Masanori Gotoh

Molecular Cloning and Characterization of a Novel UDP-GlcNAc:GalNAc-peptide β1,3-N-Acetylglucosaminyltransferase (β3Gn-T6), an Enzyme Synthesizing the Core 3 Structure of O-Glycans

The core 3 structure of theO-glycan, GlcNAcβ1–3GalNAcα1-serine/threonine, an important precursor in the biosynthesis of mucin-type glycoproteins, is synthesized by UDP-N-acetylglucosamine:GalNAc-peptide β1,3-N- acetylglucosaminyltransferase (β3Gn-T; core 3 synthase). The core 3 structure is restricted in its occurrence to mucins from specific tissues such as the stomach, small intestine, and colon. A partial sequence encoding a novel member of the human β3Gn-T family was found in one of the data bases. We cloned a complementary DNA of this gene and named it β3Gn-T6. The putative amino acid sequence of β3Gn-T6 retains the β3Gn-T motifs and is predicted to comprise a typical type II membrane protein. The soluble form of β3Gn-T6 expressed in insect cells showed β3Gn-T activity toward GalNAcα-p-nitrophenyl and GalNAcα1-serine/threonine. The β1,3-linkage between GlcNAc and GalNAc of the enzyme reaction product was confirmed by high performance liquid chromatography and NMR analyses. β3Gn-T6 effectively transferred a GlcNAc to the GalNAc residue on MUC1 mucin, resulting in the synthesis of a core 3 structure. Real time PCR analysis revealed that the β3Gn-T6 transcript was restricted in its distribution, mainly to the stomach, colon, and small intestine. We concluded that β3Gn-T6 is the most logical candidate for the core 3 synthase, which plays an important role in the synthesis of mucin-type O-glycans in digestive organs.

Differential Roles of TwoN-Acetylgalactosaminyltransferases, CSGalNAcT-1, and a Novel Enzyme, CSGalNAcT-2 INITIATION AND ELONGATION IN SYNTHESIS OF CHONDROITIN SULFATE

By a tblastn search with β1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in β1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5′-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much strongerN-acetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly- and oligosaccharides, and chondroitin sulfate poly- and oligosaccharides with a β1–4 linkage,i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e.initiation activity, than CSGalNAcT-1. Transfection of theCSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 transcripts were highly expressed in the small intestine, leukocytes, and spleen, however, both CSGalNAcTs were ubiquitously expressed in various tissues.

A novel β1,3-N-acetylglucosaminyltransferase (β3Gn-T8), which synthesizes poly-N-acetyllactosamine, is dramatically upregulated in colon cancer

A new member of the UDP‐N‐acetylglucosamine: β‐galactose β1,3‐N‐acetylglucosaminyltransferase (β3Gn‐T) family having the β3‐glycosyltransferase motifs was identified using an in silico method. This novel β3Gn‐T was cloned from a human colon cancer cell line and named β3Gn‐T8 based on its position in a phylogenetic tree and enzymatic activity. β3Gn‐T8 transfers GlcNAc to the non‐reducing terminus of the Galβ1–4GlcNAc of tetraantennary N‐glycan in vitro. HCT15 cells transfected with β3Gn‐T8 cDNA showed an increase in reactivity to both LEA and PHA‐L4 in a flow cytometric analysis. These results indicated that β3Gn‐T8 is involved in the biosynthesis of poly‐N‐acetyllactosamine chains on tetraantennary (β1,6‐branched) N‐glycan. In most of the colorectal cancer tissues examined, the level of β3Gn‐T8 transcript was significantly higher than in normal tissue. β3Gn‐T8 could be an enzyme involved in the synthesis of poly‐N‐acetyllactosamine on β1–6 branched N‐glycans in colon cancer.

