Nobuko Kawasaki

Glycosylation-Dependent Interactions of C-Type Lectin DC-SIGN with Colorectal Tumor-Associated Lewis Glycans Impair the Function and Differentiation of Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) are APCs that play an essential role by bridging innate and adaptive immunity. DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is one of the major C-type lectins expressed on DCs and exhibits high affinity for nonsialylated Lewis (Le) glycans. Recently, we reported the characterization of oligosaccharide ligands expressed on SW1116, a typical human colorectal carcinoma recognized by mannan-binding protein, which is a serum C-type lectin and has similar carbohydrate-recognition specificities as DC-SIGN. These tumor-specific oligosaccharide ligands were shown to comprise clusters of tandem repeats of Lea/Leb epitopes. In this study, we show that DC-SIGN is involved in the interaction of DCs with SW1116 cells through the recognition of aberrantly glycosylated forms of Lea/Leb glycans on carcinoembryonic Ag (CEA) and CEA-related cell adhesion molecule 1 (CEACAM1). DC-SIGN ligands containing Lea/Leb glycans are also highly expressed on primary cancer colon epithelia but not on normal colon epithelia, and DC-SIGN is suggested to be involved in the association between DCs and colorectal cancer cells in situ by DC-SIGN recognizing these cancer-related Le glycan ligands. Furthermore, when monocyte-derived DCs (MoDCs) were cocultured with SW1116 cells, LPS-induced immunosuppressive cytokines such as IL-6 and IL-10 were increased. The effects were significantly suppressed by blocking Abs against DC-SIGN. Strikingly, LPS-induced MoDC maturation was inhibited by supernatants of cocultures with SW1116 cells. Our findings imply that colorectal carcinomas affecting DC function and differentiation through interactions between DC-SIGN and colorectal tumor-associated Le glycans may induce generalized failure of a host to mount an effective antitumor response.

A novel antibody for human induced pluripotent stem cells and embryonic stem cells recognizes a type of keratan sulfate lacking oversulfated structures

We have generated a monoclonal antibody (R-10G) specific to human induced pluripotent stem (hiPS)/embryonic stem (hES) cells by using hiPS cells (Tic) as an antigen, followed by differential screening of mouse hybridomas with hiPS and human embryonal carcinoma (hEC) cells. Upon western blotting with R-10G, hiPS/ES cell lysates gave a single but an unusually diffuse band at a position corresponding to >250 kDa. The antigen protein was isolated from the induced pluripotent stem (iPS) cell lysates with an affinity column of R-10G. The R-10G positive band was resistant to digestion with peptide N-glycanase F (PNGase F), neuraminidase, fucosidase, chondrotinase ABC and heparinase mix, but it disappeared almost completely on digestion with keratanase, keratanase II and endo-β-galactosidase, indicating that the R-10G epitope is a keratan sulfate. The carrier protein of the R-10G epitope was identified as podocalyxin by liquid chromatography/mass spectrometry (LC/MS/MS) analysis of the R-10G positive-protein band material obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The R-10G epitope is a type of keratan sulfate with some unique properties. (1) The epitope is expressed only on hiPS/ES cells, i.e. not on hEC cells, unlike those recognized by the conventional hiPS/ES marker antibodies. (2) The epitope is a type of keratan sulfate lacking oversulfated structures and is not immunologically cross-reactive with high-sulfated keratan sulfate. (3) The R-10G epitope is distributed heterogeneously on hiPS cells, suggesting that a single colony of undifferentiated hiPS cells consists of different cell subtypes. Thus, R-10G is a novel antibody recognizing hiPS/ES cells, and should be a new molecular probe for disclosing the roles of glycans on these cells.

