Expression of mucin 1 possessing a 3'-sulfated core1 in recurrent and metastatic breast cancer.

Breast cancer is the most frequent cancer threatening the lives of women between the ages of 30 and 64. The cancer antigen 15-3 assay (CA15-3) has been widely used for the detection of breast cancer recurrence; however, its sensitivity and specificity are inadequate. We previously found that the breast cancer cell line YMBS secretes mucin 1 possessing 3'-sulfated core1 (3Score1-MUC1) into the medium.

High-sensitivity immunoassay with surface plasmon field-enhanced fluorescence spectroscopy using a plastic sensor chip: application to quantitative analysis of total prostate-specific antigen and GalNAcβ1-4GlcNAc-linked prostate-specific antigen for prostate cancer diagnosis.

A high-sensitivity immunoassay system with surface plasmon field-enhanced fluorescence spectrometry (SPFS) was constructed using a plastic sensor chip and then applied to the detection of total prostate-specific antigen (total PSA) and GalNAcβ1-4GlcNAc-linked prostate-specific antigen (LacdiNAc-PSA) in serum, to discriminate between prostate cancer (PC) and benign prostate hyperplasia (BPH). By using this automated SPFS immunoassay, the detection limit for total PSA in serum was as low as 0.04 pg/mL, and the dynamic range was estimated to be at least five digits.

Determination of glycan motifs using serial lectin affinity chromatography.

Serial lectin affinity chromatography is a convenient technique for characterizing glycan motifs (terminal glycan structures) of glycoproteins or released glycans. When these glycoconjugates are applied serially or in parallel to lectin-immobilized columns, information regarding the glycan motifs can be obtained. We demonstrate lectin affinity chromatographic methods for determining O-linked glycan structures of MUC1 purified from a breast cancer cell line, YMB-S, N-linked glycan structures of serum prostate-specific antigen from prostate cancer, and serum alkaline phosphatases from choriocarcinoma.

Phosphorylation and externalization of galectin-4 is controlled by Src family kinases.

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.

Elucidation of a novel lacto-N-decaose core structure in human milk using nonlinear analytical technique combinations.

Detailed structural analysis of high molecular weight human milk oligosaccharides (HMOs) is still a challenging task. Here we present a modular strategy for a flexible de novo structural characterization of this class of molecules. The protocol combines established techniques such as separation by two-dimensional high-performance liquid chromatography with different types of mass spectrometry, exoglycosidase digestion, and linkage analysis in an individual glycan-based manner.

Novel O-linked glycans containing 6'-sulfo-Gal/GalNAc of MUC1 secreted from human breast cancer YMB-S cells: possible carbohydrate epitopes of KL-6(MUC1) monoclonal antibody.

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.

Galectin-8-N-domain recognition mechanism for sialylated and sulfated glycans.

Galectin-8 has much higher affinity for 3'-O-sulfated or 3'-O-sialylated glycoconjugates and a Lewis X-containing glycan than for oligosaccharides terminating in Galβ1→3/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I.

alpha1,2-Fucosylated and beta-N-acetylgalactosaminylated prostate-specific antigen as an efficient marker of prostatic cancer.

A prostate-specific antigen (PSA) is widely used as a diagnostic marker for prostate cancer (PC) because of its high specificity. However, elevated serum PSA does not occur only in PC but also in benign prostatic hyperplasia (BPH). Since the structural changes of N-glycans during carcinogenesis are common phenomena, we investigated whether PC-specific N-glycans are linked to PSA.

Neuroblastoma GOTO cells are hypersensitive to disruption of lipid rafts.

GOTO cells, a neuroblastoma cell line retaining the ability to differentiate into neuronal or Schwann cells, were found to be rich in membrane rafts containing ganglioside GM2 and hypersensitive to lipid raft-disrupting methyl-beta-cyclodextrin (MbetaCD); the GM2-rich rafts and sensitivity to MbetaCD were markedly diminished upon their differentiation into Schwann cells. We first raised a monoclonal antibody that specifically binds to GOTO cells but not to differentiated Schwann cells and determined its target antigen as ganglioside GM2, which was shown to be highly concentrated in lipid rafts by its colocalization with flotillin, a marker protein of rafts. Disturbance of normal structure of the lipid raft by depleting its major constituent, cholesterol, with MbetaCD resulted in acute apoptotic cell death of GOTO cells, but little effects were seen on differentiated Schwann cells.

A Caenorhabditis elegans glycolipid-binding galectin functions in host defense against bacterial infection.

Galectins are a family of beta-galactoside-binding proteins that are widely found among animal species and that regulate diverse biological phenomena. To study the biological function of glycolipid-binding galectins, we purified recombinant Caenorhabditis elegans galectins (LEC-1-11) and studied their binding to C. elegans glycolipids.

Polyoxometalates as effective inhibitors for sialyl- and sulfotransferases.

Sialylated and/or sulfated carbohydrate chains in glycoproteins and glycolipids play important roles in infection by microorganisms and diseases including cancer. Inhibitors of sialyl/sulfotransferases, responsible for the biosynthesis of these carbohydrate chains, could be medical agents against such infections and diseases. Polyoxometalates (PMs) are inorganic polyanionic molecules that have been shown to exhibit activity against tumors and infectious microorganisms; however, the effects of PMs on carbohydrate biosynthesis have never been investigated.

Beta1,3-galactosyltransferases-4/5 are novel tumor markers for gynecological cancers.

