A combinatorial approach to producing sterically stabilized (Stealth) immunoliposomal drugs

We have developed a method for producing sterically stabilized immunoliposomal drugs (SIL) readily applicable to a 'mix and match' combinatorial approach for the simple manufacture of a variety of ligand-targeted liposomal drugs. Ligands coupled to the terminus of polyethylene glycol (PEG) in micelles formed from PEG-lipid derivatives (mPEG2000-DSPE) could be transferred into preformed, drug-containing liposomes from the micelles in a temperature- and time-dependent manner. Antibody densities up to 100 microg antibody/micromol of phospholipid, and up to 3 mol% of mPEG2000-DSPE, could be simultaneously transferred from the ligand-coupled micelles into the liposomal outer monolayer with negligible drug leakage from liposomes during transfer and good stability in human plasma. Transfer of anti-CD19 into SIL resulted in a three-fold increase in binding of these liposomes to CD19+ human B cell lymphoma cells.     

Indispensability of transmembrane domains of Golgi UDP-galactose transporter as revealed by analysis of genetic defects in UDP-galactose transporter-deficient murine had-1 mutant cell lines and construction of deletion mutants

UDP-galactose transporter is a membrane protein localized in the Golgi apparatus. It translocates UDP-galactose from the cytosol into the Golgi lumen, thus providing galactosyltransferases with their substrate. We characterized murine UDP-galactose transporter through molecular cloning for the following purposes: (i) to elucidate the molecular bases underlying the genetic defects of murine Had-1 mutants, which are deficient in UDP-galactose transporting activity, and (ii) to obtain information that would help us in planning rational approaches to identify functionally essential regions, based on comparison of primary structures between human and murine UDP-galactose transporters. We identified five nonsense mutations, one missense Gly178Asp mutation, and two aberrant splicing mutations. Although glycine178 is highly conserved among nucleotide-sugar transporters, a Gly178Ala variant was functional. The species-differences between human and murine UDP-galactose transporters were largely confined to the N- and C-terminal regions of the transporters. Substantial deletions in the N- and C-terminal regions did not lead to loss of UDP-galactose transporting activity, indicating that these cytosolic regions are dispensable for the transporting activity. The transporter was fused with green-fluorescent protein at the C-terminal cytosolic tail without impairing the functions of either protein. Our results demonstrate the importance of the transmembrane core region of the UDP-galactose transporter protein.     

Structural basis for the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.     

Conformational change of ascidiacyclamide caused by asymmetric modification for an isoleucine residue: structural analyses of [Gly], [Leu], and [Phe]ascidiacyclamides by x-ray diffraction and NMR spectroscopy

Ascidiacyclamide, a cytotoxic cyclic peptide from tunicate, is composed of unusual amino acids and has a repeated sequence, c[-thiazole-D-Val-oxazoline-L-Ile-]2 ([Ile]ASC). The symmetric chemical structure has been assumed to be correlated with the cytotoxicity, and it is reasonable to consider that the disturbance of its structure from the C2 symmetry results in the changes of conformation and activity. In order to quantitatively estimate the molecular conformation-activity relationship, an isoleucine residue was substituted by Gly, Leu, or Phe to disturb the C2 symmetry. The conformations of three derivatives were examined by nmr spectroscopy and the crystal structure of [Leu]ASC was also analyzed by x-ray diffraction method. The 1H-nmr experiments and the constrained molecular dynamics simulations showed the twisted "figure 8" conformers for [Gly] and [Phe]ASCs and the "square" conformer for [Leu]ASC in the DMSO solution. The x-ray crystal analysis of [Leu]ASC also revealed the square form similar to the solution structure. On the other hand, their cytotoxic activities were measured using L1210 leukemia cells and were related with the bulkiness and/or hydrophobicity of the side chain of the substituted amino acid; [Phe] > or = [Ile] > [Leu] > [Gly]ASCs. As an attempt to consider the correlation between the activity and conformer, the accessible surface area (ASA) was calculated for each derivative to estimate the size or bulkiness of its conformation. Although the ASAs of nmr structures were not directly related to the type of conformer (figure 8 or square form), it was an important probe to consider the cytotoxicity of each derivative.     

Binding analysis of Xenopus laevis translation initiation factor 4E (eIF4E) in initiation complex formation.

A translation initiation factor, eIF4E, of Xenopus laevis was purified by affinity column chromatography after the gene expression as a full-length protein in a baculovirus-insect cell system. Interaction between X. laevis eIF4E and 4E-BP2 was analyzed by affinity column chromatography, gel permeation chromatography (GPC), and surface plasmon resonance (SPR). It was found that the interaction of eIF4E with an mRNA cap-analogue enhanced the binding activity of eIF4E with 4E-BP2. Furthermore, the SPR analysis showed that the eIF4E-cap-analogue interaction was very weak regardless of complex formation of 4E-BP2 with eIF4E; the dissociation constant of eIF4E for the cap-analogue was estimated to be 10(-2)-10(-4) M. These results suggest that the participation of another initiation factor is required for eIF4E to recognize the cap structure in vivo. The results reported in this paper support "the performed complex model" of Lee et al., in which eIF4E binds to the mRNA cap structure after the initiation factors have formed the initiation complex eIF4F.     

