Akira Seko

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. Here, we found that some types of PMs can inhibit the enzymatic activities of specific sialyl/sulfotransferases. Several tungstate-type PMs inhibited Gal: alpha2,3-sialyltransferase-I (ST3Gal-I) activity at sub-nanomolar levels. The half-inhibitory concentration of the best inhibitors was 0.2 nM and the inhibition was non-competitive for both donor and acceptor substrates (Ki values approximately 0.5 nM). By certain vanadate-type PMs, ST3Gal-I and Gal 3-O-sulfotransferase-2 (Gal3ST-2) were specifically inhibited at nanomolar levels. The inhibitory effect of a tungstate-type PM on ST3Gal-I was reversible and electrostatic. A ST3Gal-I mutant protein which was converted (335)Arg residue in the C-terminal region to Glu, was rather insensitive to the PM, suggesting that specific C-terminal basic amino acid of ST3Gal-I is involved in the binding to PMs. Collectively, PMs are novel inhibitors of specific sialyl/sulfotransferases.

β1,3‐N‐Acetylglucosaminyltransferase‐7 (β3Gn‐T7) acts efficiently on keratan sulfate‐related glycans

β1,3‐N‐Acetylglucosaminyltransferase‐7 (β3Gn‐T7) has been identified as an anti‐migration factor for a lung cancer cell line but its enzymatic activity has not yet been characterized. Here we show that β3Gn‐T7 efficiently acts on keratan sulfate‐related glycans including Galβ1→4(SO3 −→6)GlcNAcβ1→3Galβ1→4(SO3 −→6)GlcNAc (L2L2), while lacto‐N‐tetraose and lacto‐N‐neo‐tetraose were poor substrates. Moreover, we found that among the other five β3Gn‐Ts and i antigen‐producing β3Gn‐T (iGn‐T), β3Gn‐T2 and iGn‐T act well on L2L2, although these specific activities were lower than those for a tetraantennary N‐glycan. These results indicate that β3Gn‐T7 is the one that most efficiently elongates L2L2 and may be involved in the biosynthesis of keratan sulfate.

Down-regulation of Gal 3–O-sulfotransferase-2 (Gal3ST-2) Expression in Human Colonic Non-mucinous Adenocarcinoma

Expression levels of sulfomucin in human colonic adenocarcinomas are lower than those in normal colonic mucosa; this should be in part caused by down‐regulation of expression of sulfotransferases, but it remains unclear which Gal 3‐O‐sulfotransferase (Gal3ST) is responsible for the bio‐ synthesis of sulfomucin. In this study, we first examined the substrate specificities of four Gal3STs cloned so far, and found that Galβ1→3GlcNAcβl→3Galβ1→4Glc (LNT) can be utilized only by Gal3ST‐2 as an acceptor substrate. The substrate specificity of Gal3ST‐2 is closely similar to those of Gal3ST activities present in human normal mucosa and adenocarcinomas, suggesting that Gal3ST‐2 is the dominant Gal3ST in colon and colonic cancer. Secondly, using LNT as a substrate, we comparatively analyzed levels of Gal3ST‐2 activities in non‐mucinous adenocarcinoma, mucinous adenocarcinomas, and the adjacent normal mucosa. We found that levels of Gal3ST‐2 activities in non‐mucinous adenocarcinoma are significantly lower than those in the adjacent normal mucosa, while those in mucinous adenocarcinomas are not significantly different from those in the adjacent normal mucosa. Moreover, we showed by a competitive RT‐PCR method that expression levels of transcript for Gal3ST‐2 in non‐mucinous adenocarcinoma are lower than those in normal mucosa. These results suggest that Gal3ST‐2 is one of the enzymes responsible for biosynthesis of sulfomucin, and that expression levels of Gal3ST‐2 are down‐regulated in non‐mucinous adenocarcinoma.

Essential Roles of 3′-Phosphoadenosine 5′-Phoshosulfate 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. In mammals, genetic defects in PAPS synthase 2, one of two PAPS synthase isozymes, cause dwarfism disorder, but little is known about the consequences of the complete loss of PAPS synthesis. To define the developmental role of sulfation, we cloned a Caenorhabditis elegans PAPS synthase-homologous gene, pps-1, and depleted expression of its product by isolating the deletion mutant and by RNA-mediated interference. PPS-1 protein exhibits specific activity to form PAPS in vitro, and disruption of the pps-1 gene by RNAi causes pleiotropic developmental defects in muscle patterning and epithelial cell shape changes with a decrease in glycosaminoglycan sulfation. Additionally, the pps-1 null mutant exhibits larval lethality. These data suggest that sulfation is essential for normal growth and integrity of epidermis in C. elegans. Furthermore, reporter analysis showed that pps-1 is expressed in the epidermis and several gland cells but not in neurons and muscles, indicating that PAPS in the neurons and muscles is provided by other cells.

Identification and characterization of N-acetylglucosamine-6-O-sulfate-specific β1,4-galactosyltransferase in human colorectal mucosa

6‐Sulfo‐sialyl Lewis X structure is attributable to recognition between lymphocytes and high endothelial venules. However, the biosynthetic pathway still remains unclear. We found that a β‐galactosyltransferase (βGalT) in human colorectal mucosa preferentially acts on GlcNAc‐6‐O‐sulfate (6S‐GN). 6S‐GN:β4GalT was partially purified by UDP‐hexanolamine‐Sepharose and asialo‐agalacto‐ovomucin‐Sepharose chromatographies. The optimum pH of this enzyme was found to be 6.5–7.5 and the Michaelis constants for 6S‐GN and UDP‐Gal were 0.43 mM and 16 μM, respectively. The enzymatic activity was dependent on divalent cations and the substrate specificity was not affected by α‐lactalbumin. This is the first demonstration of the occurrence of 6S‐GN:β4GalT.

