Tokyo, Japan
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Ideo H.,Noguchi Institute | Ideo H.,Sasaki Institute | Hoshi I.,Sasaki Institute | Hoshi I.,Yokohama City University | And 4 more authors.
Glycobiology | Year: 2013

Galectin-4 is a cytosolic protein that lacks a signal sequence but is externalized and binds to 3-O-sulfated glycoconju-gates 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 trans-fection 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. Immunofluores-cence 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 hyper-phosphorylated 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 ex-ternalization 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. © The Author 2013.


Seko A.,Tokyo Institute of Technology | Seko A.,Japan Science and Technology Agency | Ohkura T.,Sasaki Institute | Ideo H.,Tokyo Institute of Technology | Yamashita K.,Tokyo Institute of Technology
Glycobiology | Year: 2012

Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3′-sialylated, 6′-sulfated LNnT [Neu5Acα2-3(SO 3 --6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3′-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galβ1-3(SO3 --6)GalNAc] and disulfated core 1 SO3 --3Galβ1- 3(SO 3 --6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3′-sulfated core 1 derivatives [SO 3 --3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3′-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3′-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb. © 2011 The Author.

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