Glycosylation‐dependent interaction of Jacalin with CD45 induces T lymphocyte activation and Th1/Th2 cytokine secretion

Jacalin, an α‐O‐glycoside of the disaccharide Thomsen‐Friedenreich antigen (galactose β1‐3 N‐acetylgalactosamine, T‐antigen)‐specific lectin from jackfruit seeds, has been shown to induce mitogenic responses and to block infection by HIV‐1 in CD4+ T lymphocytes. The molecular mechanism underlying Jacalin‐induced T cell activation has not been elucidated completely yet. In the present study, protein tyrosine phosphatase (PTPase) CD45 was isolated from a Jurkat T cell membrane fraction as a major receptor for Jacalin through affinity chromatography and mass spectrometry. CD45, which is highly glycosylated and expressed exclusively on the surface of lymphocytes, is a key regulator of lymphocyte signaling, playing a pivotal role in activation and development. We found that the lectin induced significant IL‐2 production by a CD45‐positive Jurkat T cell line (JE6.1) and primary T cells. However, this effect did not occur in a CD45‐negative Jurkat T cell line (J45.01) and was blocked completely by a specific CD45 PTPase inhibitor in Jurkat T (JE6.1) and primary T cells. Furthermore, we also observed that Jacalin caused a marked increase in IL‐2 secretion in response to TCR ligation and CD28 costimulation and contributed to Th1/Th2 cytokine production by activating CD45. Jacalin increased CD45 tyrosine phosphatase activity, which resulted in activation of the ERK1/2 and p38 MAPK cascades. Based on these findings, we propose a new, immunoregulatory model for Jacalin, wherein glycosylation‐dependent interactions of Jacalin with CD45 on T cells elevate TCR‐mediated signaling, which thereby up‐regulate T cell activation thresholds and Th1/Th2 cytokine secretion.

Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Recognizes a Novel Ligand, Mac-2-binding Protein, Characteristically Expressed on Human Colorectal Carcinomas

Dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) is a type II transmembrane C-type lectin expressed on DCs such as myeloid DCs and monocyte-derived DCs (MoDCs). Recently, we have reported that DC-SIGN interacts with carcinoembryonic antigen (CEA) expressed on colorectal carcinoma cells. CEA is one of the most widely used tumor markers for gastrointestinal cancers such as colorectal cancer. On the other hand, other groups have reported that the level of Mac-2-binding protein (Mac-2BP) increases in patients with pancreatic, breast, and lung cancers, virus infections such as human immunodeficiency virus and hepatitis C virus, and autoimmune diseases. Here, we first identified Mac-2BP expressed on several colorectal carcinoma cell lines as a novel DC-SIGN ligand through affinity chromatography and mass spectrometry. Interestingly, we found that DC-SIGN selectively recognizes Mac-2BP derived from some colorectal carcinomas but not from the other ones. Furthermore, we found that the α1-3,4-fucose moieties of Le glycans expressed on DC-SIGN-binding Mac-2BP were important for recognition. DC-SIGN-dependent cellular interactions between immature MoDCs and colorectal carcinoma cells significantly inhibited MoDC functional maturation, suggesting that Mac-2BP may provide a tolerogenic microenvironment for colorectal carcinoma cells through DC-SIGN-dependent recognition. Importantly, Mac-2BP was detected as a predominant DC-SIGN ligand expressed on some primary colorectal cancer tissues from certain parts of patients in comparison with CEA from other parts, suggesting that DC-SIGN-binding Mac-2BP bearing tumor-associated Le glycans may become a novel potential colorectal cancer biomarker for some patients instead of CEA.

Subcellular Localization and Physiological Significance of Intracellular Mannan-binding Protein

Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

Characterization of the interaction between serum mannan-binding protein and nucleic acid ligands

