Nagako Kawashima

Tyrosine Kinase Activity of Epidermal Growth Factor Receptor Is Regulated by GM3 Binding through Carbohydrate to Carbohydrate Interactions

Epidermal growth factor receptor (EGFR), an N-glycosylated transmembrane protein with an intracellular kinase domain, undergoes dimerization by ligand binding resulting in activation of the kinase domain and phosphorylation. Ganglioside GM3 containing sialyllactose inhibits the tyrosine kinase activity of EGFR through carbohydrate to carbohydrate interactions (CCI) between N-glycans with GlcNAc termini on EGFR and oligosaccharides on GM3. In this study, we provide further evidence for CCI between EGFR and GM3. (i) In vitro and in situ, the inhibitory effect of GM3 on EGFR tyrosine kinase was much higher in A431 cells upon exposure of the GlcNAc termini of the N-glycans to glycosidase treatment (neuraminidase and β-galactosidase) than in untreated A431 cells. Furthermore, the GM3-mediated inhibition was abrogated by co-incubation with N-glycan containing terminal GlcNAc. (ii) In situ, inhibition of EGFR phosphorylation by GM3 was not observed in α-mannosidase IB (ManIB)-knocked down A431 cells that accumulate high mannose-type N-glycans. (iii) EGFR binding to GM3 was enhanced in glycosidase-treated cells that accumulated GlcNAc termini, whereas GM3 did not bind to EGFR from ManIB-knocked down cells that accumulated high mannose-type N-glycans. These results indicate that GM3-mediated inhibition of EGFR phosphorylation is caused by interaction of GM3 with GlcNAc-terminated N-glycan on EGFR.

Clathrin-Mediated Endocytosis of Quantum Dot−Peptide Conjugates in Living Cells

Efficient intracellular delivery of quantum dots (QDs) and unravelling the mechanism underlying the intracellular delivery are essential for advancing the applications of QDs toward in vivo imaging and therapeutic interventions. Here, we show that clathrin-mediated endocytosis is the most important pathway for the intracellular delivery of peptide-conjugated QDs. We selected an insect neuropeptide, namely, allatostatin (AST1, APSGAQRLYG FGL-NH(2)), conjugated it with CdSe-ZnS QDs, and investigated the intracellular delivery of the conjugate in living cells such as human epidermoid ovarian carcinoma cells (A431) and mouse embryonic fibroblast cells (3T3). We selected AST1 to investigate the intracellular delivery of QDs because we recently found it to be efficient for delivering QDs in living mammalian cells. Also, the receptors of AST1 in insects show functional and sequence similarity to G-protein-coupled galanin receptors in mammals. We employed flow cytometry and fluorescence microscopy and investigated the contributions of clathrin-mediated endocytosis, receptor-mediated endocytosis, and charge-based cell penetration or transduction to the intracellular delivery of QD-AST1 conjugates. Interestingly, the intracellular delivery was suppressed by approximately 57% when we inhibited the regulatory enzyme phosphoinositide 3-kinase (PI3K) with wortmannin and blocked the formation of clathrin-coated vesicles. In parallel, we investigated clathrin-mediated endocytosis by colocalizing QD560-labeled clathrin heavy-chain antibody and QD605-AST1. We also estimated galanin receptor-mediated endocytosis of QD-AST1 at <10% by blocking the cells with a galanin antagonist and transduction at <30% by both removing the charge of the peptide due to arginine and suppressing the cell-surface charge due to glycosaminoglycan. In short, the current work shows that multiple pathways are involved in the intracellular delivery of peptide-conjugated QDs, among which clathrin-mediated endocytosis is the most important.

Selective detection of HbA1c using surface enhanced resonance Raman spectroscopy.

In the current work, we report on selective detection of HbA1c, a marker for glycemic control in diabetic patients, using surface enhanced resonance raman spectroscopy (SERRS). We found a characteristic band around 770-830 cm(-1) in the SERRS spectrum of HbA1c which was not present in the SERRS spectrum of HbA. To examine the contribution of glucosyl moiety to the characteristic SERRS band of HbA1c, we investigated SERRS spectra for nonenzymatically glycosylated HbA. We found that the SERRS spectral features are essentially identical for both HbA1c and nonenzymatically glycosylated HbA. Furthermore, addition of HbA into colloidal solution of silver nanoparticles (Ag NPs) resulted in the formation of large aggregates of Ag NPs and subsequent sedimentation. On the other hand, aggregation of Ag NPs was considerably low in the case of HbA1c. The differential effect of HbA and HbA1c on colloidal solution of Ag NPs, probably due to their difference in hydrophilicity, enabled us to separate them in a mixture. The separation was characterized by electrophoresis and SERRS analysis. Thus, colloidal solution of Ag NPs and SERRS would be a promising tool for the selective detection of HbA1c.

