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Hans-joachim Gabius - One of the best experts on this subject based on the ideXlab platform.
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detection of malignancy associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth inhibitory Galectin 4
Iubmb Life, 2018Co-Authors: Hans-joachim Gabius, Jürgen Kopitz, Malwina Michalak, Uwe Warnken, Martina SchnölzerAbstract:Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested Galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying Galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.
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Detection of malignancy‐associated phosphoproteome changes in human colorectal cancer induced by cell surface binding of growth‐inhibitory Galectin‐4
IUBMB life, 2018Co-Authors: Malwina Michalak, Hans-joachim Gabius, Uwe Warnken, Martina Schnölzer, Jürgen KopitzAbstract:Emerging evidence on efficient tumor growth regulation by endogenous lectins directs interest to determine on a proof-of-principle level the range of information on alterations provided by full-scale analysis using phosphoproteomics. In our pilot study, we tested Galectin-4 (gal-4) that is a growth inhibitor for colon cancer cells (CRC), here working with the LS 180 line. In order to cover monitoring of short- and long-term effects stable isotope labeling by amino acids in cell culture-based quantitative phosphoproteomic analyses were conducted on LS 180 cell preparations collected 1 and 72 h after adding gal-4 to the culture medium. After short-term treatment, 981 phosphosites, all of them S/T based, were detected by phosphoproteomics. Changes higher than 1.5-fold were seen for eight sites in seven proteins. Most affected were the BET1 homolog (BET1), whose level of phosphorylation at S50 was about threefold reduced, and centromere protein F (CENPF), extent of phosphorylation at S3119 doubling in gal-4-treated cells. Phosphoproteome analysis after 72 h of treatment revealed marked changes at 33 S/T-based phosphosites from 29 proteins. Prominent increase of phosphorylation was observed for cofilin-1 at position S3. Extent of phosphorylation of the glutamine transporter SLC1A5 at position S503 was decreased by a factor of 3. Altered phosphorylation of BET1, CENPF, and cofilin-1 as well as a significant effect of gal-4 treatment on glutamine uptake by cells were substantiated by independent methods in the Vaco 432, Colo 205, CX 1, and HCT 116 cell lines. With the example of gal-4 which functions as a tumor suppressor in CRC cells, we were able to prove that cell surface binding of the lectin not only markedly influences the cell proteome, but also has a bearing on malignancy-associated intracellular protein phosphorylation. These results underscore the potential of this approach to give further work on elucidating the details of signaling underlying Galectin-triggered growth inhibition a clear direction. © 2018 IUBMB Life, 71(3):364-375, 2019.
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Galectin 4 a negative regulator of oligodendrocyte differentiation is persistently present in axons and microglia macrophages in multiple sclerosis lesions
Journal of Neuropathology and Experimental Neurology, 2018Co-Authors: Charlotte G. H. M. De Jong, Mirjana Stancic, Tineke H. Pinxterhuis, Jack Van Horssen, Anne-marie Van Dam, Hans-joachim Gabius, Wia BaronAbstract:Neuron-derived molecules are potent regulators of oligodendrocyte differentiation and myelination during brain development and upon demyelination. Their analysis will thus contribute to understanding remyelination failure in demyelinating diseases, such as multiple sclerosis (MS). Previously, we have identified neuronal Galectin-4 as a novel negative soluble regulator in the timing of developmental myelination. Here, we investigated whether Galectin-4 is re-expressed in axons upon demyelination to regulate the timing of remyelination. Our findings revealed that Galectin-4 is transiently localized to axons in demyelinated areas upon cuprizone-induced demyelination. In contrast, in chronic demyelinated MS lesions, where remyelination fails, Galectin-4 is permanently present on axons. Remarkably, microglia/macrophages in cuprizone-demyelinated areas also harbor Galectin-4, as also observed in activated microglia/macrophages that are present in active MS lesions and in inflammatory infiltrates in chronic-relapsing experimental autoimmune encephalomyelitis. In vitro analysis showed that Galectin-4 is effectively endocytosed by macrophages, and may scavenge Galectin-4 from oligodendrocytes, and that endogenous Galectin-4 levels are increased in alternatively interleukin-4-activated macrophages and microglia. Hence, similar to developmental myelination, the (re)expressed Galectin-4 upon demyelination may act as factor in the timing of oligodendrocyte differentiation, while the persistent presence of Galectin-4 on demyelinated axons may disrupt this fine-tuning of remyelination.