The Structural Basis for the Susceptibility of Gangliosides to Enzymatic Degradation

The conformational properties of GM2, GalNac-4(Neu5Ac-3) Gal-4Glc-1Cer have been compared to those of 6-GM2, in which the linkage between the GalNAc and Gal was altered from GalNac-4Gal- to GalNac-6Gal-, and to those of GD1a, Neu5Ac-3Gal-3GalNAc-4(Neu5Ac-3)Gal-4Glc-1Cer, and GalNAc-GD1a.Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions.The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Ac-3Gal-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two core GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.     

An F-box protein, FWD1, mediates ubiquitin-dependent proteolysis of beta-catenin

beta-catenin plays an essential role in the Wingless/Wnt signaling cascade and is a component of the cadherin cell adhesion complex. Deregulation of beta-catenin accumulation as a result of mutations in adenomatous polyposis coli (APC) tumor suppressor protein is believed to initiate colorectal neoplasia. beta-catenin levels are regulated by the ubiquitin-dependent proteolysis system and beta-catenin ubiquitination is preceded by phosphorylation of its N-terminal region by the glycogen synthase kinase-3beta (GSK-3beta)/Axin kinase complex. Here we show that FWD1 (the mouse homologue of Slimb/betaTrCP), an F-box/WD40-repeat protein, specifically formed a multi-molecular complex with beta-catenin, Axin, GSK-3beta and APC. Mutations at the signal-induced phosphorylation site of beta-catenin inhibited its association with FWD1. FWD1 facilitated ubiquitination and promoted degradation of beta-catenin, resulting in reduced cytoplasmic beta-catenin levels. In contrast, a dominant-negative mutant form of FWD1 inhibited the ubiquitination process and stabilized beta-catenin. These results suggest that the Skp1/Cullin/F-box protein FWD1 (SCFFWD1)-ubiquitin ligase complex is involved in beta-catenin ubiquitination and that FWD1 serves as an intracellular receptor for phosphorylated beta-catenin. FWD1 also links the phosphorylation machinery to the ubiquitin-proteasome pathway to ensure prompt and efficient proteolysis of beta-catenin in response to external signals. SCFFWD1 may be critical for tumor development and suppression through regulation of beta-catenin protein stability.     

Endothelin-1 activates p38 mitogen-activated protein kinase via endothelin-A receptor in rat myocardial cells

In myocardial cells (MCs), endothelin-1 (ET-1) exerts various effects such as hypertrophy, and causes cellular injury. Long-term treatment with an endothelin-A (ET_A) receptor antagonist improves the survival of rats with heart failure, suggesting that myocardial endothelin system contributes to the progression of heart failure. p38 mitogen-activated kinase (MAPK) is a member of the MAPK family and activated by several forms of environmental stresses. We show here the effect of ET-1 on p38 MAPK activation and the role of ET-1-activated p38 MAPK on morphological changes in MCs. ET-1-stimulated p38 MAPK phosphorylation was detectable within 2 min and maximal at 5 min and was concentration dependent. The maximum effect was obtained at 10 nM. An ET_A receptor antagonist, BQ-123, but not an endothelin-B receptor antagonist, BQ-788, inhibited these reactions. A p38 MAPK inhibitor, SB203580, failed to inhibit the morphological changes associated with ET-1-induced myocardial cell hypertrophy. These results indicate that p38 MAPK is activated by ET-1 but does not contribute to the development of ET-1-induced myocardial cell hypertrophy.     

Approach for functional analysis of glycan using RNA interference

The elucidation of the biological role of glycan is one of the most important issues to be resolved following the genome project. RNA interference is becoming an efficient reverse genetic tool for studying gene function in model organisms, including C.elegans and Drosophila melanogaster. Our molecular evolutionary study has shown that a prototype of glycosyltransferases, which synthesize a variety of glycan structures in the Golgi apparatus, was conserved between mammals and Drosophila. For analyses of the basic physiological functions of glycans, we established the Drosophila inducible RNAi knockdown system and applied it to one glycosyltransferase and one transporter, proteoglycan UDP-galactose: beta-xylose beta1,4galactosyltransferase I and the PAPS-transporter, respectively. If on the silencing of each gene induced ubiquitously under the control of a cytoplasmic actin promoter, the RNAi knockdown fly died, then the protein was indispensable for life. The expression of the target gene was disrupted specifically and the degree of interference was well correlated with the phenotype. The inducible RNAi knockdown fly obtained using the GAL4-UAS system will pave the way for the functional analysis of glycans.