Harumi Yamamoto

Germinal Center Marker GL7 Probes Activation-Dependent Repression of N-Glycolylneuraminic Acid, a Sialic Acid Species Involved in the Negative Modulation of B-Cell Activation

ABSTRACT Sialic acid (Sia) is a family of acidic nine-carbon sugars that occupies the nonreducing terminus of glycan chains. Diversity of Sia is achieved by variation in the linkage to the underlying sugar and modification of the Sia molecule. Here we identified Sia-dependent epitope specificity for GL7, a rat monoclonal antibody, to probe germinal centers upon T cell-dependent immunity. GL7 recognizes sialylated glycan(s), the α2,6-linked N-acetylneuraminic acid (Neu5Ac) on a lactosamine glycan chain(s), in both Sia modification- and Sia linkage-dependent manners. In mouse germinal center B cells, the expression of the GL7 epitope was upregulated due to the in situ repression of CMP-Neu5Ac hydroxylase (Cmah), the enzyme responsible for Sia modification of Neu5Ac to Neu5Gc. Such Cmah repression caused activation-dependent dynamic reduction of CD22 ligand expression without losing α2,6-linked sialylation in germinal centers. The in vivo function of Cmah was analyzed using gene-disrupted mice. Phenotypic analyses showed that Neu5Gc glycan functions as a negative regulator for B-cell activation in assays of T-cell-independent immunization response and splenic B-cell proliferation. Thus, Neu5Gc is required for optimal negative regulation, and the reaction is specifically suppressed in activated B cells, i.e., germinal center B cells.

Anomalous expression of Thy1 (CD90) in B-cell lymphoma cells and proliferation inhibition by anti-Thy1 antibody treatment

The anti-CD20 monoclonal antibody (Ab) rituximab is accepted to be an effective therapeutic Ab for malignant B-cell lymphoma; however, discovery of other cell surface antigens is required for the option of antibody medicine. Considering that many tumor-associated antigens are glycans, we have searched glycoconjugates for the candidate antigens that therapeutic Abs target. To this end, we first focused on the difference in the glycogenes expression in terms of Epstein-Barr virus (EBV) infection of a Burkitts lymphoma cell line, Akata. Using DNA array, flow cytometry and Western blotting, we found that Thy1 was highly expressed in EBV-positive Akata cells. Subsequently, Thy1 was found to be expressed in other B-cell lymphoma cell lines: BJAB, MutuI, and MutuIII, irrespective of EBV infection. Treatment of these cells with an anti-Thy1 monoclonal antibody inhibited proliferation more strongly than the therapeutic Ab rituximab. The B-cell lymphoma cell lines were classified based on the extent of the proliferation inhibition, which was not correlated with the expression level of Thy1. It is suggested that stable residence of receptor tyrosine kinases in lipid rafts sustains cell growth in B-cell lymphoma cells.

Correlation Index-Based Responsible-Enzyme Gene Screening (CIRES), a Novel DNA Microarray-Based Method for Enzyme Gene Involved in Glycan Biosynthesis

Background Glycan biosynthesis occurs though a multi-step process that requires a variety of enzymes ranging from glycosyltransferases to those involved in cytosolic sugar metabolism. In many cases, glycan biosynthesis follows a glycan-specific, linear pathway. As glycosyltransferases are generally regulated at the level of transcription, assessing the overall transcriptional profile for glycan biosynthesis genes seems warranted. However, a systematic approach for assessing the correlation between glycan expression and glycan-related gene expression has not been reported previously. Methodology To facilitate genetic analysis of glycan biosynthesis, we sought to correlate the expression of genes involved in cell-surface glycan formation with the expression of the glycans, as detected by glycan-recognizing probes. We performed cross-sample comparisons of gene expression profiles using a newly developed, glycan-focused cDNA microarray. Cell-surface glycan expression profiles were obtained using flow cytometry of cells stained with plant lectins. Pearsons correlation coefficients were calculated for these profiles and were used to identify enzyme genes correlated with glycan biosynthesis. Conclusions This method, designated correlation index-based responsible-enzyme gene screening (CIRES), successfully identified genes already known to be involved in the biosynthesis of certain glycans. Our evaluation of CIRES indicates that it is useful for identifying genes involved in the biosynthesis of glycan chains that can be probed with lectins using flow cytometry.