Molecular Cloning and Characterization of a Novel Human β1,4-N-Acetylgalactosaminyltransferase, β4GalNAc-T3, Responsible for the Synthesis of N,N′-Diacetyllactosediamine, GalNAcβ1–4GlcNAc

We found a novel human glycosyltransferase gene carrying a hypothetical β1,4-glycosyltransferase motif during a BLAST search, and we cloned its full-length open reading frame by using the 5′-rapid amplification of cDNA ends method. It encodes a type II transmembrane protein of 999 amino acids with homology to chondroitin sulfate synthase in its C-terminal region (GenBank™ accession number AB089940). Its putative orthologous gene was also found in mouse (accession number AB114826). The truncated form of the human enzyme was expressed in HEK293T cells as a soluble protein. The recombinant enzyme transferred GalNAc to GlcNAc β-benzyl. The product was deduced to be GalNAcβ1–4GlcNAcβ-benzyl based on mass spectrometry and NMR spectroscopy. We renamed the enzyme β1,4-N-acetylgalactosaminyltransferase-III (β4GalNAc-T3). β4GalNAc-T3 effectively synthesized N,N′-diacetylgalactosediamine, GalNAcβ1–4GlcNAc, at non-reducing termini of various acceptors derived not only from N-glycans but also from O-glycans. Quantitative real time PCR analysis showed that its transcript was highly expressed in stomach, colon, and testis. As some glycohormones contain N,N′-diacetylgalactosediamine structures in their N-glycans, we examined the ability of β4GalNAc-T3 to synthesize N,N′-diacetylgalactosediamine structures in N-glycans on a model protein. When fetal calf fetuin treated with neuraminidase and β1,4-galactosidase was utilized as an acceptor protein, β4GalNAc-T3 transferred GalNAc to it. Furthermore, the majority of the signal from GalNAc disappeared on treatment with glycopeptidase F. These results suggest that β4GalNAc-T3 could transfer GalNAc residues, producing N,N′-diacetylgalactosediamine structures at least in N-glycans and probably in both N- and O-glycans.

Chondroitin Sulfate Synthase-3 MOLECULAR CLONING AND CHARACTERIZATION

Recently, it has become evident that chondroitin sulfate (CS) glycosyltransferases, which transfer glucuronic acid and/or N-acetylgalactosamine residues from each UDP-sugar to the nonreducing terminus of the CS chain, form a gene family. We report here a novel human gene (GenBank™ accession number AB086062) that possesses a sequence homologous with the human chondroitin sulfate synthase-1 (CSS1) gene, formerly known as chondroitin synthase. The full-length open reading frame consists of 882 amino acids and encodes a typical type II membrane protein. This enzyme contains a β3-glycosyltransferase motif and a β4-glycosyltransferase motif similar to that found in CSS1. Both the enzymes were expressed in COS-7 cells as soluble proteins, and their enzymatic natures were characterized. Both glucuronyltransferase and N-acetylgalactosaminyltransferase activities were observed when chondroitin, CS polymer, and their corresponding oligosaccharides were used as the acceptor substrates, but no polymerization reaction was observed as in the case of CSS1. The new enzyme was thus designated chondroitin sulfate synthase-3 (CSS3). However, the specific activity of CSS3 was much lower than that of CSS1. The reaction products were shown to have a GlcUAβ1–3GalNAc linkage and a GalNAcβ1–4GlcUA linkage in the nonreducing terminus of chondroitin resulting from glucuronyltransferase activity and N-acetylgalactosaminyltransferase activity, respectively. Quantitative real time PCR analysis revealed that the transcript level of CSS3 was much lower than that of CSS1, although it was ubiquitously expressed in various human tissues. These results indicate that CSS3 is a glycosyltransferase having both glucuronyltransferase and N-acetylgalactosaminyltransferase activities. It may make a contribution to CS biosynthesis that differs from that of CSS1.

Chondroitin sulfate N-acetylgalactosaminyltransferase-1 plays a critical role in chondroitin sulfate synthesis in cartilage

Cartilage destruction leads to severe joint diseases, such as osteoarthritis and spinal disorders with back pain, and cartilage regeneration is very inefficient. A major component of the cartilage extracellular matrix is the proteoglycan aggrecan that contains approximately 100 chondroitin sulfate (CS) chains, which impart water absorption and resistance to compression. Here, we demonstrate that chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) plays a critical role in CS biosynthesis in cartilage. By in situ hybridization and real time reverse transcription-PCR of developing cartilage, CSGalNAcT-1 exhibited the highest level of expression. Its expression in chondrogenic ATDC5 cells correlated well with that of aggrecan core protein. In heterozygote and homozygote aggrecan-null cartilage where aggrecan transcription is decreased, CSGalNAcT-1 transcription diminished accordingly. Overexpression of the enzyme in chondrocytic cells further enhanced CS biosynthesis but not that of the aggrecan core protein, indicating that the enzyme activity is not saturated in the cells and that aggrecan synthesized in the overexpressing cells is heavier than the native molecule. Analysis of the CS chains synthesized in the overexpressing cells by gel chromatography and that of disaccharide composition revealed that the CS chains had similar length and sulfation patterns. Furthermore, adenoviral gene delivery of the enzyme into intervertebral discs displayed a substantial increase in the level of CS biosynthesis. These observations indicate that CSGalNAcT-1 overexpression increases the number of CS chains attached to aggrecan core protein. Our studies may lead to a new therapeutic intervention, ameliorating the outcome of cartilage degenerative diseases.