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Galectin-4, a Negative Regulator of Oligodendrocyte Differentiation, Is Persistently Present in Axons and Microglia/Macrophages in Multiple Sclerosis Lesions.
Journal of neuropathology and experimental neurology, 2018Co-Authors: Charlotte G. H. M. De Jong, Mirjana Stancic, Tineke H. Pinxterhuis, Jack Van Horssen, Anne-marie Van Dam, Hans-joachim Gabius, Wia BaronAbstract:Neuron-derived molecules are potent regulators of oligodendrocyte differentiation and myelination during brain development and upon demyelination. Their analysis will thus contribute to understanding remyelination failure in demyelinating diseases, such as multiple sclerosis (MS). Previously, we have identified neuronal Galectin-4 as a novel negative soluble regulator in the timing of developmental myelination. Here, we investigated whether Galectin-4 is re-expressed in axons upon demyelination to regulate the timing of remyelination. Our findings revealed that Galectin-4 is transiently localized to axons in demyelinated areas upon cuprizone-induced demyelination. In contrast, in chronic demyelinated MS lesions, where remyelination fails, Galectin-4 is permanently present on axons. Remarkably, microglia/macrophages in cuprizone-demyelinated areas also harbor Galectin-4, as also observed in activated microglia/macrophages that are present in active MS lesions and in inflammatory infiltrates in chronic-relapsing experimental autoimmune encephalomyelitis. In vitro analysis showed that Galectin-4 is effectively endocytosed by macrophages, and may scavenge Galectin-4 from oligodendrocytes, and that endogenous Galectin-4 levels are increased in alternatively interleukin-4-activated macrophages and microglia. Hence, similar to developmental myelination, the (re)expressed Galectin-4 upon demyelination may act as factor in the timing of oligodendrocyte differentiation, while the persistent presence of Galectin-4 on demyelinated axons may disrupt this fine-tuning of remyelination.
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Neurons define non-myelinated axon segments by the regulation of Galectin-4-containing axon membrane domains.
Scientific reports, 2017Co-Authors: Natalia Díez-revuelta, Hans-joachim Gabius, Alonso M. Higuero, Silvia Velasco, Maria Penas-de-la-iglesia, José Abad-rodríguezAbstract:The mechanism underlying selective myelination of axons versus dendrites or neuronal somata relies on the expression of somatodendritic membrane myelination inhibitors (i.e. JAM2). However, axons still present long unmyelinated segments proposed to contribute to axonal plasticity and higher order brain functions. Why these segments remain unmyelinated is still an unresolved issue. The bifunctional lectin Galectin-4 (Gal-4) organizes the transport of axon glycoproteins by binding to N-acetyllactosamine (LacNac) termini of N-glycans. We have shown that Gal-4 is sorted to segmental domains (G4Ds) along the axon surface, reminiscent of these long unmyelinated axon segments in cortical neurons. We report here that oligodendrocytes (OLGs) do not deposit myelin on Gal-4 covered surfaces or myelinate axonal G4Ds. In addition, Gal-4 interacts and co-localizes in G4Ds with contactin-1, a marker of another type of non-myelinated segments, the nodes of Ranvier. Neither Gal-4 expression nor G4D dimensions are affected by myelin extracts or myelinating OLGs, but are reduced with neuron maturation. As in vitro, Gal-4 is consistently segregated from myelinated structures in the brain. Our data shape the novel concept that neurons establish axon membrane domains expressing Gal-4, the first inhibitor of myelination identified in axons, whose regulated boundaries delineate myelination-incompetent axon segments along development.
Sabine André - One of the best experts on this subject based on the ideXlab platform.
