Takashi Ohkura

Polylactosamine on glycoproteins influences basal levels of lymphocyte and macrophage activation

β1,3-N-acetylglucosaminyltransferase 2 (β3GnT2) is a polylactosamine synthase that synthesizes a backbone structure of carbohydrate structures onto glycoproteins. Here we generated β3GnT2-deficient (β3GnT2−/−) mice and showed that polylactosamine on N-glycans was markedly reduced in their immunological tissues. In WT mice, polylactosamine was present on CD28 and CD19, both known immune costimulatory molecules. However, polylactosamine levels on these molecules were reduced in β3GnT2−/− mice. β3GnT2−/− T cells lacking polylactosamine were more sensitive to the induction of intracellular calcium flux on stimulation with anti-CD3ε/CD28 and proliferated more strongly than T cells from WT mice. β3GnT2−/− B cells also showed hyperproliferation on BCR stimulation. Macrophages from β3GnT2−/− mice had higher cell surface CD14 levels and enhanced responses to endotoxin. These results indicate that polylactosamine on N-glycans is a putative immune regulatory factor presumably suppressing excessive responses during immune reactions.

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.

Glycoproteomic discovery of serological biomarker candidates for HCV/HBV infection-associated liver fibrosis and hepatocellular carcinoma.

We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. FEBS J.2010, 277(1), 95-105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC-MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner.

β3GNT2 (B3GNT2), A MAJOR POLYLACTOSAMINE SYNTHASE: ANALYSIS OF B3GNT2-DEFICIENT MICE

The polylactosamine structure is a fundamental structure of carbohydrate chains and carries a lot of biofunctional carbohydrate epitopes. To investigate the biological function of polylactosamine chains, here we generated and analyzed knockout mice lacking the gene B3gnt2, which encodes a major polylactosamine synthase. In beta1,3-N-acetylglucosaminyltransferase (B3gnt2) B3gnt2-deficient (B3gnt2-/-) mice, the number of polylactosamine structures was markedly lower than in wild-type mice. Flow cytometry, LEL lectin-blotting, and glycan analysis by metabolic labeling demonstrated that the amount of polylactosamine chains on N-glycans was greatly reduced in the tissues of B3gnt2-/- mice. We examined whether immunological abnormalities were present in B3gnt2-/- mice. We screened polylactosamine-carrying molecules of wild-type mice by lectin microarray analysis and found that polylactosamine was present on CD28 and CD19, two established immune co-stimulatory molecules. Polylactosamine levels on these molecules were lower in B3gnt2-/- mice than in wild-type mice. B3gnt2-/- T cells were more sensitive to the induction of intracellular Ca2+ flux on stimulation with anti-CD3epsilon/CD28 antibodies and proliferated more strongly than wild-type T cells. B3gnt2-/- B cells also showed hyperproliferation on BCR stimulation. These results showed that hyperactivation of lymphocytes occurred due to a lack of polylactosamine on receptor molecules in B3gnt2-/- mice. This finding indicates that polylactosamine has an important role in immunological biofunctions. We can therefore attempt to identify the in vivo biological function of glycans using glycogene-deficient mice.

Glycoproteomics Approach for Identifying Glycobiomarker Candidate Molecules for Tissue Type Classification of Non-small Cell Lung Carcinoma

Histopathological classification of lung cancer has important implications in the application of clinical practice guidelines and the prediction of patient prognosis. Thus, we focused on discovering glycobiomarker candidates to classify the types of lung cancer tissue. First, we performed lectin microarray analysis of lung cancer tissue specimens and cell lines and identified Aleuria aurantia lectin (AAL), Hippeastrum hybrid lectin (HHL), and Concanavalia ensiformis agglutinin (ConA) as lectin probes specific to non-small cell lung carcinoma (NSCLC). LC-MS-based analysis was performed for the comprehensive identification of glycoproteins and N-linked glycosylation sites using lectin affinity capture of NSCLC-specific glycoforms of glycoproteins. This analysis identified 1092 AAL-bound glycoproteins (316 gene symbols) and 948 HHL/ConA-bound glycoproteins (279 gene symbols). The lectin microarray-assisted verification using 15 lung cancer cell lines revealed the NSCLC-specific expression of fibronectin. The glycosylation profiling of fibronectin indicated that the peanut agglutinin (PNA) signal appeared to differentiate two NSCLC types, adenocarcinoma and large cell carcinoma, whereas the protein expression level was similar between these types. Our glycoproteomics approach together with the concurrent use of an antibody and lectin is applicable to the quantitative and qualitative monitoring of variations in glycosylation of fibronectin specific to certain types of lung cancer tissue.