Yutaro Azuma

Altered glycosylation of α1-acid glycoprotein in patients with inflammation and diabetes mellitus

Background: In certain pathophysiological conditions, such as inflammation rheumatoid arthritis and diabetes mellitus (DM), alterations in asparagine-linked glycan (N-glycan) patterns of the acute-phase protein, α1-acid glycoprotein (AGP), have been reported. In this study, we investigated N-glycan structures of AGP purified from the sera of patients with acute inflammation (n=5), type 2 diabetes mellitus (n=5), and healthy individuals (n=5). Methods: N-Glycans were released with peptide N-glycosidase F (PNGase F) from denatured AGP and purified with cellulose cartridge. N-glycans were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) in combination with exoglycosidase digestion. Results: We revealed increases in bi-antennary complex glycans and in α1–3 fucosylated bi-, tri-, and tetra-antennary glycans and a decrease in tri-antennary glycans in inflammation patients. These results support increases in bindings to concanavalin A (ConA) and Aleuria aurantia lectins (AALs). In diabetic patients, the pathogenesis-specific change in N-glycan patterns of AGP was not significant. Conclusions: The MALDI-TOFMS method is sensitive and suitable for profiling analysis of N-glycans in clinical samples.

Interleukin-1β induces sialyl Lewis X on hepatocellular carcinoma HuH-7 cells via enhanced expression of ST3Gal IV and FUT VI gene

We previously demonstrated that human hepatocellular carcinoma‐derived HuH‐7 cells stimulated with interleukin‐1β (IL‐1β) produce α1‐acid glycoprotein (AGP) with increased amounts of sialyl Lewis X (sLeX) antigen, although the mechanism remained obscure. Here, we report our investigation of the mechanism. sLeX expression on HuH‐7 cells was induced 2.5 times more after 48 h stimulation with 100 U/mL IL‐1β compared with control, as indicated by anti‐sLeX antibody binding. Furthermore, expression of 2,3‐sialylated N‐acetyllactosamine increased gradually up to 48 h after IL‐1β stimulation; this preceded the increase in sLeX expression. Increases in α2,3‐sialyltransferase activity also preceded increases in α1,3‐fucosyltransferase activity. Furthermore, mRNA levels of ST3Gal IV, FUT IV and VI in HuH‐7 cells stimulated with IL‐1β were increased at 2–4 h, while increases in FUT VI mRNA level occurred gradually after 24 h. IL‐1β‐induced sLeX expression on HuH‐7 cells was suppressed by transfection of gene‐specific small interference RNAs against FUT VI and ST3Gal IV but not against FUT IV and ST3Gal III. These data results that IL‐1β induces expression of sLeX on HuH‐7 cells by enhanced expression of FUT VI and ST3Gal IV gene.

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3[emsp4 ]h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized α2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied α2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2[emsp4 ]h of etoposide treatment. Moreover, the decrease in α2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.

Alteration of sugar chains on α1-acid glycoprotein secreted following cytokine stimulation of HuH-7 cells in vitro

In inflammation, hepatocytes secrete several proteins into serum, the so-called acute phase proteins. In addition to increased serum levels of alpha(1)-acid glycoprotein (AGP), there are also changes in the composition of its sugar chains. To investigate the cytokine-stimulated alteration of sugar chains on AGP, we cultured the human hepatoma cell line HuH-7 cells in serum-free medium (IS-RPMI) with and without IL-1beta and IL-6, and analyzed AGP secretion into the medium. AGP was increased during stimulation with both IL-1beta and IL-6, although the effect of IL-1beta was more pronounced. Lectin-binding assay for secreted AGP also indicated significant increases in the binding activities to Aleuria aurantia lectin (AAL), Sambucus sieboldiana agglutinin (SSA), and concanavalin-A (ConA). In particular, AAL binding activity increased with higher expression of the sialyl Lewis X (sLe(X)) antigen, NeuAc alpha2-3 Gal beta1-4 (Fuc alpha1-3) GlcNAc-R. Thus, the increase in AGP fucosylation may be correlated with the increase of sLe(X). The present results indicate that the serum-free culture of HuH-7 cells provides a useful model for investigating the secretion of proteins from hepatocytes, and the effects of cytokines on the changes in sugar chains of glycoproteins in vitro.