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Detection of Proteome Changes in Human Colon Cancer Induced by Cell Surface Binding of Growth-Inhibitory Human Galectin-4 Using Quantitative SILAC-Based Proteomics
Journal of proteome research, 2016Co-Authors: Malwina Michalak, Sabine André, Hans-joachim Gabius, Uwe Warnken, Martina Schnölzer, Juergen KopitzAbstract:Endogenous lectins have the capacity to translate glycan-encoded information on the cell surface into effects on cell growth. As test cases to examine changes in protein presence associated with tumor growth inhibition, we applied SILAC-based proteomics on human colon carcinoma cells treated with Galectin-4 (Gal-4). The five tested lines—LS 180, Vaco 432, Colo 205, CX 1, and HCT 116—responded with differentiation and reduced proliferation to Gal-4 binding. In proteomic analysis (mass spectral data deposited with PRIDE, PXD003489), 2654 proteins were quantified, of which 190 were down-regulated and 115 were up-regulated (>2-fold). 1D annotation analysis of the results indicated down-regulation of DNA replication-associated processes, while protein presence for secretory and transport functions appeared increased. The strongest induction was found for CALB2 (calretinin; ∼24-fold), TGM2 (protein-glutamine γ-glutamyltransferase 2; ∼11-fold), S100A3 (∼10-fold), and GSN (gelsolin; 9.5-fold), and the most pronou...
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Combining glycocluster synthesis with protein engineering: an approach to probe into the significance of linker length in a tandem-repeat-type lectin (Galectin-4).
Carbohydrate research, 2014Co-Authors: Sabine André, Hans-joachim Gabius, Guan-nan Wang, Paul V. MurphyAbstract:Abstract Complementarity in lectin–glycan interactions in situ is assumed to involve spatial features in both the lectin and the glycan, giving a functional meaning to structural aspects of the lectin beyond its carbohydrate-binding site. In combining protein engineering with glycocluster synthesis, it is shown that the natural linker length of a tandem-repeat-type human lectin (Galectin-4) determines binding properties in two binding assays (using surface-presented glycoprotein and cell surface assays). The types of glycocluster tested included bivalent lactosides based on tertiary amides of terephthalic, isophthalic, 2,6-naphthalic and oxalic acids as well as bivalent H(type 2) trisaccharides grafted on secondary/tertiary terephthalamides and two triazole-linker-containing cores. The presented data reveal a marked change in susceptibility to the test compounds when turning the tandem-repeat-type to a proto-type-like display. The testing of glycoclusters is suggested as a general strategy to help to delineate the significance of distinct structural features of lectins beyond their contact sites to the glycan.
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Neuronal Galectin-4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering.
Journal of neurochemistry, 2013Co-Authors: Silvia Velasco, Sabine André, Hans-joachim Gabius, Natalia Díez-revuelta, Teresa Hernández-iglesias, Herbert Kaltner, José Abad-rodríguezAbstract:Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that Galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule- and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.
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887 Intraluminal Release of Galectin 4 in EHEC Infection Provides a Novel Host Defense Mechanism
Gastroenterology, 2013Co-Authors: Sabine André, Hans-joachim Gabius, Olga KovbasnjukAbstract:G A A b st ra ct s no significant difference in pancreatic MPO (unit mg of protein) in wild type 2981+734; CB(-/-)3042+767;T7(-/-) 3070+292 (P.0.05) treated with L-arginine, but it was significantly higher compared to saline treated animals(149+12). We also did not find a significant difference in lung MPO in unit /mg of protein in (Wild type 7766+1852; CB(-/)6234+602 and T7(-/-)7185+1236 treated with L-arginine HCL(P .0.05) but significantly higher than saline treated(2555+285) animals. These findings correlate with our finding in cerulein model of acute pancreatitis. Conclusion: Regardless of the model of acute pancreatitis trypsinogen activation significantly decreases acinar cell necrosis, but local and systemic inflammation is independent of trypsin activation.
