Yohko U. Katagiri

Analysis of N-linked oligosaccharide chains of glycoproteins on nitrocellulose sheets using lectin-peroxidase reagents

A rapid and convenient method was established for analysis of the N-linked carbohydrate chains of glycoproteins on nitrocellulose sheets. Proteins were separated by polyacrylamide gel electrophoresis, transferred to nitrocellulose sheets, reacted with peroxidase-coupled lectins, and detected by color development of the enzyme reaction. Four glycoproteins having N-linked oligosaccharide chains were used as test materials: Taka-amylase A (which has a high-mannose-type chain), ovalbumin (high-mannose-type chains and hybrid-type chains), transferrin (biantennary chains of complex type), and fetuin (triantennary chains of complex type and O-linked-type chains). Concanavalin A interacted with Taka-amylase A, transferrin, and ovalbumin but barely interacted with fetuin. After treatment of the glycoproteins on a nitrocellulose sheet with endo-beta-N-acetylglucosaminidase H, transferrin reacted with concanavalin A but Taka-amylase A and ovalbumin did not. Wheat germ agglutinin interacted with Taka-amylase A but not ovalbumin; therefore, they were distinguishable from each other. Fetuin and transferrin were detected by Ricinus communis agglutinin or peanut agglutinin after removal of sialic acid by treatment with neuraminidase or by weak-acid hydrolysis. Erythroagglutinating Phaseolus vulgaris agglutinin detected fetuin and transferrin. Thus, the combined use of these procedures distinguished the four different types of N-linked glycoproteins. This method was also applied to the analysis of membrane glycoproteins from sheep red blood cells. The terminally positioned sugars of sialic acid, alpha-fucose, alpha-galactose, and alpha-N-acetylgalactosamine were also detected with lectins from Limulus polyphemus, Lotus tetragonolobus, Maclura pomifera, and Dolichos biflorus, respectively.

CD24 Induces Apoptosis in Human B Cells Via the Glycolipid-Enriched Membrane Domains/Rafts-Mediated Signaling System

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt’s lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.

Kinetic Analysis of Binding between Shiga Toxin and Receptor Glycolipid Gb3Cer by Surface Plasmon Resonance

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the “bivalent analyte” model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

Osteopontin, a coordinator of host defense system: a cytokine or an extracellular adhesive protein?

Introduction Specific cellular adhesion and migration of cells are important themes in embryonic development, tumor cell metastasis, wound healing and inflammation. Recent advances in our understanding of the molecular basis of cell adhesion and migratory interactions with extracellular matrix proteins have resulted in the concept that many cell interactions are dependent on specific adhesive recognition amino acid sequences. One such adhesive recognition sequence is an Arg-Gly-Asp (RGD) tripeptide sequence found in various proteins such as extracellular matrix proteins (fibronectin, vitronectin, osteopontin and collagen) and coagulation factors (fibrinogen, thrombospondin and von Willebrand factor) (60). Among these, osteopontin (OPN) is originally identified as a major sialylated noncollagenous matrix protein in bone (12). Most studies to date have focused on the role of OPN in the process of mineralization and bone morphogenesis (7, 9, 42, 43, 53, 57). An extensive study of OPN expression in adult human tissue by Brown et al (6) revealed that the OPN was a major constituent of the luminal surface of epithelial cells in the gastrointestinal tract, gall bladder, pancreas, urinary and reproductive tracts, lung bronchi, mammary and salivary glands, and sweat duct where epithelial cells communicate with the external environment. OPN is also secreted into various body fluids, including milk, serum, urine and cochlear fluid. OPN is very unique in the sense that it can be classified not only as extracellular matrix but also a lymphokine since it possesses a signal sequence and is secreted by activated T cells and regulates various immune functions (25, 32, 38, 39, 48). Thus, the purpose of this minireview is to provide an important aspect of our current knowledge regarding the function of OPN as a coordinator of the host defense system and the potential relationship of this protein to several pathological conditions.

Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells

ABSTRACT Ewings family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlled EWS/ETS-inducible system in human bone marrow-derived mesenchymal progenitor cells (MPCs). Ectopic expression of both EWS/FLI1 and EWS/ERG proteins resulted in a dramatic change of morphology, i.e., from a mesenchymal spindle shape to a small round-to-polygonal cell, one of the characteristics of EFT. EWS/ETS also induced immunophenotypic changes in MPCs, including the disappearance of the mesenchyme-positive markers CD10 and CD13 and the up-regulation of the EFT-positive markers CD54, CD99, CD117, and CD271. Furthermore, a prominent shift from the gene expression profile of MPCs to that of EFT was observed in the presence of EWS/ETS. Together with the observation that EWS/ETS enhances the ability of cells to invade Matrigel, these results suggest that EWS/ETS proteins contribute to alterations of cellular features and confer an EFT-like phenotype to human MPCs.

Globotriaosyl ceramide (CD77/Gb3) in the glycolipid-enriched membrane domain participates in B-cell receptor–mediated apoptosis by regulating Lyn kinase activity in human B cells

The role of CD77 expressed on a fraction of germinal center B cells, also known as glycosphyngolipid Gb3, and as a functional receptor for Shiga toxins (Stx) in B-cell receptor (BCR)-mediated apoptosis was investigated. Using Stx1-sensitive Burkitts lymphoma Ramos cells as an in vitro model of CD77(+) germinal center B cells, intracellular signaling events mediated by either Stx1 or anti-CD77 antibody were examined immunobiochemically and immunocytologically. We observed prompt activation of Lyn and Syk kinases leading to increased binding of these proteins to surface IgM (sIgM) in Ramos cells after Stx1 treatment. We also observed microscopic colocalization of CD77 and sIgM after stimulation with Stx1. Along with the synergism between the cross-linking of CD77 and that of sIgM in their effect on apoptosis induction, it was highly probable that CD77 cross-linking induces activation of the BCR signaling cascade. Analysis using sucrose density gradient centrifugation suggested that Stx1 binding to CD77 induced recruitment and activation of Lyn in the glycolipid-enriched membrane (GEM) fractions. Once activated, however, Lyn seemed to acquire an increased detergent solubility and moved outside of the GEM fractions. This study describes the participation of the GEM domain in BCR-signaling cascade and suggests a possible role of CD77 as a regulator of BCR-induced apoptosis in human B cells.

