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 theMalasseziaspecies among 50 different fungal species tested.Malasseziais 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 abrogatedMalasseziarecognition. 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 onMalasseziaspecies and use this to distinguish them from other fungi.Malasseziaactivated 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 againstMalasseziawas also impaired in Mincle^−/−mice. These results indicate that Mincle is the first specific receptor forMalasseziaspecies to be reported and plays a crucial role in immune responses to this fungus.

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.

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.

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.

Mouse sialic acid-binding immunoglobulin-like lectin F (Siglec-F) is an eosinophil surface receptor, which contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain, implicating it as a regulator of cell signaling as documented for other siglecs. Here, we show that the sialoside sequence 6′-sulfo-sLe^X (Neu5Acα2–3[6-SO₄] Galβ1–4[Fucα1–3]GlcNAc) is a preferred ligand for Siglec-F. In glycan array analysis of 172 glycans, recombinant Siglec-F-Fc chimeras bound with the highest avidity to 6′-sulfo-sLe^X. Secondary analysis showed that related structures, sialyl-Lewis X (sLe^X) and 6-sulfo-sLe^X containing 6-GlcNAc-SO₄ showed much lower binding avidity, indicating significant contribution of 6-Gal-SO₄ on Siglec-F binding to 6′-sulfo-sLe^x. The lectin activity of Siglec-F on mouse eosinophils was “masked” by endogenous cis ligands and could be unmasked by treatment with sialidase. Unmasked Siglec-F mediated mouse eosinophil binding and adhesion to multivalent 6′‐sulfo-sLe^X structure, and these interactions were inhibited by anti-Siglec-F monoclonal antibody (mAb). Although there is no clear-cut human ortholog of Siglec-F, Siglec-8 is encoded by a paralogous gene that is expressed selectively by human eosinophils and has recently been found to recognize 6′-sulfo-sLe^X. These observations suggest that mouse Siglec-F and human Siglec-8 have undergone functional convergence during evolution and implicate a role for the interaction of these siglecs with their preferred 6′-sulfo-sLe^X ligand in eosinophil biology.

The glycome represents the total set of glycans expressed in a cell. The glycome has been assumed to vary between cell types, stages of development and differentiation, and during malignant transformation. Analysis of the glycome provides a basis for understanding the functions of glycans in these cellular processes. Recently, a technique called lectin microarray was developed for rapid profiling of glycosylation, although its use was mainly restricted to glycoproteins of cell lysates, and thus unable to profile the intact cell surface glycans. Here we report a simple and sensitive procedure based on this technology for direct analysis of the live mammalian cell-surface glycome. Fluorescent-labeled live cells were applied in situ to the established lectin microarray consisting of 43 immobilized lectins with distinctive binding specificities. After washing, bound cells were directly detected by an evanescent-field fluorescence scanner in a liquid phase without fixing and permeabilization. The results obtained by differential profiling of CHO and its glycosylation-defective mutant cells, and splenocytes of wild-type and β1-3-N-acetylglucosaminyltransferase II knockout mice performed as model experiments agreed well with their glycosylation phenotypes. We also compared cell surface glycans of K562 cells before and after differentiation and found a significant increase in the expression of O-glycans on differentiated cells. These results demonstrate that the technique provides a novel strategy for profiling global changes of the mammalian cell surface glycome.