Reversible dimerization of EGFR revealed by single-molecule fluorescence imaging using quantum dots.

The current work explores intermolecular interactions involved in the lateral propagation of cell-signaling by epidermal growth factor receptors (EGFRs). Activation of EGFRs by binding an EGF ligand in the extracellular domain of the EGFR and subsequent dimerization of the EGFR initiates cell-signaling. We investigated interactions between EGFRs in living cells by using single-molecule microscopy, Förster resonance energy transfer (FRET), and atomic force microscopy. By analyzing time-correlated intensity and propagation trajectories of quantum dot (QD)-labeled EGFR single molecules, we found that signaling dimers of EGFR [(EGF-EGFR)(2)] are continuously formed in cell membrane through reversible association of heterodimers [EGF(EGFR)(2)]. Also, we found that the lateral propagation of EGFR activation takes place through transient association of a heterodimer with predimers [(EGFR)(2)]. We varified the transient association between activated EGFR and predimers using FRET from QD-labeled heterodimers to Cy5-labeled predimers and correlated topography and fluorescence imaging. Without extended single-molecule fluorescence imaging and by using bio-conjugated QDs, reversible receptor dimerization in the lateral activation of EGFR remained obscured.

Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis.

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the β‐subunit of N‐acetyl‐β‐hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N‐acetyl‐β‐hexosaminidase β‐subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild‐type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal‐regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N‐acetyl‐β‐hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up‐regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease.

Abnormal production of macrophage inflammatory protein‐1α by microglial cell lines derived from neonatal brains of Sandhoff disease model mice

Sandhoff disease (SD) is a lysosomal β‐hexosaminidase deficiency involving excessive accumulation of undegraded substrates, including terminal N‐acetylglucosamine‐oligosaccharides and GM2 ganglioside, and progressive neurodegeneration. Our previous study demonstrated remarkable induction of macrophage inflammatory factor‐1α (MIP‐1α) in microglia in the brains of SD model mice as a putative pathogenic factor for SD via microglia‐mediated neuroinflammation. In this study, we established microglial cell lines (WT‐ and SD‐Mg) from wild‐type and SD mice, and first demonstrated the enhanced production of MIP‐1α in SD‐Mg. Inhibitors of protein kinase C (PKC) and Akt reduced the production of MIP‐1α by SD‐Mg. Elevated activation of Akt and partial translocation of PKC isozymes (α, βI, βII, and δ) from the cytoplasm to the membrane in SD‐Mg were also revealed by means of immunoblotting. Furthermore, it was demonstrated that intracellular extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase, and phospholipase C (PLC), but not phosphoinositide 3‐kinase, should contribute to the induction of MIP‐1α in SD‐Mg, and that PLC could independently regulate the activation of both PKC and Akt. We proposed here that the deregulated activation of PLC should cause the enhanced MIP‐1α production via plural signaling pathways mediated by PKC and Akt, followed by extracellular signal‐regulated kinase and c‐Jun N‐terminal kinase, in SD‐Mg.

Induction of Glycosphingolipid GM3 Expression by Valproic Acid Suppresses Cancer Cell Growth

Glycosphingolipid GM3, a known suppressor of epidermal growth factor receptor (EGFR) phosphorylation, inhibits cell proliferation. Valproic acid, conversely, is known as an up-regulator of GM3 synthase gene (ST3GAL5). To test the possibility that valproic acid could inhibit EGFR phosphorylation by increasing the level of GM3 in cells, we treated A431 epidermoid carcinoma cells with valproic acid and found that valproic acid treatment caused an about 6-fold increase in the GM3 level but only a marginal increase in the GM2 level in these cells and that the observed increase in GM3 level was valproic acid dose-dependent. Consistent with this observation, valproic acid treatment induced GM3 synthase gene expression by about 8-fold. Furthermore, phosphorylation of EGFR was reduced, and cell proliferation was inhibited following valproic acid treatment. Consistent with these results, transient expression of GM3 synthase gene in A431 cells also increased cellular level of GM3, reduced phosphorylation of EGFR, and inhibited cell proliferation. Treatment with l-phenyl-2-decanoylamino-3-morpholino-l-propanol, an inhibitor of glucosylceramide synthesis, decreased the cellular level of GM3 and reduced the inhibitory effects of valproic acid on EGFR phosphorylation and cell proliferation. These results suggested that induction of GM3 synthesis was enough to inhibit proliferation of cancer cells by suppressing EGFR activity. Valproic acid treatment similarly increased the GM3 level and reduced phosphorylation of EGFR in U87MG glioma cells and inhibited their proliferation. These results suggested that up-regulators of GM3 synthase gene, such as valproic acid, are potential suppressors of cancer cell proliferation.