BBA - General Subjects (v.1790, #2)

Glycobiology in the cytosol: The bitter side of a sweet world by Yoko Funakoshi; Tadashi Suzuki (81-94).
Progress in glycobiology has undergone explosive growth over the past decade with more of the researchers now realizing the importance of glycan chains in various inter- and intracellular processes. However, there is still an area of glycobiology awaiting exploration. This is especially the case for the field of “glycobiology in the cytosol” which remains rather poorly understood. Yet evidence is accumulating to demonstrate that the glycoconjugates and their recognition molecules (i.e. lectins) are often present in this subcellular compartment.
Keywords: Cytosol; Glycosylation; Lectin; Glycosidase; Glycosyltransferase; Glycobiology;

Simultaneous recognition of nucleobase and sites of DNA damage: Effect of tethered cation on the binding affinity by Arivazhagan Rajendran; Viruthachalam Thiagarajan; Burki Rajendar; Seiichi Nishizawa; Norio Teramae (95-100).
The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH2) has been synthesized and characterized by Mass and 1H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T m) and fluorescence measurements.Thermal denaturation study of DNA duplex containing T target base revealed the ΔT m of 5.1 °C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH2 is quenched only upon adding the DNA containing T target base.The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (± 0.3) × 106 M− 1. The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex.Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.
Keywords: Single nucleotide polymorphism; Nucleobase recognition; Abasic site; Flanking base; Molecular modeling;

Binding of Silurus asotus lectin to Gb3 on Raji cells causes disappearance of membrane-bound form of HSP70 by Shigeki Sugawara; Tasuku Kawano; Takashi Omoto; Masahiro Hosono; Takeo Tatsuta; Kazuo Nitta (101-109).
Heat shock proteins (HSPs) are divided into stress-inducible and constitutive types. Generally, HSP70 (stress inducible) and HSC70 (constitutive) are representative of their types, respectively. From the results of immunocytochemical analysis, both HSP70 and HSC70 were constitutively expressed in globotriaosylceramide (Gb3)-expressing Raji cells as well as Gb3-negative K562 cells. Furthermore, the membrane-bound form of HSP70 was present on the surfaces of two cell lines as patch and cap-like structures, and was recovered in the cholesterol rich microdomains (CRM) prepared from them. On the other hand, HSP70 was partially co-localized with Gb3 on the surface of Raji cells. This result suggested that HSP70 was not associated with all of Gb3 molecules but with Gb3 specifically located in the particular environment. The effect of Silurus asotus lectin (SAL), which is one of the rhamnose-binding lectins and specifically binds to Gb3, on the disappearance of membrane-bound HSP70 was dependent on whether Gb3 was present or not. These results suggested that the disappearance of membrane-bound HSP70 was caused by SAL binding to Gb3, that the reduction of membrane-bound HSP70 might result in the decrease in cell volume observed, and that the mechanism of SAL-induced HSP70 expression may differ from that of heat shock in Raji cells.
Keywords: Heat shock protein; Membrane-bound form; Cholesterol rich microdomain; Burkitt's lymphoma cell; Rhamnose-binding lectin; Catfish (Silurus asotus); Globotriaosylceramide;

Dynamics of gene expression during bone matrix formation in osteogenic cultures derived from human embryonic stem cells in vitro by Elerin Kärner; Carl-Magnus Bäckesjö; Jessica Cedervall; Rachael V. Sugars; Lars Ährlund-Richter; Mikael Wendel (110-118).
Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using pre-osteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, β-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a pre-maturational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern.
Keywords: Osteogenesis; Differentiation; Extracellular matrix; Gene expression; Embryonic stem cell;

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis by C.B.B. Lira; K.E. Gui; A.M. Perez; R.C.V. da Silveira; L.M. Gava; C.H.I. Ramos; M.I.N. Cano (119-125).
Replication protein A (RPA) is a single-stranded DNA-binding protein that has been implicated in DNA metabolism and telomere maintenance. Subunit 1 of RPA from Leishmania amazonensis (LaRPA-1) has previously been affinity-purified on a column containing a G-rich telomeric DNA. LaRPA-1 binds and co-localizes with parasite telomeres in vivo. Here we describe the purification and characterization of native recombinant LaRPA-1 (rLaRPA-1). The protein was initially re-solubilized from inclusion bodies by using urea. After dialysis, rLaRPA-1 was soluble but contaminated with DNA, which was removed by an anion-exchange chromatography of the protein solubilized in urea. However, rLaRPA-1 precipitated after dialysis to remove urea. To investigate whether the contaminating DNA was involved in chaperoning the refolding of rLaRPA-1, salmon sperm DNA or heparin was added to the solution before dialysis. The addition of either of these substances prevented the precipitation of rLaRPA-1. The resulting rLaRPA-1 was soluble, correctly folded, and able to bind telomeric DNA. This is the first report showing the characterization of rLaRPA1 and of the importance of additives in chaperoning the refolding of this protein. The availability of rLaRPA-1 should be helpful in assessing the importance of this protein as a potential drug target.
Keywords: Replication protein A subunit 1; Leishmania amazonensis; Telomere-binding protein; Recombinant protein refolding; Heparin; Spectroscopic analysis;