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Ganglioside GM1/Galectin-Dependent Growth Regulation in Human Neuroblastoma Cells: Special Properties of Bivalent Galectin-4 and Significance of Linker Length for Ligand Selection
Neurochemical research, 2012Co-Authors: Jürgen Kopitz, Sabine André, Seda Ballikaya, Hans-joachim GabiusAbstract:Orchestrated upregulation of cell surface presentation of ganglioside GM1 and homodimeric Galectin-1 is the molecular basis for growth regulation of human neuroblastoma (SK-N-MC) cells. Further study led to the discovery of competitive inhibition by Galectin-3, prompting us to test tandem-repeat-type Galectin-4 (two different lectin domains connected by a 42-amino-acid linker). This lectin bound to cells at comparably high affinity without involvement of the ganglioside, as disclosed by assays in the presence of cholera toxin B-subunit or Galectin-1 and blocking glucosylceramide synthesis. Notably, when tested separately, binding of both lectin domains showed partial sensitivity to the bacterial agglutinin. Despite its ability for cross-linking surface association of Galectin-4 did not affect proliferation, in contrast to homodimeric Galectins. The truncation of linker length from 42 to 16 amino acids altered binding properties to let partial sensitivity to the bacterial lectin emerge. Cross-competition between parental and engineered proteins did not exceed 40%. No effect on cell growth was detected. This study reveals complete functional divergence between Galectins differing in the spatial mode of lectin-site presentation and dependence of reactivity to distinct counter-receptor(s) on linker length. Due to the documented presence of Galectin-4 in the nervous system and its affinity for sulfatide these in vitro results indicate the potential for a distinct functionality profile of this lectin in vivo, giving further research direction.
Katsuko Yamashita - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation and externalization of Galectin-4 is controlled by Src family kinases
Glycobiology, 2013Co-Authors: Hiroko Ideo, Katsuko Yamashita, Ikue Hoshi, Masaru SakamotoAbstract:Galectin-4 is a cytosolic protein that lacks a signal sequence but is externalized and binds to 3-O-sulfated glycoconjugates extracellularly. The mechanism of subcellular localization and externalization of Galectin-4 has not yet been determined. A preliminary experiment using pervanadate (PV) showed that Galectin-4 is tyrosine-phosphorylated in cells and suggested that Src kinases are involved. Cell transfection with Galectin-4 and active Src plasmids showed that Galectin-4 can be tyrosine phosphorylated by members of the Src kinase family. The C-terminal peptide YVQI of Galectin-4 was found to play an important role in its tyrosine phosphorylation, and the SH2 domains of Src and SHP2 were found to bind to this peptide. Immunofluorescence analysis showed that Galectin-4 and phosphorylated proteins were intensely stained in the area of membrane protrusions of PV-treated or Src-activated cells. Furthermore, MUC1 derived from NUGC-4 cells was observed to bind to Galectin-4, and externalization of the bound molecules from the cell to the medium increased in the hyperphosphorylated condition. Study of the transfection of the mutant Galectin-4 which lacks the C-terminal peptide revealed that the phosphorylation status is important for externalization of Galectin-4. These results suggest that externalization of Galectin-4 can be regulated by signaling molecules and that it may function intracellularly as an adaptor protein serving to modulate the trafficking of glycoproteins.
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recognition mechanism of Galectin 4 for cholesterol 3 sulfate
Journal of Biological Chemistry, 2007Co-Authors: Hiroko Ideo, Akira Seko, Katsuko YamashitaAbstract:Abstract Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, and it co-localizes on the cell surface of human colonic adenocarcinoma cells with glycosphingolipids carrying SO–3→3Galβ1→3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K. (2005) J. Biol. Chem. 280, 4730–4737). In the present study, it was found that Galectin-4 also binds to cholesterol 3-sulfate, which has no β-galactoside moiety. This characteristic of Galectin-4 is unique within the Galectin family. The site-directed mutated Galectin-4-R45A had diminished binding ability toward cholesterol 3-sulfate, suggesting that Arg45 of Galectin-4 is indispensable for cholesterol 3-sulfate recognition. Gel filtration and chemical cross-linking experiments revealed that some Galectin-4 exists as dimers, and this multivalency seemed to enhance its avidity for cholesterol 3-sulfate binding. Cholesterol 3-sulfate and sulfatide co-existed with Galectin-4 in detergent-insoluble fractions of porcine esophagus and intestine, respectively. These results suggested that not only sulfated glycosphingolipids but also cholesterol 3-sulfate are endogenous ligands for Galectin-4 in vivo.