Shiga toxin binding to globotriaosyl ceramide induces intracellular signals that mediate cytoskeleton remodeling in human renal carcinoma-derived cells

Shiga toxin is a bacterial toxin consisting of A and B subunits. Generally, the essential cytotoxicity of the toxin is thought to be mediated by the A subunit, which possesses RNA cleavage activity and thus induces protein synthesis inhibition. We previously reported, however, that the binding of the Shiga toxin 1-B subunit to globotriaosyl ceramide, a functional receptor for Shiga toxin, induces intracellular signals in a manner that is dependent on glycolipid-enriched membrane domains, or lipid rafts. Although the precise role of this signaling mechanism is not known, here we report that Shiga-toxin-mediated intracellular signals induce cytoskeleton remodeling in ACHN cells derived from renal tubular epithelial carcinoma. Using confocal laser scanning microscopy, we observed that Shiga toxin 1-B treatment induces morphological changes in ACHN cells in a time-dependent manner. In addition, the morphological changes were accompanied by the redistribution of a number of proteins, including actin, ezrin, CD44, vimentin, cytokeratin, paxillin, FAK, and α- and γ-tubulins, all of which are involved in cytoskeletal organization. The transient phosphorylation of ezrin and paxillin was also observed during the course of protein redistribution. Experiments using inhibitors for a variety of kinases suggested the involvement of lipid rafts, Src family protein kinase, PI 3-kinase, and RHO-associated kinase in Shiga toxin 1-B-induced ezrin phosphorylation. Shiga toxin 1-B-induced cytoskeletal remodeling should provide an in vitro model that can be used to increase our understanding of the pathogenesis of Shiga-toxin-mediated cell injury and the role of lipid-raft-mediated cell signaling in cytoskeletal remodeling.

A role for lipid rafts in immune cell signaling.

Cross‐linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src‐family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.

Non‐RGD domains of osteopontin promote cell adhesion without involving αv integrins

Osteopontin (OPN) is an integrin‐binding secreted protein that contains an Arg‐Gly‐Asp (RGD) amino acid sequence and binds to various cell types via RGD‐mediated interaction with the αvβ3 integrin. We have identified a cell line whose binding to OPN does not require RGD or αv interactions. We compared the ability of two murine cell lines, L929 fibroblastic cells and B16‐BL6 melanoma cells, to interact with OPN (from human milk, and recombinant human and mouse OPN) as well as recombinant OPN prepared to include either the N‐terminal or C‐terminal halves but lacking the RGD sequence. Both cell lines adhered to GRGDS peptides coupled to BSA, and these interactions were inhibited by addition of GRGDS (but not GRGES) peptides or a monoclonal antibody specific to the αv integrin subunit. Adhesion of L929 cells to OPN was also dependent on the RGD sequence and the αv integrin subunit. However, the binding of B16‐BL6 cells was not inhibited by either GRGDS peptides or the anti‐αv antibody. B16‐BL6 (but not L929) cells were also able to adhere to and spread on both N‐terminal and C‐terminal OPN proteins that lack the RGD sequence, and these interactions were not inhibited by either GRGDS peptides or anti‐αv antibody. Together these results indicate that B16‐BL6 cells can adhere to OPN by interactions that are independent of either the RGD sequence or the αv integrin subunit, and suggest that some cells can interact with additional, non‐RGD binding sites in OPN.

Pre-B Cell Antigen Receptor-Mediated Signal Inhibits CD24-Induced Apoptosis in Human Pre-B Cells

We previously reported that the cross-linking of cluster of differentiation (CD)24 induces apoptosis in Burkitt’s lymphoma cells and that this phenomenon can be enhanced by a B cell Ag receptor (BCR)-mediated signal. In this study, we extend our previous observation and report that CD24 also mediated apoptosis in human precursor-B acute lymphoblastic leukemia cell lines in the pro-B and pre-B stages accompanying activation of multiple caspases. Interestingly, simultaneous cross-linking of pre-BCR clearly inhibited CD24-mediated apoptosis in pre-B cells. We also observed that mitogen-activated protein kinases (MAPKs) were involved in the regulation of this apoptotic process. Pre-BCR cross-linking induced prompt and strong activation of extracellular signal-regulated kinase 1, whereas CD24 cross-linking induced the sustained activation of p38 MAPK, following weak extracellular signal-regulated kinase 1 activation. SC68376, a specific inhibitor of p38 MAPK, inhibited apoptosis induction by CD24 cross-linking, whereas anisomycin, an activator of p38 MAPK, enhanced the apoptosis. In addition, PD98059, a specific inhibitor of MEK-1, enhanced apoptosis induction by CD24 cross-linking and reduced the antiapoptotic effects of pre-BCR cross-linking. Collectively, whether pre-B cells survive or die may be determined by the magnitude of MAPK activation, which is regulated by cell surface molecules. Our findings should be important to understanding the role of CD24-mediated cell signaling in early B cell development.