Finding of a zero linking number topoisomer by You Cheng Xu (126-133).
It is generally assumed that native deoxyribonucleic acid (DNA) is a right-handed double helix. A reasonable deduction is that during replication, the two parental strands have to unwind very quickly. However, this surmised quick unwinding is problematic and has never been proven experimentally. It is hypothesized that the two strands of DNA are winding with each other ambidextrously rather than plectonemically. The successful assembling and disassembling of a zero linking number topoisomer supports this hypothesis. It was further proven by quick separation of singly nicked DNA. The new DNA model was also verified by the “figure 8” structure, which is the annealing product of two single-stranded circular DNA with a 2 kb complementary insert in opposite directions. These experimental results are hard to be explained by the traditional Watson–Crick model. The significance of this finding in the understanding of DNA replication is briefly discussed.
Keywords: pBR322; Gyrase; Left-handed DNA; DNA replication; Linking number; Figure 8 structure;

The fluorinated surfactant sodium perfluorooctanoate (SPFO) could bind onto ubiquitin (UBQ) and induce the unfolding of UBQ. By using 15N-edited heteronuclear single-quantum coherence (HSQC) NMR and 19F NMR to monitor 15N-labeled UBQ and SPFO, respectively, the binding sites and the aggregation process of SPFO on UBQ at various SPFO concentrations were observed. A detailed process from specific binding to cooperative binding of SPFO on UBQ, and a detailed structure change of UBQ upon the increase of SPFO concentration were obtained. The refolding of UBQ in UBQ–SPFO complex was carried out by adding cationic surfactant. It was shown that added cationic surfactants formed mixed micelles with SPFO and resulted in the dissociation of the UBQ–SPFO complex, and consequently, most ubiquitin could be refolded to its native state.
Keywords: Ubiquitin; Sodium perfluorooctanoate; Dodecyltrimethylammonium chloride; Unfolding; Binding sites;

Affinity of low molecular weight fucoidan for P-selectin triggers its binding to activated human platelets by Laure Bachelet; Isabelle Bertholon; Damien Lavigne; Roger Vassy; Martine Jandrot-Perrus; Frédéric Chaubet; Didier Letourneur (141-146).
Background: P-selectin is an adhesion receptor expressed on activated platelets and endothelial cells. Its natural ligand, P-selectin glycoprotein ligand-1, is expressed on leucocytes and the P-selectin/PSGL-1 interaction is involved in leukocyte rolling. We have compared the interaction of P-selectin with several low molecular weight polysaccharides: fucoidan, heparin and dextran sulfate. Methods: Binding assays were obtained from the interaction of the polysaccharides with Sialyl Lewis X and PSGL-1 based constructs onto microtiter plates coated with P-selectin. SELDI TOF mass spectrometry was performed with anionic chips arrays coated with P-selectin in the absence or in the presence of polysaccharides. Kd were obtained from surface plasmon resonance experiments with immobilized P-selectin constructs, polysaccharides being injected in the mobile phase. Human whole blood flow cytometry experiments were performed with fluorescein isothiocyanate labelled polysaccharides with or without platelets activators. Results: The fucoidan prevented P-selectin binding to Sialyl Lewis X with an IC50 of 20 nM as compared to 400 nM for heparin and < 25000 nM for dextran sulfate. It exhibited the highest affinity for immobilized P-selectin with a KD of 1.2 nM, two orders of magnitude greater than the KD of the other polysaccharides. Mass spectrometry evidenced the formation of a complex between P-selectin and fucoidan. The intensity of the fucoidan binding to platelets was dependent on the level of platelet activation. Competition between fucoidan and an anti P-selectin antibody demonstrated the specificity of the interaction. General significance: Low molecular weight fucoidan is a promising therapeutic agent of natural origin for biomedical applications.
Keywords: Fucoidan; P-selectin; Platelet; Binding; Surface plasmon resonance; Flow cytometry;

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells by Apostolos Koutsokeras; Panagiotis S. Kabouridis (147-153).
Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.
Keywords: Protein transduction domain; TAT; Cell penetrating peptide; Lipid raft; Signaling inhibitor;