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Galectin-4 Binds to Sulfated Glycosphingolipids and Carcinoembryonic Antigen in Patches on the Cell Surface of Human Colon Adenocarcinoma Cells
The Journal of biological chemistry, 2004Co-Authors: Hiroko Ideo, Akira Seko, Katsuko YamashitaAbstract:Abstract Galectin-4, a member of the Galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly repeated carbohydrate recognition domains and specifically binds to an pyranoside with high affinity (Ideo, H., Seko, A., Ohkura, T., Matta, K. L., and Yamashita, K. (2002) Glycobiology 12, 199–208). In this study, we found that Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that Galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized Galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to Galectin-4 with KD value of 2 × 10-8 m by surface plasmon resonance and coimmunoprecipitated with Galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biologically important ligands for Galectin-4.
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High-affinity binding of recombinant human Galectin-4 to SO3–→3Galβ1→3GalNAc pyranoside
Glycobiology, 2002Co-Authors: Hiroko Ideo, Akira Seko, Takashi Ohkura, Khushi L. Matta, Katsuko YamashitaAbstract:Galectin-4 is a member of Galectin family and has two carbohydrate recognition domains. Although Galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of Galectin-4 comparatively with that of Galectin-3, using surface plasmon resonance, Galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for Galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, Galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to Galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to Galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that Galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.
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high affinity binding of recombinant human Galectin 4 to so3 3galβ1 3galnac pyranoside
Glycobiology, 2002Co-Authors: Hiroko Ideo, Akira Seko, Takashi Ohkura, Khushi L. Matta, Katsuko YamashitaAbstract:Galectin-4 is a member of Galectin family and has two carbohydrate recognition domains. Although Galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of Galectin-4 comparatively with that of Galectin-3, using surface plasmon resonance, Galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for Galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, Galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to Galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to Galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that Galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.
Herbert Kaltner - One of the best experts on this subject based on the ideXlab platform.
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Neuronal Galectin-4 is required for axon growth and for the organization of axonal membrane L1 delivery and clustering.
Journal of neurochemistry, 2013Co-Authors: Silvia Velasco, Sabine André, Hans-joachim Gabius, Natalia Díez-revuelta, Teresa Hernández-iglesias, Herbert Kaltner, José Abad-rodríguezAbstract:Axon membrane glycoproteins are essential for neuronal differentiation, although the mechanisms underlying their polarized sorting and organization are poorly understood. We describe here that Galectin-4 (Gal-4), a lectin highly expressed in gastrointestinal tissues and involved in epithelial glycoprotein transport, is expressed by hippocampal and cortical neurons where it is sorted to discrete segments of the axonal membrane in a microtubule- and sulfatide-dependent manner. Gal-4 knockdown retards axon growth, an effect that can be rescued by recombinant Gal-4 addition. This Gal-4 reduction, as inhibition of sulfatide synthesis does, lowers the presence and clustered organization of axon growth-promoting molecule NCAM L1 at the axon membrane. Furthermore, we find that Gal-4 interacts with L1 by specifically binding to LacNAc branch ends of L1 N-glycans. Impairing the maturation of these N-glycans precludes Gal-4/L1 association resulting in a failure of L1 membrane cluster organization. In all, Gal-4 sorts to axon plasma membrane segments by binding to sulfatide-containing microtubule-associated carriers and being bivalent, it organizes the transport of L1, and likely other axonal glycoproteins, by attaching them to the carriers through their LacNAc termini. This mechanism would underlie L1 functional organization on the plasma membrane, required for proper axon growth.
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Gene-expression signature of adhesion/growth-regulatory tissue lectins (Galectins) in transitional cell cancer and its prognostic relevance.