Serum MBP, also known as MBL, is a C‐type lectin that is known to be a soluble host defense factor involved in innate immunity. It has been well established that dying microbes and apoptotic cells release highly viscous DNA that induces inflammation and septic shock, and apoptotic cells display fragmented DNA on their surfaces. However, PRRs that mediate the recognition and clearance of free DNA and fragmented DNA in apoptotic cells have not been characterized clearly. Although MBP was reported recently to bind DNA as a novel ligand, binding characterization and the recognition implications have not been addressed yet. In this study, we show that MBP can bind DNA and RNA in a calcium‐dependent manner from a variety of origins, including bacteria, plasmids, synthetic oligonucleotides, and fragmented DNA of apoptotic cells. Direct binding and competition studies indicate that MBP binds nucleic acids via its CRD to varying degrees and that MBP binds dsDNA more effectively than ssDNA and ssRNA. Furthermore, we reveal that the MBP‐DNA complex does not trigger complement activation via the MBP lectin pathway, and the lectin pathway of complement activation is required for MBP‐mediated enhancement of phagocytosis of targets bearing MBP ligands and that MBP can recognize the fragmented DNA presented on apoptotic cells. Therefore, we propose that the MBP lectin pathway may support effective recognition and clearance of cellular debris by facilitating phagocytosis, possibly through immunomodulatory mechanisms, thus preventing autoimmunity.

Highly fucosylated N-glycan ligands for mannan-binding protein expressed specifically on CD26 (DPPVI) isolated from a human colorectal carcinoma cell line, SW1116

The serum mannan-binding protein (MBP) is a host defense C-type lectin specific for mannose, N-acetylglucosamine, and fucose residues, and exhibits growth inhibitory activity toward human colorectal carcinoma cells. The MBP-ligand oligosaccharides (MLO) isolated from a human colorectal carcinoma cell line, SW1116, are large, multiantennary N-glycans with highly fucosylated polylactosamine-type structures having Le(b)-Le(a) or tandem repeats of the Le(a) structure at their nonreducing ends. In this study, we isolated the major MBP-ligand glycoproteins from SW1116 cell lysates with an MBP column and identified them as CD26/dipeptidyl peptidase IV (DPPIV) (110 kDa) and CD98 heavy chain (CD98hc)/4F2hc (82 kDa). Glycosidase digestion revealed that CD26 contained such complex-type N-glycans that appear to mediate the MBP binding. MALDI-MS of the N-glycans released from CD26 by PNGase F demonstrated conclusively that CD26 is the major MLO-carrying protein. More interestingly, a comparison of the N-glycans released from the MBP-binding and non-MBP-binding glycopeptides suggested that complex-type N-glycans carrying a minimum of 4 Le(a)/Le(b) epitopes arranged either as multimeric tandem repeats or terminal epitopes on multiantennary structures are critically important for the high affinity binding to MBP. Analysis of the N-glycan attachment sites demonstrated that the high affinity MLO was expressed preferentially at some N-glycosylation sites, but this site preference was not so stringent. Finally, hypothetical 3D models of tandem repeats of the Le(a) epitope and the MBP-Lewis oligosaccharide complex were presented.

Mannose/N-acetylglucosamine-binding proteins from mammalian sera.

Publisher Summary Sera from various mammals are known to contain a binding protein that can recognize and bind mannose and N-acetylglucosamine residues. This protein—called “serum mannan-binding protein,” (S-MBP)—has been shown to activate the complement system through the classical pathway. Bovine serum is known to contain a unique protein, called conglutinin, that binds to a complement component but does not activate the complement system. Conglutinin is also shown to be a carbohydrate-binding protein specific for N-acetylglucosamine. This chapter is concerned with isolation of S-MBP from human, rat, rabbit, and bovine sera as well as isolation of conglutinin from bovine serum. The binding properties of conglutinin and MBP are generally similar. However, there are important differences in their binding specificity. Conglutinin is inhibited almost exclusively by N-acetylglucosamine both in binding to 125 I-labeled mannan and in the conglutination reaction, while MBP is potently inhibited by mannose as well as N-acetylglucosamine.

