-3Gal beta 1-3(NeuAc alpha 2-6)GalNAc serine/threonine-linked tetrasaccharide chains and their sequentially deglycosylated derivatives were also applied to this column. Glycopeptides carrying three constitutive GalNAc-Ser/Thr(Tn-antigen) had the strongest affinity, whereas those with fully sialylated carbohydrate tetrasaccharide chains showed weak interaction. The association kinetics of Asn-linked glycopeptides from bovine asialofetuin to recombinant macrophage lectin was determined by surface plasmon resonance spectroscopy. The results indicate k(assoc) value of 1.63 x 10(4) M-1 s-1. The calculated value for Ka was 6.20 x 10(7) M.

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The complete amino acid sequence of the Lotus tetragonolobus lectin was determined by a protein sequencer after digestion with endoproteinases of Lys‐C and Asp‐N, and compared with those of other leguminous plant lectins.