Histopathology, 2007Co-Authors: Langbein S, Sabine André, Herbert Kaltner, Brade J, Jasmin Katrin Badawi, M. Hatzinger, Martin Lensch, Specht K, Ulrich Brinck, Peter AlkenAbstract:Aims: Lectins, and especially Galectins, appear to be important in malignancy-associated processes. The aim was to analyse comprehensively the presence of Galectins in urothelial tumours. Methods and results: Non-cross-reactive antibodies against seven family members from the three subgroups (prototype: Galectin-1, -2 and -7; chimera type: Galectin-3; tandem-repeat type: Galectin-4, -8 and -9) were used. Gene expression was monitored in specimens of normal urothelium, fresh tumour tissue and cell lines by real-time polymerase chain reaction (PCR). The presence and evidence of tumour-associated up-regulation were shown for Galectin-1 and -3. This was less clear-cut for Galectin-4 and -8. Galectin-7 was expressed in all cell lines; Galectin-2 and -9 were detected at comparatively low levels. Galectin-2, -3 and -8 up-regulation was observed in superficial tumours, but not in muscle-invasive tumours (P
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Effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the Galβ1-terminated core saccharides on the binding of domain-I of recombinant tandem-repeat-type Galectin-4 from rat gastrointestinal tract (G4-
Biochimie, 2004Co-Authors: Jia-hau Liu, Sabine André, Herbert Kaltner, Tanuja Singh, Hans-joachim GabiusAbstract:In our recent publication, we defined core aspects of the carbohydrate specificity of domain-I of recombinant tandem-repeat-type Galectin-4 from rat gastrointestinal tract (G4-N), especially its potent interaction with the linear tetrasaccharide Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (Ibeta1-3L). The assumed role of Galectin-4 as a microvillar raft stabilizer/organizer and as a malignancy-associated factor in hepatocellular and gastrointestinal carcinomas called for further refinement of its binding specificity. Thus, the effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the terminal Galbeta1-core saccharides were thoroughly examined by the enzyme-linked lectinosorbent and lectin-glycan inhibition assays. The results indicate that (a) a high-density of polyvalent Galbeta1-3/4GlcNAc (I/II), Galbeta1-3GalNAc (T) and/or GalNAcalpha1-Ser/Thr (Tn) strongly favors G4-N/glycoform binding. These glycans were up to 2.3 x 10(6), 1.4 x 10(6), 8.8 x 10(5), and 1.4 x 10(5) more active than Gal, GalNAc, monomeric I/II and T, respectively; (b) while lFuc is a poor inhibitor, its presence as alpha1-2 linked to terminal Galbeta1-containing oligosaccharides, such as H active Ibeta1-3L, markedly enhances the reactivities of these ligands; (c) when blood group A (GalNAcalpha1-) or B (Galalpha1-) determinants are attached to terminal Galbeta1-3/4GlcNAc (or Glc) oligosaccharides, the reactivities are also increased; (d) with lFucalpha1-3/4 linked to sub-terminal GlcNAc, the reactivities of these haptens are reduced; and (e) short chain Le(a)/Le(x)/Le(y) and the short chains of sialyl Le(a)/Le(x) are poor inhibitors. These distinct binding features of G4-N establish the important concept of affinity enhancement by high density polyvalencies of glycotopes (vs. multi-antennary I/II) and by introduction of an ABH key sugar to Galbeta1-terminated core glycotopes. The polyvalent ligand binding properties of G4-N may help our understanding of its crucial role for cell membrane raft stability and provide salient information for the optimal design of blocking substances such as anti-tumoral glycodendrimers.
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effects of polyvalency of glycotopes and natural modifications of human blood group abh lewis sugars at the galβ1 terminated core saccharides on the binding of domain i of recombinant tandem repeat type Galectin 4 from rat gastrointestinal tract g4 n
Biochimie, 2004Co-Authors: Jia-hau Liu, Sabine André, Herbert Kaltner, Tanuja Singh, Hans-joachim GabiusAbstract:In our recent publication, we defined core aspects of the carbohydrate specificity of domain-I of recombinant tandem-repeat-type Galectin-4 from rat gastrointestinal tract (G4-N), especially its potent interaction with the linear tetrasaccharide Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (Ibeta1-3L). The assumed role of Galectin-4 as a microvillar raft stabilizer/organizer and as a malignancy-associated factor in hepatocellular and gastrointestinal carcinomas called for further refinement of its binding specificity. Thus, the effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the terminal Galbeta1-core saccharides were thoroughly examined by the enzyme-linked lectinosorbent and lectin-glycan inhibition assays. The results indicate that (a) a high-density of polyvalent Galbeta1-3/4GlcNAc (I/II), Galbeta1-3GalNAc (T) and/or GalNAcalpha1-Ser/Thr (Tn) strongly favors G4-N/glycoform binding. These glycans were up to 2.3 x 10(6), 1.4 x 10(6), 8.8 x 10(5), and 1.4 x 10(5) more active than Gal, GalNAc, monomeric I/II and T, respectively; (b) while lFuc is a poor inhibitor, its presence as alpha1-2 linked to terminal Galbeta1-containing oligosaccharides, such as H active Ibeta1-3L, markedly enhances the reactivities of these ligands; (c) when blood group A (GalNAcalpha1-) or B (Galalpha1-) determinants are attached to terminal Galbeta1-3/4GlcNAc (or Glc) oligosaccharides, the reactivities are also increased; (d) with lFucalpha1-3/4 linked to sub-terminal GlcNAc, the reactivities of these haptens are reduced; and (e) short chain Le(a)/Le(x)/Le(y) and the short chains of sialyl Le(a)/Le(x) are poor inhibitors. These distinct binding features of G4-N establish the important concept of affinity enhancement by high density polyvalencies of glycotopes (vs. multi-antennary I/II) and by introduction of an ABH key sugar to Galbeta1-terminated core glycotopes. The polyvalent ligand binding properties of G4-N may help our understanding of its crucial role for cell membrane raft stability and provide salient information for the optimal design of blocking substances such as anti-tumoral glycodendrimers.