β1,3-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 β1,3-galactosyltransferase-4 and/or 5 (β3Gal-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 β3Gal-T5 and constructed a sandwich ELISA method. Western blot analysis and immunoprecipitation revealed that this ELISA recognizes both β3Gal-T4 and β3Gal-T5. Results: We found β3Gal-T4 and T5 enzymatic activity in ovarian cancer tissues, indicating that these enzymes are expressed at least in ovarian cancer. The cutoff value was determined by ROC analysis to be 5.4 ng/ml in the sera. The β3Gal-T4/T5-positive rates for the sera from ovarian cancer and uterine cervical cancer patients were comparable with the CA 125- and SCC antigen-positive rates for these cancers, respectively. Significantly, the β3Gal-T4/T5-positive rate was higher for uterine corpus cancer (64%) than the CA 125 (37%)- and CA 19-9 (24%)-positive rates. The stage I uterine corpus cancers had particularly high β3Gal-T4/T5-positive rates (57%). Conclusion: β3Gal-T4/T5 is a novel tumor marker for uterine corpus cancer and other gynecological cancers.

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. et al. (2002) Glycobiology12, 379–388). Here, we prepared a GlcNAc6ST-2-specific polyclonal antibody for immunohistochemical analysis and found that GlcNAc6ST-2 is ectopically expressed by not only colonic mucinous adenocarcinomas but also ovarian mucinous, clear cell and papillary serous adenocarcinomas. In contrast, solid serous adenocarcinomas, endometrioid adenocarcinomas, and mucinous adenomas expressed GlcNAc6ST-2 much less frequently or not at all. RT-PCR analysis confirmed that GlcNAc6ST-2 transcripts are expressed in ovarian mucinous adenocarcinomas but not in mucinous adenomas. In addition, immunohistochemical analysis using sulfated glycan-specific monoclonal antibodies showed that ovarian adenocarcinoma cells express GlcNAc 6-O-sulfated glycans, including an L-selectin ligand and its related glycans. These results indicate that GlcNAc6ST-2 would be a novel tumor antigen that is specifically expressed in ovarian mucinous, clear cell and papillary serous adenocarcinomas.

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. Interestingly, the purified pGal3ST required submillimolar concentrations of spermine or basic lipids, such as D-sphingosine and N,N-dimethylsphingosine, for enzymatic activity. pGal3ST recognized Galbeta1-->3GalNAc (core 1) as an optimal substrate, and had weaker activity for Galbeta1-->3GlcNAc (type 1) and Galbeta1-->4GlcNAc (type 2). Substrate competition experiments proved that a single enzyme catalyses sulphation of all three oligosaccharides. Among the four human Gal3STs cloned to date, the substrate specificity of pGal3ST is most similar to that of human Gal3ST-2, which is also strongly expressed in colonic mucosa, although the kinetics of pGal3ST and human Gal3ST-2 were rather different. To determine whether pGal3ST is the orthologue of human Gal3ST-2, a cDNA encoding porcine Gal3ST-2 was isolated and the enzyme was expressed in COS-7 cells for analysis of substrate specificity. This revealed that porcine Gal3ST-2 has the same specificity as pGal3ST, indicating that pGal3ST is indeed the porcine equivalent of Gal3ST-2. The substrate specificity of mouse Gal3ST-2 was also different from those of human and porcine Gal3ST-2 enzymes. Mouse Gal3ST-2 preferred core 1 and type 2 glycans to type 1, and the K(m) values were much higher than those of human Gal3ST-2. These results suggest that porcine Gal3ST-2 requires basic compounds for catalytic activity and that human, mouse and porcine Gal3ST-2 orthologues have diverse substrate specificities.

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. First, we created a monoclonal antibody and polyclonal antiserum against GlcNAc6ST2. These antibodies were specific for GlcNAc6ST2, as shown by Western blot analysis and immunoprecipitation. Using these antibodies, we constructed a sandwich ELISA method for detecting GlcNAc6ST2 in the serum. GlcNAc6ST2 provided lower positive rates for ovarian cancer than CA125, but higher positive rates for uterine cervical and corpus cancer than SCC antigens and CA125, respectively. A significantly higher percentage of stage I uterine cervical and corpus cancers were positive for GlcNAc6ST2 than for SCC antigens and CA125, respectively. GlcNAc6ST2 could therefore be a good serological marker for detecting early-stage uterine cervical and corpus cancers.

Carbohydrate recognition of vesicular integral protein of 36 kDa (ViP36) in intracellular transport of newly synthesized glycoproteins.

Publisher Summary This chapter focuses on the methods to determine the carbohydrate-binding specificity of the vesicular integral protein of 36 kDa (VIP36) to be used in analyzing its roles. VIP36 recognizes high mannose-type glycans and is involved in the transport and sorting of glycoproteins carrying high mannose-type glycans. The methodology for measuring lectin activity of VIP36 discussed includes preparation of recombinant VIP36, preparation of radiolabeled cell lysate, preparation of high mannose-type aspartylglycans (Man7–9GlcNAc2Asn), in vitro binding assay, and preparation of mutant recombinant VIP36. Biological significance of lectin activities of VIP36 in intracellular transport of clusterin is discussed which includes the assay of interaction of VIP36 with clusterin through high mannose-type glycan, plasmid construction and isolation of clones showing stable overproduction, besides thetransport assay of clusterin.