Hiroaki Tateno

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRγ-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRγ, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to α-mannose but not mannan. Thus, Mincle may recognize specific geometry of α-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle−/− mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle−/− mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.

Lectin microarrays: concept, principle and applications

The lectin microarray is a novel platform for glycan analysis, having emerged only in recent years. Unlike other conventional methods, e.g., liquid chromatography and mass spectrometry, it enables rapid and high-sensitivity profiling of complex glycan features without the need for liberation of glycans. Target samples include an extensive range of glycoconjugates involved in cells, tissues, body fluids, as well as synthetic glycans and their mimics. Various procedures for rapid differential glycan profiling have been developed for glycan-related biomarkers. Such glycoproteomics targeting allows precise diagnosis of chronic diseases potentially related to cancer. Application of this method to evaluation of various types of stem cells resulted in the discovery of a new pluripotent cell-specific glycan marker. To explore this technology a more fundamental and extensive understanding of lectins is necessary in relation to the structural uniqueness of glycans. In this chapter, the essence of the lectin microarray is described with some focus on an evanescent-field-activated fluorescence detection principle as a system to achieve in situ (i.e., washing free) aqueous-phase observation under equilibrium conditions. The developed lectin microarray system allows even researchers with poor experience in glycan profiling to perform extensive high-throughput analysis targeting various forms of glycans and even cells.

Galectin-9 increases Tim-3+ dendritic cells and CD8+ T cells and enhances antitumor immunity via galectin-9-Tim-3 interactions.

A Tim-3 ligand, galectin-9 (Gal-9), modulates various functions of innate and adaptive immune responses. In this study, we demonstrate that Gal-9 prolongs the survival of Meth-A tumor-bearing mice in a dose- and time-dependent manner. Although Gal-9 did not prolong the survival of tumor-bearing nude mice, transfer of naive spleen cells restored a prolonged Gal-9-induced survival in nude mice, indicating possible involvement of T cell-mediated immune responses in Gal-9-mediated antitumor activity. Gal-9 administration increased the number of IFN-γ-producing Tim-3+ CD8+ T cells with enhanced granzyme B and perforin expression, although it induced CD4+ T cell apoptosis. It simultaneously increased the number of Tim-3+CD86+ mature dendritic cells (DCs) in vivo and in vitro. Coculture of CD8+ T cells with DCs from Gal-9-treated mice increased the number of IFN-γ producing cells and IFN-γ production. Depletion of Tim-3+ DCs from DCs of Gal-9-treated tumor-bearing mice decreased the number of IFN-γ-producing CD8+ T cells. Such DC activity was significantly abrogated by Tim-3-Ig, suggesting that Gal-9 potentiates CD8+ T cell-mediated antitumor immunity via Gal-9-Tim-3 interactions between DCs and CD8+ T cells.

Glycome diagnosis of human induced pluripotent stem cells using lectin microarray

Induced pluripotent stem cells (iPSCs) can now be produced from various somatic cell (SC) lines by ectopic expression of the four transcription factors. Although the procedure has been demonstrated to induce global change in gene and microRNA expressions and even epigenetic modification, it remains largely unknown how this transcription factor-induced reprogramming affects the total glycan repertoire expressed on the cells. Here we performed a comprehensive glycan analysis using 114 types of human iPSCs generated from five different SCs and compared their glycomes with those of human embryonic stem cells (ESCs; nine cell types) using a high density lectin microarray. In unsupervised cluster analysis of the results obtained by lectin microarray, both undifferentiated iPSCs and ESCs were clustered as one large group. However, they were clearly separated from the group of differentiated SCs, whereas all of the four SCs had apparently distinct glycome profiles from one another, demonstrating that SCs with originally distinct glycan profiles have acquired those similar to ESCs upon induction of pluripotency. Thirty-eight lectins discriminating between SCs and iPSCs/ESCs were statistically selected, and characteristic features of the pluripotent state were then obtained at the level of the cellular glycome. The expression profiles of relevant glycosyltransferase genes agreed well with the results obtained by lectin microarray. Among the 38 lectins, rBC2LCN was found to detect only undifferentiated iPSCs/ESCs and not differentiated SCs. Hence, the high density lectin microarray has proved to be valid for not only comprehensive analysis of glycans but also diagnosis of stem cells under the concept of the cellular glycome.

Structural analysis of the Laetiporus sulphureus hemolytic pore-forming lectin in complex with sugars.

LSL is a lectin produced by the parasitic mushroom Laetiporus sulphureus, which exhibits hemolytic and hemagglutinating activities. Here, we report the crystal structure of LSL refined to 2.6-Å resolution determined by the single isomorphous replacement method with the anomalous scatter (SIRAS) signal of a platinum derivative. The structure reveals that LSL is hexameric, which was also shown by analytical ultracentrifugation. The monomeric protein (35 kDa) consists of two distinct modules: an N-terminal lectin module and a pore-forming module. The lectin module has a β-trefoil scaffold that bears structural similarities to those present in toxins known to interact with galactose-related carbohydrates such as the hemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum, abrin, and ricin. On the other hand, the C-terminal pore-forming module (composed of domains 2 and 3) exhibits three-dimensional structural resemblances with domains 3 and 4 of the β-pore-forming toxin aerolysin from the Gram-negative bacterium Aeromonas hydrophila, and domains 2 and 3 from the ϵ-toxin from Clostridium perfringens. This finding reveals the existence of common structural elements within the aerolysin-like family of toxins that could be directly involved in membrane-pore formation. The crystal structures of the complexes of LSL with lactose and N-acetyllactosamine reveal two dissacharide-binding sites per subunit and permits the identification of critical residues involved in sugar binding.

Glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of glycan-binding proteins

The extensive involvement of glycan-binding proteins (GBPs) as regulators in diverse biological phenomena provides a fundamental reason to investigate their glycan-binding specificities. Here, we developed a glycoconjugate microarray based on an evanescent-field fluorescence-assisted detection principle for investigation of GBPs. Eighty-nine selected multivalent glycoconjugates comprising natural glycoproteins, neo-glycoproteins, and polyacrylamide (PAA)-conjugated glycan epitopes were immobilized on an epoxy-activated glass slide. The GBP binding was monitored by an evanescent-field fluorescence-assisted scanner at equilibrium without washing steps. The detection principle also allows direct application of unpurified GBPs with the aid of specific antibodies. Model experiments using plant lectins (RCA120, ConA, and SNA), galectins (3 and 8), a C-type lectin (DC-SIGN) and a siglec (CD22) provided data consistent with previous work within 4 h using less than 40 ng of GBPs per analysis. As an application, serum profiling of antiglycan antibodies (IgG and IgM) was performed with Cy3-labeled secondary antibodies. Moreover, novel carbohydrate-binding ability was demonstrated for a human IL-18 binding protein. Thus, the developed glycan array is useful for investigation of various types of GBPs, with the added advantage of wash-free analysis.

Distinct endocytic mechanisms of CD22 (siglec-2) and siglec-F reflect roles in cell signaling and innate immunity

ABSTRACT Sialic acid-binding immunoglobulin-like lectins (siglecs) are predominately expressed on immune cells. They are best known as regulators of cell signaling mediated by cytoplasmic tyrosine motifs and are increasingly recognized as receptors for pathogens that bear sialic acid-containing glycans. Most siglec proteins undergo endocytosis, an activity tied to their roles in cell signaling and innate immunity. Here, we investigate the endocytic pathways of two siglec proteins, CD22 (Siglec-2), a regulator of B-cell signaling, and mouse eosinophil Siglec-F, a member of the rapidly evolving CD33-related siglec subfamily that are expressed on cells of the innate immune system. CD22 exhibits hallmarks of clathrin-mediated endocytosis and traffics to recycling compartments, consistent with previous reports demonstrating its localization to clathrin domains. Like CD22, Siglec-F mediates endocytosis of anti-Siglec-F and sialoside ligands, a function requiring intact tyrosine-based motifs. In contrast, however, we find that Siglec-F endocytosis is clathrin and dynamin independent, requires ADP ribosylation factor 6, and traffics to lysosomes. The results suggest that these two siglec proteins have evolved distinct endocytic mechanisms consistent with roles in cell signaling and innate immunity.

Comparative analysis of core-fucose-binding lectins from Lens culinaris and Pisum sativum using frontal affinity chromatography.

Lens culinaris lectin (LCA) is a useful probe for the detection in serum of a core-fucosylated alpha-fetoprotein, called AFP-L3 fraction, which is a well-known marker for the diagnosis and prognosis of hepatocellular carcinoma. Here we performed a systematic quantitative interaction analysis of LCA and its close homolog, Pisum sativum lectin (PSA), by frontal affinity chromatography with 143 pyridylaminated (PA) glycans including a series of core-fucosylated glycans. Both lectins showed binding affinity to core-fucosylated, mono- and bi-antennary N-glycans, but not to their tri- and tetra-antennary forms, indicating that the addition of the GlcNAc residue at the N-acetylglucosaminyltransferase IV position abrogates the binding affinity. However, their specificities are distinguishable: while LCA showed the highest affinity to the core-fucosylated, agalactosylated, bi-antennary N-glycan (K(a)=1.1 x 10(5) M(-1)), PSA showed the highest affinity to the core-fucosylated, trimannosyl structure (K(a)=1.2 x 10(5) M(-1)). Glycan-binding specificities of LCA and PSA were also analyzed by glycoconjugate microarray compared to other core-fucose-binding lectins from Aspergillus oryzae (AOL) and Aleuria auratia (AAL). LCA and PSA bound specifically to core fucose, whereas AOL and AAL exhibited broad specificity to fucosylated glycans. These results explain why LCA is appropriate as a specific probe for AFP-L3, which mainly contains a core-fucosylated, biantennary N-glycan, but not its highly branched forms.

Lectin-based structural glycomics: a practical approach to complex glycans.

Glycans exist in nature in various forms of glycoconjugates, i.e., glycoproteins, glycolipids, and glycosaminoglycans, either in soluble or membrane‐bound forms. One of their prominent properties distinguished from nucleic acids and proteins is “heterogeneity” largely attributed to their inherent features of biosynthesis. In general, various methods based on the physicochemical principles have been taken for their separation and structural determination although all of them require prior liberation of glycans and appropriate labeling. On the other hand, a series of carbohydrate‐binding proteins, or “lectins,” have extensively been used in a more direct manner for cell typing, histochemical staining, and glycoprotein fractionation. Although most procedures conventionally used are useful, unfortunately they lack “throughput” comparable to a performance required for current omics studies. Recently, a novel technique called lectin microarray has attracted increasing attention from not only glycoscientists but also researchers in other fields, because it is straightforward and also informative. The method is innovating in that it enables direct approach to glycoconjugates such as glycoproteins and even cells without liberation of glycans from the core substrate, and therefore can be effectively applied for the sake of differential profiling in various fields. Concept, strategy, and technical advancement of lectin microarray are described. Also, as an introduction to glycomics, the authors explain the motivation to challenge this theme.

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.