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Effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the Galbeta1-terminated core saccharides on the binding of domain-I of recombinant tandem-repeat-type Galectin-4 from rat gastrointestinal tract (
Biochimie, 2004Co-Authors: Jia-hau Liu, Sabine André, Herbert Kaltner, Tanuja Singh, Hans-joachim GabiusAbstract:In our recent publication, we defined core aspects of the carbohydrate specificity of domain-I of recombinant tandem-repeat-type Galectin-4 from rat gastrointestinal tract (G4-N), especially its potent interaction with the linear tetrasaccharide Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (Ibeta1-3L). The assumed role of Galectin-4 as a microvillar raft stabilizer/organizer and as a malignancy-associated factor in hepatocellular and gastrointestinal carcinomas called for further refinement of its binding specificity. Thus, the effects of polyvalency of glycotopes and natural modifications of human blood group ABH/Lewis sugars at the terminal Galbeta1-core saccharides were thoroughly examined by the enzyme-linked lectinosorbent and lectin-glycan inhibition assays. The results indicate that (a) a high-density of polyvalent Galbeta1-3/4GlcNAc (I/II), Galbeta1-3GalNAc (T) and/or GalNAcalpha1-Ser/Thr (Tn) strongly favors G4-N/glycoform binding. These glycans were up to 2.3 x 10(6), 1.4 x 10(6), 8.8 x 10(5), and 1.4 x 10(5) more active than Gal, GalNAc, monomeric I/II and T, respectively; (b) while lFuc is a poor inhibitor, its presence as alpha1-2 linked to terminal Galbeta1-containing oligosaccharides, such as H active Ibeta1-3L, markedly enhances the reactivities of these ligands; (c) when blood group A (GalNAcalpha1-) or B (Galalpha1-) determinants are attached to terminal Galbeta1-3/4GlcNAc (or Glc) oligosaccharides, the reactivities are also increased; (d) with lFucalpha1-3/4 linked to sub-terminal GlcNAc, the reactivities of these haptens are reduced; and (e) short chain Le(a)/Le(x)/Le(y) and the short chains of sialyl Le(a)/Le(x) are poor inhibitors. These distinct binding features of G4-N establish the important concept of affinity enhancement by high density polyvalencies of glycotopes (vs. multi-antennary I/II) and by introduction of an ABH key sugar to Galbeta1-terminated core glycotopes. The polyvalent ligand binding properties of G4-N may help our understanding of its crucial role for cell membrane raft stability and provide salient information for the optimal design of blocking substances such as anti-tumoral glycodendrimers.