Wisteria floribunda agglutinin positive human Mac‐2‐binding protein as a predictor of hepatocellular carcinoma development in chronic hepatitis C patients

Wisteria floribunda agglutinin (WFA)‐positive human Mac‐2‐binding protein (WFA+‐M2BP) is a new glycol marker related to liver fibrosis. The aim of the present study was to evaluate WFA+‐M2BP as a predictor of hepatocellular carcinoma (HCC) development in patients with chronic hepatitis C.

Novel Glycobiomarker for Ovarian Cancer That Detects Clear Cell Carcinoma

Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.

Application of a glycoproteomics-based biomarker development method: alteration in glycan structure on colony stimulating factor 1 receptor as a possible glycobiomarker candidate for evaluation of liver cirrhosis.

The importance of diagnosis and therapies for liver cirrhosis (LC) is indisputable. Thus, a reliable method for monitoring the progression of liver fibrosis and resultant LC is urgently needed. Previously, using a lectin-assisted glycoproteomic method, we identified 26 serum glycoproteins as promising glycobiomarker candidates for monitoring the progression of liver diseases. In this study, we identified colony stimulating factor 1 receptor (CSF1R) as a promising LC marker candidate and then established Wisteria floribunda agglutinin (WFA)-reactive CSF1R (WFA(+)-CSF1R) as a novel possible glycobiomarker candidate by utilizing a glycoproteomics-based strategy. The serum level of WFA(+)-CSF1R in patients with hepatitis C virus (HCV)-infected liver disease was measured by an antibody-lectin sandwich ELISA. In a proof-of-concept experiment of the strategy preceding to future clinical studies, LC patients showed a high serum WFA(+)-CSF1R level in selected samples (P = 1.3 × 10(-17)). This result suggests WFA(+)-CSF1R is a possible biomarker candidate for evaluation of LC. Our results verified feasibility of this strategy for glycobiomarker development.

Chondroitin sulfate synthase-2/chondroitin polymerizing factor has two variants with distinct function

Chondroitin sulfate (CS) is a polysaccharide consisting of repeating disaccharide units of N-acetyl-d-galactosamine and d-glucuronic acid residues, modified with sulfated residues at various positions. To date six glycosyltransferases for chondroitin synthesis have been identified, and the complex of chondroitin sulfate synthase-1 (CSS1)/chondroitin synthase-1 (ChSy-1) and chondroitin sulfate synthase-2 (CSS2)/chondroitin polymerizing factor is assumed to play a major role in CS biosynthesis. We found an alternative splice variant of mouse CSS2 in a data base that lacks the N-terminal transmembrane domain, contrasting to the original CSS2. Here, we investigated the roles of CSS2 variants. Both the original enzyme and the splice variant, designated CSS2A and CSS2B, respectively, were expressed at different levels and ratios in tissues. Western blot analysis of cultured mouse embryonic fibroblasts confirmed that both enzymes were actually synthesized as proteins and were localized in both the endoplasmic reticulum and the Golgi apparatus. Pulldown assays revealed that either of CSS2A, CSS2B, and CSS1/ChSy-1 heterogeneously and homogeneously interacts with each other, suggesting that they form a complex of multimers. In vitro glycosyltransferase assays demonstrated a reduced glucuronyltransferase activity in CSS2B and no polymerizing activity in CSS2B co-expressed with CSS1, in contrast to CSS2A co-expressed with CSS1. Radiolabeling analysis of cultured COS-7 cells overexpressing each variant revealed that, whereas CSS2A facilitated CS biosynthesis, CSS2B inhibited it. Molecular modeling of CSS2A and CSS2B provided support for their properties. These findings, implicating regulation of CS chain polymerization by CSS2 variants, provide insight in elucidating the mechanisms of CS biosynthesis.