Transport of N-acetylchitooligosaccharides and fluorescent N-acetylchitooligosaccharide analogs into rat liver lysosomes.

Free polymannose-type oligosaccharides (fOS) are processed by cytosolic enzymes to generate Man5GlcNAc which is transferred to lysosomes and degraded. Lysosomal fOS import was demonstrated in vitro but is poorly characterized in part due to lack of convenient substrates. As chitooligosaccharides (COS, oligomers ß1,4-linked GlcNAc) block [3H]Man5GlcNAc transport into lysosomes, we asked if COS are themselves transported and if so, can they be chemically modified to generate fluorescent substrates. We show that COS are degraded by lysosomal hydrolases to generate GlcNAc, and robust ATP-dependent transport of [3H]COS2/4 di and tetrasaccharides into intact rat liver lysosomes was observed only after blocking lysosomal [3H]GlcNAc efflux with cytochalasin B. As oligosaccharides with unmodified reducing termini are the most efficient inhibitors of [3H]COS2/4 and [3H]Man5GlcNAc transport, the non-reducing GlcNAc residue of COS2-4 was de-N-acetylated using Sinorhizobium meliloti NodB, and the resulting amine substituted with rhodamine B (RB) to yield RB-COS2-4. The fluorescent compounds inhibit [3H]Man5GlcNAc transport and display temperature-sensitive, ATP-dependent transport into a sedimentable compartment that is ruptured with the lysosomotropic agent L-methyl methionine ester. Once in this compartment, RB-COS3 is converted to RB-COS2 further identifying it as the lysosomal compartment. RB-COS2/3 and [3H]Man5GlcNAc transports are blocked similarly by competing sugars, and are partially inhibited by the vacuolar ATPase inhibitor bafilomycin and high concentrations of the P-type ATPase inhibitor orthovanadate. These data show that Man5GlcNAc, COS2/4 and RB-COS2/3 are transported into lysosomes by the same or closely related mechanism and demonstrate the utility of COS modified at their non-reducing terminus to study lysosomal oligosaccharide transport.

CEBA: A new heterobifunctional reagent for plasmid DNA functionalization by click chemistry.

Here, we report the synthesis of 3,6,9-trioxaundecan-1-{4-[(2-Chloroethyl)Ethylamino)]-Benzylamino},11-Azide (CEBA). CEBA alkylates the N7 of guanine of DNA thanks its chloroethyl group and can be coupled by a strain-promoted azide-alkyne cycloaddition to an alkynylated molecule. The optimization of the alkylation level of pDNA reveals that the expression of the encoded gene is preserved when it is randomly modified with at most 1 CEBA molecule per 150 bp. We show that the azido group of CEBA allows the linkage via click chemistry of CEBA-pDNA with a fluorophore or a peptide containing a dibenzocyclooctyne (DBCO) function. This new heterobifunctional reagent opens new ways to equip pDNA easily with signal molecules including peptides and nucleic acids without side products providing great interest for non-viral gene therapy.

Comparative binding and uptake of liposomes decorated with mannose oligosaccharides by cells expressing the mannose receptor or DC-SIGN.

Mannose Receptor (MR) and DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) are two mannose-specific targets for antigens carried by liposomes but DC-SIGN is more specific of DCs. Here, DC targeting is addressed by using DPPC/DOPE liposomes decorated with a series of diether lipids with a polar head of either a mannose (Man), tri-antenna of α-d-mannopyranoside (Tri-Man), [Manα1-3(Manα1-6)Man] (Man-tri), pseudo-Man4 (PMan4) or pseudo-Man5 (PMan5). Liposomes decorated with Man-Tri show the highest binding and internalization in cells expressing DC-SIGN and in human monocytes-derived DCs. Conversely, cells expressing MR bind and take up Tri-Man liposomes 3-fold higher than Man-tri liposomes. Comparatively, liposomes decorated with PMan4 and PMan5 do not show any advantages. Overall, the results indicate that liposomes decorated with Man-tri residues are more selective toward DCs than those with Tri-Man thanks to better recognition by DC-SIGN.

Shiga Toxin Induces Lipid Compression: A Mechanism for Generating Membrane Curvature.

Biomembranes are hard to compress laterally, and membrane area compressibility has not been associated with biological processes. Using X-ray surface scattering, we observed that bacterial Shiga toxin compresses lipid packing in a gel phase monolayer upon binding to its cellular receptor, the glycolipid Gb3. This toxin-induced reorganization of lipid packing reached beyond the immediate membrane patch that the protein was bound to, and linkers separating the Gb3 carbohydrate and ceramide moieties modulated the toxin's capacity to compress the membrane. Within a natural membrane, asymmetric compression of the toxin-bound leaflet could provide a mechanism to initiate narrow membrane bending, as observed upon toxin entry into cells. Such lipid compression and long-range membrane reorganization by glycolipid-binding proteins represent novel concepts in membrane biology that have direct implications for the construction of endocytic pits in clathrin-independent endocytosis.

