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. Therefore, we here evaluated whether 3Score1-MUC1 is secreted into the blood streams of breast cancer patients, and whether it can serve as an improved breast cancer marker. We developed a lectin-sandwich immunoassay, called Gal4/MUC1, using a 3'-sulfated core1-specific galectin-4 and a MUC1 monoclonal antibody. Using the Gal4/MUC1 assay method, we found that 3Score1-MUC1 was profoundly expressed in the blood streams of patients with recurrent and/or metastatic breast cancer. The positive ratio of the Gal4/MUC1 assay was higher than that of the CA15-3 assay in both primary (n = 240) and relapsed (n = 43) patients, especially in the latter of which the positive ratio of Gal4/MUC1 was 86%. whereas that of CA15-3 was 47%. Furthermore, serum Gal4/MUC1 levels could more sensitively reflect the recurrence of primary breast cancer patients after surgery. Therefore, the Gal4/MUC1 assay should be an excellent alternative to the CA15-3 tumor marker for tracking the recurrence and metastasis of breast cancer.

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 GalNAcß1-4GlcNAc-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 GalNAcß1-4GlcNAc-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. A two-step sandwich SPFS immunoassay for LacdiNAc-PSA was constructed using both the anti-PSA IgG antibody to capture PSA and Wisteria floribunda agglutinin (WFA) for the detection of LacdiNAc. The results of the LacdiNAc-PSA immunoassay with SPFS showed that the assay had a sensitivity of 20.0 pg/mL and permitted the specific distinction between PC and BPH within the PSA gray zone. These results suggested that high-sensitivity automated SPFS immunoassay systems might become a powerful tool for the diagnosis of PC and other diseases.

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. These lectin-fractionated samples are analyzed quantitatively by measuring radioactivity, antigen contents are analyzed using enzyme-linked immunosorbent assay, and enzymatic activities are assessed.

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. 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.

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. 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)Galß1-4GlcNAcß1-3Galß1-4Glc], 3'-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galß1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galß1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives [SO(3)(-)-3Galß1-3(±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.

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. As a proof of principle, this approach was applied to two distinct HMO isomers representing a difucosylated octaose core and a trifucosylated decaose core. Obtained data revealed the presence of one terminal Lewis A and one internal Lewis X epitope in the case of the octaose and led to the identification of this molecule as a difucosylated iso-lacto-N-octaose. The trifucosylated, doubly branched lacto-N-neo-decaose was shown to represent a new type of HMO core structure in which the branched antenna is linked to carbon atom 3 of the innermost galactosyl residue. Hence, using this analytical protocol a novel HMO structure could be defined. Our results further demonstrate that a combination of different techniques may be required for de novo structural analysis of these molecules.

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 Galß1→3/4GlcNAc, and this specificity is mainly attributed to the N-terminal carbohydrate recognition domain (N-domain, CRD) (Ideo, H., Seko, A., Ishizuka, I., and Yamashita, K. (2003) Glycobiology 13, 713-723). In this study, we elucidated the crystal structures of the human galectin-8-N-domain (-8N) in the absence or presence of 4 ligands. The apo molecule forms a dimer, which is different from the canonical 2-fold symmetric dimer observed for galectin-1 and -2. In a galectin-8N-lactose complex, the lactose-recognizing amino acids are highly conserved among the galectins. However, Arg(45), Gln(47), Arg(59), and the long loop region between the S3 and S4 ß-strands are unique to galectin-8N. These amino acids directly or indirectly interact with the sulfate or sialic acid moieties of 3'-sialyl- and 3'-sulfolactose complexed with galectin-8N. Furthermore, in the LNF-III-galectin-8N complex, van der Waals interactions occur between the α1-3-branched fucose and galactose and between galactose and Tyr(141), and these interactions increase the affinity toward galectin-8N. Based on the findings of these x-ray crystallographic analyses, a mutagenesis study using surface plasmon resonance showed that Arg(45), Gln(47), and Arg(59) of galectin-8N are indispensable and coordinately contribute to the strong binding of galectins-8N to sialylated and sulfated oligosaccharides. Arg(59) is the most critical amino acid for binding in the S3-S4 loop region.

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. We first analyzed the carbohydrate structures of PSA derived from seminal fluid, serum of BPH and PC patients, and PC cell line, namely, LNCaP using eight lectin-immobilized columns and then with enzyme-linked immunosorbent assay (ELISA). The fraction of serum PSA from PC patients bound to both Fucalpha1-2Gal and betaGalNAc binding Trichosanthes japonica agglutinin-II (TJA-II) column, while that from BPH patients did not exhibit this binding ability, thereby implying that there is elevated expression of alpha1,2-fucosylation and beta-N-acetylgalactosaminylation of PSA during carcinogenesis. We then performed a real-time polymerase chain reaction (PCR) and confirmed that these structural changes were responsible for the elevated expression of fucosyltransferase I (FUT1) and beta-N-acetylgalactosaminyltransferase 4(B4GALNT4). Second, we measured TJA-II-bound PSA contents and the binding ratios of TJA-II column chromatography in serum PSA samples from 40 patients of both PC and BPH. The results indicated that both TJA-II-bound PSA content and TJA-II binding ratios (%) could be used to discriminate between PC and BPH with more than 95% probability, and TJA-II-bound PSA can be regarded as a potential marker of PC.