Binding of natural cytotoxicity receptor NKp46 to sulfate- and α2,3-NeuAc-containing glycans and its mutagenesis

Natural cytotoxicity receptor 1 (NCR1, NKp46) binds to heparin and heparan sulfate; however, other natural ligands for NKp46 have yet to be elucidated. Using the recombinant extracellular region (coding for AA 22-258) of NKp46 tagged with 6× His (NKp46-H6), and mutants K136Q, R139Q, H142Q, R145Q, and K149Q, we determined their binding affinities to sulfate- and NeuAc-containing glycans-coated plates. NKp46-H6 directly bound to plates coated with heparin- and heparan sulfate-conjugated bovine serum albumin with K(d) values of 770 and 850 nM, respectively. The binding of NKp46-H6 to heparin-BSA was suppressed by soluble heparin, herparan sulfate, fucoidan, λ-carrageenan, and dextran sulfate, but not by 2-O-, 6-O-, and N-desulfated heparin. NKp46-H6 also bound to multimeric sialyl Lewis X expressing transferrin secreted by human hepatoma HepG2 cells (HepTF) with a K(d) value of 530 nM, but not to desialylated HepTF, commercially available TF, or 1-acid glycoprotein. Moreover, mutants R139Q, R145Q, and K149Q had significantly reduced binding to these sulfate-containing glycans, and K136Q and K149Q to HepTF, indicating that NKp46 binds to sulfate- and 2,3-NeuAc-containing glycans mainly via ionic interactions. However, the binding sites of NKp46 were different.

NKG2D and CD94 bind to multimeric α2,3-linked N-acetylneuraminic acid

Killer lectin-like receptors on natural killer cells mediate cytotoxicity through glycans on target cells including the sialyl Lewis X antigen (sLeX). We investigated whether NK group 2D (NKG2D) and CD94 can bind to sialylated N-linked glycans, using recombinant glutathione S-transferase-fused extracellular lectin-like domains of NKG2D (rNKG2Dlec) and CD94 (rCD94lec). Both rNKG2Dlec and rCD94lec bound to plates coated with high-sLeX-expressing transferrin secreted by HepG2 cells (HepTF). The binding of rNKG2Dlec and rCD94lec to HepTF was markedly suppressed by treatment of HepTF with neuraminidase and in the presence of N-acetylneuraminic acid. Moreover, rNKG2Dlec and rCD94lec bound to alpha2,3-sialylated human alpha(1)-acid glycoprotein (AGP) but not to alpha2,6-sialylated AGP. Mutagenesis revealed that (152)Y of NKG2D and (144)F and (160)N of CD94 were critical for HepTF binding. This is the first report that NKG2D and CD94 bind to alpha2,3-sialylated but not to alpha2,6-sialylated multi-antennary N-glycans.

NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Killer lectin-like receptors NKG2D and CD94 on natural killer cells trigger cytotoxicity through binding of glycans on target cells including sialyl Lewis X antigen. We previously reported that NKG2D and CD94 recognize alpha2,3-linked NeuAc on multi-antennary N-glycans. Here we further investigated polysaccharide binding by these receptors, using glutathione-S-transferase-fused extracellular domains of NKG2D AA 73-216 (rNKG2Dlec) and CD94 AA 68-179 (rCD94lec). We found that rNKG2Dlec and rCD94lec bind in a dose-dependent manner to plates coated with heparin-conjugated bovine serum albumin (heparin-BSA). Binding to heparin-BSA was suppressed by soluble sulfate-containing polysaccharides, but minimally impacted by 2-O-, 6-O-, and 2-N-desulfated heparin. Mutagenesis revealed that (152)Y and (199)Y of NKG2D and (144)F, (160)N, and (166)C of CD94 were critical for binding to heparin-BSA. The present manuscript provides the first evidence that NKG2D and CD94 bind to heparin and sulfate-containing polysaccharides.