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Phosphorylation and externalization of Galectin-4 is controlled by Src family kinases
Glycobiology, 2013Co-Authors: Hiroko Ideo, Katsuko Yamashita, Ikue Hoshi, Masaru SakamotoAbstract:Galectin-4 is a cytosolic protein that lacks a signal sequence but is externalized and binds to 3-O-sulfated glycoconjugates extracellularly. The mechanism of subcellular localization and externalization of Galectin-4 has not yet been determined. A preliminary experiment using pervanadate (PV) showed that Galectin-4 is tyrosine-phosphorylated in cells and suggested that Src kinases are involved. Cell transfection with Galectin-4 and active Src plasmids showed that Galectin-4 can be tyrosine phosphorylated by members of the Src kinase family. The C-terminal peptide YVQI of Galectin-4 was found to play an important role in its tyrosine phosphorylation, and the SH2 domains of Src and SHP2 were found to bind to this peptide. Immunofluorescence analysis showed that Galectin-4 and phosphorylated proteins were intensely stained in the area of membrane protrusions of PV-treated or Src-activated cells. Furthermore, MUC1 derived from NUGC-4 cells was observed to bind to Galectin-4, and externalization of the bound molecules from the cell to the medium increased in the hyperphosphorylated condition. Study of the transfection of the mutant Galectin-4 which lacks the C-terminal peptide revealed that the phosphorylation status is important for externalization of Galectin-4. These results suggest that externalization of Galectin-4 can be regulated by signaling molecules and that it may function intracellularly as an adaptor protein serving to modulate the trafficking of glycoproteins.
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recognition mechanism of Galectin 4 for cholesterol 3 sulfate
Journal of Biological Chemistry, 2007Co-Authors: Hiroko Ideo, Akira Seko, Katsuko YamashitaAbstract:Abstract Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, and it co-localizes on the cell surface of human colonic adenocarcinoma cells with glycosphingolipids carrying SO–3→3Galβ1→3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K. (2005) J. Biol. Chem. 280, 4730–4737). In the present study, it was found that Galectin-4 also binds to cholesterol 3-sulfate, which has no β-galactoside moiety. This characteristic of Galectin-4 is unique within the Galectin family. The site-directed mutated Galectin-4-R45A had diminished binding ability toward cholesterol 3-sulfate, suggesting that Arg45 of Galectin-4 is indispensable for cholesterol 3-sulfate recognition. Gel filtration and chemical cross-linking experiments revealed that some Galectin-4 exists as dimers, and this multivalency seemed to enhance its avidity for cholesterol 3-sulfate binding. Cholesterol 3-sulfate and sulfatide co-existed with Galectin-4 in detergent-insoluble fractions of porcine esophagus and intestine, respectively. These results suggested that not only sulfated glycosphingolipids but also cholesterol 3-sulfate are endogenous ligands for Galectin-4 in vivo.
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Galectin-4 Binds to Sulfated Glycosphingolipids and Carcinoembryonic Antigen in Patches on the Cell Surface of Human Colon Adenocarcinoma Cells
The Journal of biological chemistry, 2004Co-Authors: Hiroko Ideo, Akira Seko, Katsuko YamashitaAbstract:Abstract Galectin-4, a member of the Galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly repeated carbohydrate recognition domains and specifically binds to an pyranoside with high affinity (Ideo, H., Seko, A., Ohkura, T., Matta, K. L., and Yamashita, K. (2002) Glycobiology 12, 199–208). In this study, we found that Galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that Galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized Galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to Galectin-4 with KD value of 2 × 10-8 m by surface plasmon resonance and coimmunoprecipitated with Galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biologically important ligands for Galectin-4.
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High-affinity binding of recombinant human Galectin-4 to SO3–→3Galβ1→3GalNAc pyranoside
Glycobiology, 2002Co-Authors: Hiroko Ideo, Akira Seko, Takashi Ohkura, Khushi L. Matta, Katsuko YamashitaAbstract:Galectin-4 is a member of Galectin family and has two carbohydrate recognition domains. Although Galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of Galectin-4 comparatively with that of Galectin-3, using surface plasmon resonance, Galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for Galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, Galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to Galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to Galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that Galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.
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high affinity binding of recombinant human Galectin 4 to so3 3galβ1 3galnac pyranoside
Glycobiology, 2002Co-Authors: Hiroko Ideo, Akira Seko, Takashi Ohkura, Khushi L. Matta, Katsuko YamashitaAbstract:Galectin-4 is a member of Galectin family and has two carbohydrate recognition domains. Although Galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of Galectin-4 comparatively with that of Galectin-3, using surface plasmon resonance, Galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for Galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, Galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to Galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to Galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that Galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.