Wheat germ agglutinin stains dispersed post-golgi vesicles after treatment with the cytokinesis inhibitor psychosine

The galactosylsphingosine psychosine (Psy) is one of the sphingolipids and induce the formation of multinuclear cells in several cell lines by inhibiting cytokinesis. In the present report, we show that intracellular organelles, including wheat germ agglutinin (WGA)‐positive vesicles and early endosomes, are selectively dispersed by Psy. WGA is a conventional Golgi marker and WGA‐positive vesicles appeared to co‐localize with the Golgi apparatus in untreated cells. Psy treatment induced the dispersal of WGA‐positive vesicles without affecting the structure of the Golgi apparatus, resulting in discrimination of WGA‐positive vesicles from the Golgi apparatus. In sharp contrast to this effect of Psy, WGA‐positive vesicles were not affected by brefeldin A treatment, which induced the disappearance of the Golgi apparatus. Immunostaining with anti‐TGN46 antibodies revealed that a large portion of the WGA‐positive vesicles were derived from the trans‐Golgi network. Notably, the dispersed WGA‐positive vesicles did not stain with anti‐syntaxin 6, another marker of the trans‐Golgi network. During cytokinesis, WGA‐positive vesicles in the cytoplasm decreased, and WGA staining accumulated at the cleavage furrow, which was apparently inhibited by the presence of Psy. These data suggest that the transport of WGA‐positive vesicles to the cleavage furrow is associated with the progression of cytokinesis. J. Cell. Physiol. 215: 517–525, 2008.

Quantitative Transcriptomic Profiling of Branching in a Glycosphingolipid Biosynthetic Pathway

Cellular biosynthesis of macromolecules often involves highly branched enzyme pathways, thus cellular regulation of such pathways could be rather difficult. To understand the regulatory mechanism, a systematic approach could be useful. We genetically analyzed a branched biosynthetic pathway for glycosphingolipid (GSL) GM1 using correlation index-based responsible enzyme gene screening (CIRES), a novel quantitative phenotype-genotype correlation analysis. CIRES utilizes transcriptomic profiles obtained from multiple cells. Among a panel of B cell lines, expression of GM1 was negatively correlated with and suppressed by gene expression of CD77 synthase (CD77Syn), whereas no significant positive correlation was found for enzymes actually biosynthesizing GM1. Unexpectedly, a GM1-suppressive phenotype was also observed in the expression of catalytically inactive CD77Syn, ruling out catalytic consumption of lactosylceramide (LacCer) as the main cause for such negative regulation. Rather, CD77Syn seemed to limit other branching reaction(s) by targeting LacCer synthase (LacCerSyn), a proximal enzyme in the pathway, because they were closely localized in the Golgi apparatus and formed a complex. Moreover, turnover of LacCerSyn was accelerated upon CD77Syn expression to globally change the GSL species expressed. Collectively, these data suggest that transcriptomic assessment of macromolecule biosynthetic pathways can disclose a global regulatory mechanism(s) even when unexpected.

Superoxide production from human polymorphonuclear leukocytes by human mannan-binding protein (MBP)

Mannan-binding protein (MBP) is a Ca2+-dependent mammalian lectin that plays an important role in innate immunity. In this study, we found that ligand-bound MBP stimulates polymorphonuclear leukocytes (PMN) to induce cell aggregation and superoxide production. The biological response of PMN to ligand-bound MBP was dose- and time-dependent. The PMN aggregation and superoxide production induced by ligand-bound MBP was blocked completely by pertussis toxin, and partially blocked by a platelet activation factor receptor antagonist, TCV-309. These findings suggest that the ligand-bound MBP stimulates PMN through a putative MBP receptor(s) on PMN. Published in 2004.

DNA Microarray in Glycobiology

DNA microarray analysis enables us to provide a considerable amount of information on glyco-chain expression (Brazma et al. 2001; Ide et al. 2006). DNA microarray experiments are developed to obtain overall transcriptome of cells using DNAs spotted on a slide glass as probes and fluorescently labeled DNAs derived from mRNA samples. DNA microarray experiments require high-quality RNA. Thus, it is advisable to prepare RNA as intact as possible. The quality of RNA is usually assessed by the intactness of the peaks and the lack of degradation of RNA by capillary electrophoresis. This step is essential for the reliable final readout of microarray experiments. Following is the normal procedure operated in our laboratory for the DNA microarray experiments using RIKEN human and mouse glyco-chain-related DNA microarrays version 2 (human version: GEO platform GPL 3465 http://www.ncbi.nlm.nih.gov/geo/), which were designed by us and spotted by Takara Bio Inc.