Prolonged high glucose suppresses phorbol 12-myristate 13-acetate and ionomycin-induced interleukin-2 mRNA expression in Jurkat cells.

BACKGROUND The disturbance of immunological responses is a complication of diabetes mellitus. METHODS AND RESULTS We cultured Jurkat cells in 11.1 (normal) and 22.2 mmol/l (high) glucose for 12 weeks and stimulated them with 10 nmol/l phorbol 12-myristate 13-acetate (PMA) and 500 nmol/l ionomycin. RT-PCR revealed that induced interleukin (IL)-2 mRNA expression levels were suppressed in high glucose cultures compared to those in normal glucose. Promoter activities of IL-2, nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1), after 6 h stimulation with PMA and ionomycin, gradually decreased in high glucose cultures to approximately 20% of those in normal glucose at 12 weeks. The prolonged culture in high glucose increased inducible cAMP early repressor (ICER) II mRNA and protein levels, and overexpression of ICER II dose-dependently suppressed promoter activities of IL-2, NFAT, and AP-1. Moreover, ICER II mRNA expression was transiently induced by stimulation with PMA and ionomycin in normal glucose cultures; however, with high glucose, the induction disappeared. CONCLUSION These results indicate that ICER II protein accumulates during prolonged culture in high glucose and suppresses IL-2 mRNA expression in Jurkat cells.

Transcriptional Regulation of Fucosyltransferase 1 Gene Expression in Colon Cancer Cells

The α1,2-fucosyltransferase I (FUT1) enzyme is important for the biosynthesis of H antigens, Lewis B, and Lewis Y. In this study, we clarified the transcriptional regulation of FUT1 in the DLD-1 colon cancer cell line, which has high expression of Lewis B and Lewis Y antigens, expresses the FUT1 gene, and shows α1,2-fucosyltransferase (FUT) activity. 5′-rapid amplification of cDNA ends revealed a FUT1 transcriptional start site −10 nucleotides upstream of the site registered at NM_000148 in the DataBase of Human Transcription Start Sites (DBTSS). Using the dual luciferase assay, FUT1 gene expression was shown to be regulated at the region −91 to −81 nt to the transcriptional start site, which contains the Elk-1 binding site. Site-directed mutagenesis of this region revealed the Elk-1 binding site to be essential for FUT1 transcription. Furthermore, transfection of the dominant negative Elk-1 gene, and the chromatin immunoprecipitation (CHIp) assay, supported Elk-1-dependent transcriptional regulation of FUT1 gene expression in DLD-1 cells. These results suggest that a defined region in the 5′-flanking region of FUT1 is critical for FUT1 transcription and that constitutive gene expression of FUT1 is regulated by Elk-1 in DLD-1 cells.

The effect of alpha2,6-linked sialic acid on anti-IgM antibody-induced apoptosis in Ramos cells.

Apoptosis in B cells is induced through the B cell antigen receptor (BCR) and affects the sialic acid recognition molecules on B cells. We investigated the effects of α1-acid glycoprotein (AGP), which mainly contains α2,6-linked sialic acid, on anti-IgM antibody (Ab)-induced apoptosis in Ramos cells, which are derived from Burkitts lymphoma. When Ramos cells were incubated with anti-IgM-Ab in plates coated with AGP, neuraminidase-digested AGP (asAGP) or α2,3-sialylated AGP (2,3AGP), apoptosis was suppressed only in those coated with AGP. We also studied the effects of CD22, which is expressed on the surface of mature B cells and binds to sugar chains containing α2,6-linked sialic acid, with anti-CD22 monoclonal antibody (mAb). Anti-CD22mAb enhanced anti-IgM Ab-induced apoptosis in Ramos cells. These contradictory results suggested that the recognition molecules for α2,6-linked sialic acid on AGP, which inhibits B-cell apoptosis, is distinct from CD22, or that different binding domains of CD22 between α2,6-linked sialic acid and anti-CD22 mAb exert opposite functions of suppression or enhancement to anti-IgM Ab-induced B cells.