Quantitative analysis of EGFR affinity to immobilized glycolipids by surface plasmon resonance

EGF-induced activation of EGFR tyrosine kinase is known to be inhibited by ganglioside GM3, its dimer, and other mimetics. However, details of the interaction, such as kinetic properties, have not yet been clarified. The direct interaction is now defined by the surface plasmon resonance (SPR) technique. To determine the affinity of EGFR for lyso-GM3 or lyso-GM3 mimetic, these glycolipid ligands were covalently immobilized onto a sensor chip, and binding affinities were investigated. Results of these studies confirmed the direct interaction of lyso-GM3 or its mimetic with EGFR. A strong interaction between EGFR and lyso-GM3 or its mimetic was indicated by increased binding of EGFR to glycolipid-immobilized surface, in an EGFR dose-dependent manner.     

Gene cloning and enzymatic characteristics of a novel gamma-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364

A new gene, cda, was found in the downstream region of the cgt gene encoding cyclodextrin (CD) glucanotransferase from Bacillus clarkii 7364. Cda encoded by the cda was a cyclodextrinase that has extremely high specificity for gamma-CD. The rates of hydrolysis toward alpha- and beta-CD, maltooctaose and polysaccharides were less than 4% of that toward gamma-CD. Cda also has a transglycosylation activity, by which the maltotriose moiety was transferred from maltohexaose and maltopentaose. The comparison of the amino acid sequences between Cda and CD-degrading enzymes revealed the sequence of Cda has unique features. One of them is Gly247 next to the catalytic nucleophile Asp246. Most enzymes in GH family 13 have more bulky amino acids at this position. Other features in Cda are the lack of the N-domain in CD-degrading enzymes involving in the dimerization contributing to the preference of CDs and the existence of a long extra sequence in the C-terminus. Despite the lack of N-domain, Cda showed a dodecameric structure. The long extra sequence in the C-terminus might contribute to the oligomerization of Cda through a new mechanism. These unique features indicate that Cda is a novel type of CD-degrading enzyme.     

Effect of lipid mimetics of GM3 and lyso-GM3 dimer on EGF receptor tyrosine kinase and EGF-induced signal transduction

The tyrosine kinase activity associated with epidermal growth factor receptor (EGFR) has been a target in studies of pharmacological reagents to inhibit growth of cancer cells, which are mostly of epidermal origin. Lyso-GM3 dimer showed stronger inhibitory effect on the tyrosine kinase of EGFR than GM3, with minimal cytotoxicity [Y. Murozuka, et al. Lyso-GM3, its dimer, and multimer: their synthesis, and their effect on epidermal growth factor-induced receptor tyrosine kinase. Glycoconj. J. 24 (2007) 551-563]. Synthesis of lipids with sphingosine requires many steps, and the yield is low. A biocombinatory approach overcame this difficulty; however, products required a C(12) aliphatic chain, rather than the sphingosine head group [Y. Murozuka, et al. Efficient sialylation on azidododecyl lactosides by using B16 melanoma cells. Chemistry & Biodiversity 2 (2005) 1063-1078]. The present study was to clarify the effects of these lipid mimetics of GM3 and lyso-GM3 dimer on EGFR tyrosine kinase activity, and consequent changes of the A431 cell phenotype, as follows. (i) A lipid mimetic of lyso-GM3 dimer showed similar strong inhibitory effect on EGF-induced EGFR tyrosine kinase activity, and similar low cytotoxicity, as the authentic lyso-GM3 dimer. (ii) A lipid mimetic of lyso-GM3 dimer inhibited tyrosine phosphorylation of EGFR or its dimer to a level similar to that of the authentic lyso-GM3 dimer, but more strongly than GM3 or a lipid mimetic of GM3. (iii) Associated with the inhibitory effect of a lipid mimetic of lyso-GM3 dimer on EGF-induced EGFR kinase activity, only Akt kinase activity was significantly inhibited, but kinases associated with other signal transducers were not affected. (iv) The cell cycle of A431 cells, and the effects of GM3 and a lipid mimetic of lyso-GM3 dimer, were studied by flow cytometry, measuring the rate of DNA synthesis with propidium iodide. Fetal bovine serum greatly enhanced S phase and G(2)/M phase. Enhanced G(2)/M phase was selectively inhibited by pre-incubation of A431 cells with a lipid mimetic of lyso-GM3 dimer, whereas GM3 had only a minimal effect.     

Tumor autocrine motility factor is an angiogenic factor hat stimulates endothelial cell motility.

Autocrine motility factor (AMF) is a type of tumor-secreted cytokine that primarily stimulates tumor cell motility via receptor-mediated signaling pathways and is thought to be connected to tumor progression and metastasis. Using in vivo models, we showed that critical neovascularization responded to a biological amount of AMF. This angiogenic activity was fixed by specific inhibitors against AMF. AMF stimulated in vitro motility of human umbilical vein endothelial cells (HUVECs), inducing the expression of cell surface AMF receptor localizing a single predominant perinuclear pattern closely correlated with its motile ability. AMF also elicited the formation of tube-like structures mimicking angiogenesis when HUVECs were grown in three-dimensional type I collagen gels. We further immunohistochemically detected AMF receptors on the surrounding sites of newborn microvessels. These findings suggest that AMF is a possible tumor progressive angiogenic factor which may act in a paracrine manner for the endothelial cells in the clinical neoplasm, and it will be a new target for anti-angiogenic treatment.     

Comprehensive DNA microarray expression profiles of tumors in tenascin-C-knockout mice

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays a pivotal role in tumor growth. However, the mechanism whereby TNC affects tumor biology remains unclear. To investigate the exact role of TNC in primary tumor growth, a mouse mammary tumor cell line, GLMT1, was first developed. Subsequently, global gene expression in GLMT1-derived tumors was compared between wild-type (WT) and TNC-knockout (TNKO) mice. Tumors in WT mice were significantly larger than those in TNKO mice. DNA microarray analysis revealed 447 up and 667 downregulated in the tumors inoculated into TNKO mice as compared to tumors in WT mice. Validation by quantitative gene expression analysis showed that Tnc, Cxcl1, Cxcl2, and Cxcr2 were significantly upregulated in WT mice. We hypothesize that TNC stimulates the CXCL1/2-CXCR2 pathway involved in cancer cell proliferation.     

Localization of α-Galactosidase in an Alkalophilic Strain of Micrococcus†

Localization of α-galactosidase in an alkalophilic strain of Micrococcus was investigated in relation to the cell membrane as a permeability barrier. The most α-galactosidase appered to be intracellular; only about 4% of α-galactosidase was released by lysozyme or freeze-thaw treatments of the whole cells. The enzyme activity was not inhibited by treatment of the whole cells with diazo-7-amino-1,3-naphthalene disulfonic acid (NDS) which penetrated the cell wall but not the cytoplasmic membrane. The enzyme activity of the whole cells increased about four-fold by toluene-acetone treatment which caused an alteration in the membrane permeability. The enzyme in such cells became to be relatively sensitive to pH. These results showed that cell membrane played a protective role as a permeability barrier against alkaline environment.     

A Portable Lipid Bilayer System for Environmental Sensing with a Transmembrane Protein

This paper describes a portable measurement system for current signals of an ion channel that is composed of a planar lipid bilayer. A stable and reproducible lipid bilayer is formed in outdoor environments by using a droplet contact method with a micropipette. Using this system, we demonstrated that the single-channel recording of a transmembrane protein (alpha-hemolysin) was achieved in the field at a high-altitude (∼3623 m). This system would be broadly applicable for obtaining environmental measurements using membrane proteins as a highly sensitive sensor.