Mechanism of Shiga Toxin Clustering on Membranes.

The bacterial Shiga toxin interacts with its cellular receptor, the glycosphingolipid globotriaosylceramide (Gb3 or CD77), as a first step to entering target cells. Previous studies have shown that toxin molecules cluster on the plasma membrane, despite the apparent lack of direct interactions between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface. The most likely interpretation of these findings is that a membrane fluctuation-induced force generates an effective attraction between toxin molecules. Such force would be of similar strength to the electrostatic force at separations around 1 nm, remain strong at distances up to the size of toxin molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications.

Synthesis of a trimannosylated-equipped archaeal diether lipid for the development of novel glycoliposomes.

An archaeal diether lipid possessing a tri-antenna of α-D-mannopyranoside linked via an oligoethylene spacer to a (2S)-2-(phytanyloxy)-3-(hexadecyloxy)propanoic acid backbone (TriMan-Diether) was designed and synthesized. This new mannosylated lipid inserted in liposomes would show both DC-targeting and adjuvant properties thanks to the TriMan structure and the diether tail part, respectively.

Carbohydrate conformation and lipid condensation in monolayers containing glycosphingolipid Gb3: influence of acyl chain structure.

Globotriaosylceramide (Gb3), a glycosphingolipid found in the plasma membrane of animal cells, is the endocytic receptor of the bacterial Shiga toxin. Using x-ray reflectivity (XR) and grazing incidence x-ray diffraction (GIXD), lipid monolayers containing Gb3 were investigated at the air-water interface. XR probed Gb3 carbohydrate conformation normal to the interface, whereas GIXD precisely characterized Gb3's influence on acyl chain in-plane packing and area per molecule (APM). Two phospholipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), were used to study Gb3 packing in different lipid environments. Furthermore, the impact on monolayer structure of a naturally extracted Gb3 mixture was compared to synthetic Gb3 species with uniquely defined acyl chain structures. XR results showed that lipid environment and Gb3 acyl chain structure impact carbohydrate conformation with greater solvent accessibility observed for smaller phospholipid headgroups and long Gb3 acyl chains. In general, GIXD showed that Gb3 condensed phospholipid packing resulting in smaller APM than predicted by ideal mixing. Gb3's capacity to condense APM was larger for DSPC monolayers and exhibited different dependencies on acyl chain structure depending on the lipid environment. The interplay between Gb3-induced changes in lipid packing and the lipid environment's impact on carbohydrate conformation has broad implications for glycosphingolipid macromolecule recognition and ligand binding.

Nonlinear acoustic fields in acoustic metamaterial based on a cylindrical pipe with periodically arranged side holes.

Nonlinear acoustic fields in transmission-line acoustic metamaterials based on a cylindrical pipe with periodically arranged side holes are studied, in which the dispersions and characteristic parameters of the nonlinear acoustic waves are obtained with the Bloch theory, and meanwhile the distributions of the fundamental wave (FW) and second harmonic wave (SHW) in the metamaterial are simulated. Three characteristic frequency bands are defined according to the relations between the frequencies of the FW, SHW, and the low-frequency forbidden band (LFB) in the metamaterial. Especially, when the FW is in the LFB while the SHW is outside the LFB, the SHW can transmit through the metamaterial although the FW is blocked, which exhibits the possibility to extract the information from the SHW instead of the FW. In addition, experiments are carried out to measure the distributions of the acoustic pressures for the FW and SHW along the metamaterial and the experimental results are in agreement with the theory.

Synthesis, in vitro biological evaluation and molecular docking studies of benzimidamides as potential BACE1 inhibitors.

A series of 3, 5-disubstituted benzimidamides were synthesized and biologically evaluated as potential BACE1 inhibitors. Both the targeted compounds (benzimidamides) and the synthetic intermediates (benzonitriles) were tested for their BACE1 inhibitory activities in a cell-free FRET assay. All the synthesized benzimidamides were active as BACE1 inhibitors and compound 6d showed the lowest IC(50) value of 3.35 µm. Molecular docking study proposed a binding mode, which would help to the further optimization on 6d to achieve more potent, BBB penetrant BACE1 inhibitors.

The discovery of novel ß-secretase inhibitors: pharmacophore modeling, virtual screening, and docking studies.

This article describes the identification of two small molecular inhibitors for ß-secretase by integrating virtual screening with fluorescence resonance energy transfer bioassay. A ligand-based pharmacophore model was developed, and the sequential virtual screening of ZINC database was performed using the acquired pharmacophore model and molecular docking. Biological evaluation of 10 virtual hits led to the identification of two novel inhibitors with IC(50) values of 4.76 and 0.31 µm, respectively. These two moderate inhibitors could represent new potentials for the development of anti-Alzheimer's disease agents.