Background/aims: While the CA 125 and SCC antigens are used as tumor markers for ovarian cancer and uterine cervical cancer, respectively, an effective marker for uterine corpus cancer has not been identified. We asked whether beta1,3-galactosyltransferase-4 and/or 5 (beta3Gal-T4/T5) could serve as novel tumor markers for detecting gynecological carcinomas, especially those of the uterine corpus.

Methods: We obtained a monoclonal antibody and a polyclonal antiserum against beta3Gal-T5 and constructed a sandwich ELISA method.

N-Acetylglucosamine 6-O-sulfotransferase-2 as a tumor marker for uterine cervical and corpus cancer.

N-Acetylglucosamine 6-O-sulfotransferase-2 (GlcNAc6ST2) is ectopically expressed in ovarian mucinous and clear cell adenocarcinoma [Kanoh et al., Glycoconj J 23:453-460, 2006]. Here we studied whether GlcNAc6ST2 protein can be detected in sera from patients with gynecological cancers and could serve as a tumor marker.

Activation of beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) by beta3Gn-T8. Possible involvement of beta3Gn-T8 in increasing poly-N-acetyllactosamine chains in differentiated HL-60 cells.

Enzymatic activities of some glycosyltransferases are markedly increased via complex formation with other transferases or cofactor proteins. We previously showed that beta1,3-N-acetylglucosaminyltransferase-2 (beta3Gn-T2) and beta3Gn-T8 can form a heterodimer in vitro and that the complex exhibits much higher enzymatic activity than either enzyme alone (Seko, A., and Yamashita, K.

Structural basis for recognition of high mannose type glycoproteins by mammalian transport lectin VIP36.

VIP36 functions as a transport lectin for trafficking certain high mannose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca(2+) and mannosyl ligands are also described.

Recognition mechanism of galectin-4 for cholesterol 3-sulfate.

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)-->3Galbeta1-->3(GalNAc) residues (Ideo, H., Seko, A., and Yamashita, K.

Ectopic expression of N-acetylglucosamine 6-O-sulfotransferase 2 in chemotherapy-resistant ovarian adenocarcinomas.

Mucinous and clear cell adenocarcinomas are the major histological types of ovarian epithelial cancer and are associated with a poor prognosis due to their resistance to chemotherapy. A novel tumor marker specific for ovarian mucinous and clear cell adenocarcinomas would be helpful for overcoming these serious diseases. We showed previously by enzymological characterization and RT-PCR that colonic mucinous adenocarcinoma tissues ectopically express GlcNAc6ST-2, a member of the carbohydrate 6-O-sulfotransferase family (Seko, A.

Essential roles of 3'-phosphoadenosine 5'-phosphosulfate synthase in embryonic and larval development of the nematode Caenorhabditis elegans.

Sulfation of biomolecules, which is widely observed from bacteria to humans, plays critical roles in many biological processes. All sulfation reactions in all organisms require activated sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS), as a universal donor. In animals, PAPS is synthesized from ATP and inorganic sulfate by the bifunctional enzyme, PAPS synthase.

Structures of the carbohydrate recognition domain of Ca2+-independent cargo receptors Emp46p and Emp47p.

Emp46p and Emp47p are type I membrane proteins, which cycle between the endoplasmic reticulum (ER) and the Golgi apparatus by vesicles coated with coat protein complexes I and II (COPI and COPII). They are considered to function as cargo receptors for exporting N-linked glycoproteins from the ER. We have determined crystal structures of the carbohydrate recognition domains (CRDs) of Emp46p and Emp47p of Saccharomyces cerevisiae, in the absence and presence of metal ions.

Effects of antisecretory agents on angiogenesis during healing of gastric ulcers.

Background: We studied the effects of a proton pump inhibitor (PPI) and an H2-receptor antagonist (H2-blocker) on angiogenesis during gastric ulcer healing, by examining stromal cell-derived factor (SDF-1) and CXC chemokine receptor 4 (CXCR4) expression in the gastric mucosa.

Methods: Patients with gastric ulcers were allocated to an untreated control group, consisting of patients with active ulcers (GA), healing ulcers (GH), and ulcer scars (GS) or a PPI group (P; given rabeprazole at 20 mg/day), or an H2-blocker group (H; given nizatidine at 800 mg/day). Frozen sections of biopsy specimens were examined by reverse transcription-polymerase chain reaction (RT-PCR) to analyze SDF-1 and CXCR4 mRNA.

Porcine, mouse and human galactose 3-O-sulphotransferase-2 enzymes have different substrate specificities; the porcine enzyme requires basic compounds for its catalytic activity.

Sulphation of galactose at the C-3 position is one of the major post-translational modifications of colorectal mucin. Thus we partially purified a Gal 3-O-sulphotransferase from porcine colonic mucosa (pGal3ST) and studied its enzymatic characteristics. The enzyme was purified 48500-fold by sequential chromatographies on hydroxyapatite, Con A (concanavalin A)-Sepharose, porcine colonic mucin-Sepharose, Cu2+-chelating Sepharose and AMP-agarose.

Characterization of a novel galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T8): the complex formation of beta3Gn-T2 and beta3Gn-T8 enhances enzymatic activity.

We characterized a novel member of the beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) gene family, beta3Gn-T8. A recombinant soluble form of beta3Gn-T8 was expressed in Pichia pastoris (P. pastoris), and its substrate specificity was compared with that of beta3Gn-T2.