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. Until this study, GM2-rich rafts are poorly characterized and MbetaCD hypersensitivity, which may have clinical implications, has not been reported.

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. We found that LEC-8 binds to glycolipids in C. elegans through carbohydrate recognition. It has been reported that Cry5B-producing Bacillus thuringiensis strains can infect C. elegans and that the C. elegans Cry5B receptor molecules are glycolipids. We found that Cry5B and LEC-8 bound to C. elegans glycolipid-coated plates in a dose-dependent manner and that Cry5B binding to glycolipids was inhibited by the addition of LEC-8. LEC-8 is usually expressed strongly in the pharyngeal-intestinal valve and intestinal-rectal valve and is expressed weakly in intestine. However, when C. elegans were fed Escherichia coli expressing Cry5B, intestinal LEC-8::EGFP protein levels increased markedly. In contrast, LEC-8::EGFP expression triggered by Cry5B was reduced in toxin-resistant C. elegans mutants, which had mutations in genes involved in biosynthesis of glycolipids. Moreover, the LEC-8-deficient mutant was more susceptible to Cry5B than wild-type worms. These results suggest that the glycolipid-binding lectin LEC-8 contributes to host defense against bacterial infection by competitive binding to target glycolipid molecules.

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.

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. Western blot analysis and immunoprecipitation revealed that this ELISA recognizes both beta3Gal-T4 and beta3Gal-T5. RESULTS: We found beta3Gal-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 beta3Gal-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 beta3Gal-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 beta3Gal-T4/T5-positive rates (57%). CONCLUSION: beta3Gal-T4/T5 is a novel tumor marker for uterine corpus cancer and other gynecological cancers.

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.

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. (2005) Glycobiology 15, 943-951). Here we examined this activation and the biological significance of complex formation in differentiated HL-60 cells. beta3Gn-T2 and -T8 were co-immunoprecipitated from the lysates of both-transfected COS-7 cells, indicating their association in vivo. We prepared inactive mutants of both enzymes by destroying the DXD motifs. The mixture of mutated beta3Gn-T2 and intact beta3Gn-T8 did not exhibit any activation, whereas the mixture of intact beta3Gn-T2 and mutated beta3Gn-T8 had increased activity, indicating the activation of beta3Gn-T2 via complex formation. Next, we compared expression levels of beta3Gn-T1-T8 in HL-60 cells and DMSO-treated differentiated HL-60 cells, which produce larger poly-N-acetyllactosamine chains. The expression level of beta3Gn-T8 in the differentiated cells was 2.6-fold higher than in the untreated cells. Overexpression of beta3Gn-T8, but not beta3Gn-T2, induced an increase in poly-N-acetyllactosamine chains in HL-60 cells. These results raise a possibility that up-regulation of beta3Gn-T8 in differentiated HL-60 cells increases poly-N-acetyllactosamine chains by activating intrinsic beta3Gn-T2.

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. The carbohydrate recognition domain is composed of a 17-stranded antiparallel beta-sandwich and binds one Ca(2+) adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca(2+) ion orients the side chains of Asp(131), Asn(166), and His(190) for carbohydrate binding. This result explains the Ca(2+)-dependent carbohydrate binding of this protein. The Man-alpha-1,2-Man-alpha-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high mannose type glycoprotein recognition by VIP36 in a Ca(2+)-dependent and D1 arm-specific manner.

Nonsteroidal anti-inflammatory drug use does not affect short-term endoscopic and histologic outcomes after Helicobacter pylori eradication in patients with rheumatoid arthritis.

We evaluated the effects of the use of nonsteroidal anti-inflammatory drugs (NSAIDs) on endoscopic and histological findings in patients with rheumatoid arthritis (RA) before and after the eradication of Helicobacter pylori infection. Helicobacter pylori (H. pylori) eradication using lansoprazole 30 mg, amoxicillin 750 mg, and clarithromycin 200 mg twice daily for 1 week was conducted in 44 patients (mean age: 56.5 years) with RA. Using the updated Sydney system, endoscopic and histological findings of the greater curvature of the antrum, the greater curvature of the upper corpus, and the lesser curvature of the lower corpus were compared before and after eradication, for a mean follow-up period of 3.5 months. Overall, H. pylori eradication was successful in 32 patients (72.7%). Of these 32 patients, 23 were NSAID users. In the successful eradication group, (1) there was no significant change on endoscopic findings, including gastric erythema and erosion in all three regions irrespective of NSAIDs use; (2) of 17 active ulcers before eradication in NSAIDs users, all healed except for one duodenal ulcer that persisted, where one patient newly developed a gastric ulcer, one developed erosive duodenitis, and two developed reflux esophagitis, all in NSAID users; (3) neutrophil infiltration and chronic inflammation were significantly improved in all three regions after H. pylori eradication irrespective of use of NSAIDs, while atrophic change and intestinal metaplasia did not change. In the eradication failure group; (1) there was no significant change on endoscopic and histological findings in the three regions; (2) two of three ulcers present before eradication on NSAID users persisted even after eradication, and no new cases of gastric ulcer or erosive duodenitis occurred. In conclusion, over a mean follow-up period of 3.5 months, use of NSAIDs in Japanese patients with RA did not impair the healing process of gastric and duodenal ulcers nor did it affect the endoscopic and histological improvements associated with H. pylori eradication.

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. (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 beta-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 Arg(45) 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.

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) Glycobiology 12, 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.

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. 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.