Junko Kominami

Phylogenetic and specificity studies of two-domain GNA-related lectins: Generation of multispecificity through domain duplication and divergent evolution

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.

The function of rhamnose-binding lectin in innate immunity by restricted binding to Gb3

L-rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3, on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs.

Mannose-binding lectin from yam (Dioscorea batatas) tubers with insecticidal properties against Helicoverpa armigera (Lepidoptera: Noctuidae).

The amino acid sequence of mannose-binding lectin, named DB1, from the yam (Dioscorea batatas, synonym Dioscorea polystachya) tubers was determined. The lectin was composed of two isoforms DB1(Cys86) and DB1(Leu86) consisting of 108 amino acid residues with 90% sequence homology between them. DB1 showed a high sequence similarity to snowdrop (Galanthus nivalis) bulb lectin, GNA; especially, the carbohydrate-binding sites of GNA were highly conserved in DB1. DB1 interacted with D-mannose residues of oligosaccharides, and the oligosaccharides carrying two mannose-alpha-1,3-D-mannose units showed high binding affinity. DB1 was examined for insecticidal activity against Helicoverpa armigera (Lepidoptera: Noctuidae) larvae at different stages of development. The rate of adults successfully emerging from pupae fed on DB1 was 33%, when incorporated into an artificial diet at a level of 0.01% (w/w). Although DB1 had no or marginal inhibitory effects on gut proteolytic and glycolic enzymes, the lectin strongly bound to larval brush border and peritrophic membrane detected by immunostaining. The results show that DB1 may fulfill a defense role against insect pests.

Comparative analysis of oligosaccharide specificities of fucose-specific lectins from Aspergillus oryzae and Aleuria aurantia using frontal affinity chromatography

Aleuria aurantia lectin (AAL) is widely used to estimate the extent of alpha1,6-fucosylated oligosaccharides and to fractionate glycoproteins for the detection of specific biomarkers for developmental antigens. Our previous studies have shown that Aspergillus oryzae lectin (AOL) reflects the extent of alpha1,6-fucosylation more clearly than AAL. However, the subtle specificities of these lectins to fucose linked to oligosaccharides through the 2-, 3-, 4-, or 6-position remain unclear, because large amounts of oligosaccharides are required for the systematic comparative analysis using surface plasmon resonance. Here we show a direct comparison of the dissociation constants (K(d)) of AOL and AAL using 113 pyridylaminated oligosaccharides with frontal affinity chromatography. As a result, AOL showed a similar specificity as AAL in terms of the high affinity for alpha1,6-fucosylated oligosaccharides, for smaller fucosylated oligosaccharides, and for oligosaccharides fucosylated at the reducing terminal core GlcNAc. On the other hand, AOL showed 2.9-6.2 times higher affinity constants (K(a)) for alpha1,6-fucosylated oligosaccharides than AAL and only AAL additionally recognized oligosaccharides which were alpha1,3-fucosylated at the reducing terminal GlcNAc. These results explain why AOL reflects the extent of alpha1,6-fucosylation on glycoproteins more clearly than AAL. This systematic comparative analysis made from a quantitative viewpoint enabled a clear physical interpretation of these fucose-specific lectins with multivalent fucose-binding sites.

Desulfated galactosaminoglycans are potential ligands for galectins: Evidence from frontal affinity chromatography

Galectins, a group of beta-galactoside-binding lectins, are involved in multiple functions through specific binding to their oligosaccharide ligands. No previous work has focused on their interaction with glycosaminoglycans (GAGs). In the present work, affinities of established members of human galectins toward a series of GAGs were investigated, using frontal affinity chromatography. Structurally-defined keratan sulfate (KS) oligosaccharides showed significant affinity to a wide range of galectins if Gal residue(s) remained unsulfated, while GlcNAc sulfation had relatively little effect. Consistently, galectins showed much higher affinity to corneal type I than cartilageous type II KS. Unexpectedly, galectin-3, -7, and -9 also exerted significant affinity to desulfated, GalNAc-containing GAGs, i.e., chondroitin and dermatan, but not at all to hyaluronan and N-acetylheparosan. These observations revealed that the integrity of 6-OH of betaGalNAc is important for galectin recognition of these galactosaminoglycans, which were shown, for the first time, to be implicated as potential ligands of galectins.

Carbohydrate Specificity of Lectins from Boletopsis leucomelas and Aralia cordate

The carbohydrate specificity of three novel lectins, Boletopsis leucomelas lectin (BLL), Aralia cordate lectin (ACL), and Wasabia japonica lectin (WJL), was examined by frontal affinity chromatography using a panel of fluorescently labeled 47 oligosaccharides. The results indicate that BLL recognizes an agalacto structure of the biantennary chain and its bisecting structure. ACL showed strong affinity for triantennary oligosaccharides, but no affinity for tetraantennary structure. WJL showed no appreciable affinity for any of the 47 glycans examined. These lectins with a unique affinity specificity might be useful for examining alterations in the glycan structures of the glycoconjugates in association with development and various diseases.

Sugar-Binding Profiles of Chitin-Binding Lectins from the Hevein Family: A Comprehensive Study

Chitin-binding lectins form the hevein family in plants, which are defined by the presence of single or multiple structurally conserved GlcNAc (N-acetylglucosamine)-binding domains. Although they have been used as probes for chito-oligosaccharides, their detailed specificities remain to be investigated. In this study, we analyzed six chitin-binding lectins, DSA, LEL, PWM, STL, UDA, and WGA, by quantitative frontal affinity chromatography. Some novel features were evident: WGA showed almost comparable affinity for pyridylaminated chitotriose and chitotetraose, while LEL and UDA showed much weaker affinity, and DSA, PWM, and STL had no substantial affinity for the former. WGA showed selective affinity for hybrid-type N-glycans harboring a bisecting GlcNAc residue. UDA showed extensive binding to high-mannose type N-glycans, with affinity increasing with the number of Man residues. DSA showed the highest affinity for highly branched N-glycans consisting of type II LacNAc (N-acetyllactosamine). Further, multivalent features of these lectins were investigated by using glycoconjugate and lectin microarrays. The lectins showed substantial binding to immobilized LacNAc as well as chito-oligosaccharides, although the extents to which they bound varied among them. WGA showed strong binding to heavily sialylated glycoproteins. The above observations will help interpret lectin-glycoprotein interactions in histochemical studies and glyco-biomarker investigations.

Chapter 10 – Frontal Affinity Chromatography: Systematization for Quantitative Interaction Analysis Between Lectins and Glycans

Publisher Summary Sugar-binding proteins, i.e., lectins, have been utilized for more than 100 years to investigate the diversity and quantity of glycans expressed on the cell surface. In order to understand the fundamental features of lectin affinity, systematic analysis of lectin—glycan interactions is an absolute requirement. It is widely known that binding affinities of lectins to oligosaccharides are relatively low, particularly in comparison with antibodies. It is therefore often difficult to obtain precise dissociation constants, or affinity constants in a systematic manner. For this purpose, frontal affinity chromatography (FAC) has emerged as perhaps the best choice among various interaction analysis methods. This chapter first briefly describes the overall features and principle of FAC, and then discusses various applications of the technique in analysis of lectins and glycans such as profiling the sugar-binding specificity of a particular lectin, pair-wise comparison of lectin sugar-binding specificities, and profiling the branching features of N-linked glycans.