Mannan-Binding Protein, a C-Type Serum Lectin, Recognizes Primary Colorectal Carcinomas through Tumor-Associated Lewis Glycans

Mannan (mannose)-binding protein (MBP) is a C-type serum lectin that plays a key role in innate immunity. MBP forms large multimers (200–600 kDa) and exhibits broad specificity for mannose, N-acetylglucosamine, and fucose. MBP exhibits high affinity for unique oligosaccharides that have been isolated from human colorectal carcinoma (SW1116) cells and characterized as highly fucosylated high m.w. type 1 Lewis glycans. In this study, we first demonstrated that MBP recognizes human primary colorectal carcinoma tissues through tumor-associated MBP ligands. We performed fluorescence-based histochemistry of MBP in human colorectal carcinoma tissues and showed that MBP clearly stained cancer mucosae in a Ca2+-dependent manner. Coincubation with plant (Aleuria aurantia) lectin, but not Con A, blocked MBP staining, indicating that fucose, rather than mannose, is involved in this interaction. The expression of MBP ligands was detected in 127 of 330 patients (38.5%), whereas, most significantly, there was no expression in 69 nonmalignant tissues. The MBP-staining pattern in cancer mucosae significantly overlapped with that of Lewis b [Fucα1-2Galβ1-3(Fucα1-4)GlcNAc] staining, but the Lewis b staining in normal tissues was not associated with MBP staining. In addition, the MBP staining correlated inversely with the expression of CA19-9 Ag, and MBP stained 11 of 25 (44%) CA19-9 (sialyl Lewis a [NeuAc(α2-3)Galβ1-3(Fucα1-4)GlcNAc])− colorectal carcinoma tissues. We found a favorable prognosis in patients with MBP ligand+ tumors. These results suggest that selective recognition of cancer cells by endogenous MBP seems to be associated with an antitumor effect and that tissue staining with MBP in combination with CA19-9 may serve as a novel indicator of colorectal carcinoma tissues.

A Cytotoxic Antibody Recognizing Lacto-N-fucopentaose I (LNFP I) on Human Induced Pluripotent Stem (hiPS) Cells.

Background: Carbohydrate epitopes are often used as markers for characterization of hiPS cells. Results: A mouse IgG1 antibody (R-17F) was raised using hiPS cells as an antigen. Conclusion: R-17F recognizes lacto-N-fucopentaose I on glycolipid and exhibits a cytotoxic effect on hiPS/ES cells. Significance: R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS/ES cells, which are a risk factor for carcinogenesis. We have generated a mouse monoclonal antibody (R-17F, IgG1 subtype) specific to human induced pluripotent stem (hiPS)/embryonic stem (ES) cells by using a hiPS cell line as an antigen. Triple-color confocal immunostaining images of hiPS cells with R-17F indicated that the R-17F epitope was expressed exclusively and intensively on the cell membranes of hiPS cells and co-localized partially with those of SSEA-4 and SSEA-3. Lines of evidence suggested that the predominant part of the R-17F epitope was a glycolipid. Upon TLC blot of total lipid extracts from hiPS cells with R-17F, one major R-17F-positive band was observed at a slow migration position close to that of anti-blood group H1(O) antigen. MALDI-TOF-MS and MSn analyses of the purified antigen indicated that the presumptive structure of the R-17F antigen was Fuc-Hex-HexNAc-Hex-Hex-Cer. Glycan microarray analysis involving 13 different synthetic oligosaccharides indicated that R-17F bound selectively to LNFP I (Fucα1–2Galβ1–3GlcNAcβ1–3Galβ1–4Glc). A critical role of the terminal Fucα1–2 residue was confirmed by the selective disappearance of R-17F binding to the purified antigen upon α1–2 fucosidase digestion. Most interestingly, R-17F, when added to hiPS/ES cell suspensions, exhibited potent dose-dependent cytotoxicity. The cytotoxic effect was augmented markedly upon the addition of the secondary antibody (goat anti-mouse IgG1 antibody). R-17F may be beneficial for safer regenerative medicine by eliminating residual undifferentiated hiPS cells in hiPS-derived regenerative tissues, which are considered to be a strong risk factor for carcinogenesis.