BBA - General Subjects (v.1568, #3)
Oligonucleotide lipoplexes: the influence of oligonucleotide composition on complexation by Victor M Meidan; Judith Glezer; Ninette Amariglio; Jack S Cohen; Yechezkel Barenholz (177-182).
Despite extensive investigations into oligonucleotide lipoplexes, virtually no work has addressed whether the physicochemical properties of these assemblies vary as a function of the constituent oligonucleotide (ODN) sequence and/or composition. The present study was aimed at answering this question. To this end, we complexed N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) liposomes, in dispersion, with either 18-mer phosphorothiote homo-oligonucleotides composed of either adenine, thymidine or cytosine; or one of three structurally related 18-mer phosphorothioate oligonucleotides (S-ODNs) (G3139, its reverse sequence and its two-base mismatch). After ODN addition to vesicles at different mole ratios, changes in pH and electrical surface potential at the lipid–water interface were analyzed by using the fluorophore heptadecyl-7-hydroxycoumarin while particle size distributions were analyzed by static-light scattering. The results indicate that each homo-oligonucleotide does indeed exhibit different complexation behavior. In particular, the maximal level of DOTAP neutralization by the polyadenine S-ODN is much lower than that for the two other homo-oligonucleotides and hence its lipoplex is much more positively charged. Much smaller electrostatic differences are also apparent between lipoplexes formed from each of the G3139-related ODNs. This paper identifies nucleotide base selection and sequence as a variable that can affect the physicochemical properties of oligonucleotide lipoplexes and hence probably their transfection competency.
Keywords: Base sequence; Cationic liposome; Lipoplex; Oligonucleotide; 4-Heptadecyl-7-hydroxycoumarin; Fluorescence;
Oxidized low-density lipoprotein-binding specificity of Asp-hemolysin from Aspergillus fumigatus by Yoichi Kudo; Yuji Fukuchi; Takeshi Kumagai; Keiichi Ebina; Katsushi Yokota (183-188).
Oxidized low-density lipoprotein (Ox-LDL) is known to be involved in the generation and progression of atherosclerosis. Ox-LDL has a number of potentially atherogenic effects on vascular cells, including the uncontrolled uptake by scavenger receptors. We have previously shown that Asp-hemolysin binds to Ox-LDL in a concentration-dependent manner. The present study was undertaken to clarify the binding specificity of Asp-hemolysin to Ox-LDL. We examined the binding specificity of Asp-hemolysin to Ox-LDL using several modified lipoproteins and scavenger receptor ligands. Asp-hemolysin bound to Ox-LDL with shorter LDL oxidation times. However, Asp-hemolysin did not bind to the acetylated LDL. The native high-density lipoprotein (n-HDL) and modified HDL (e.g., acetylated HDL, oxidized HDL) also had no Asp-hemolysin binding. Furthermore, inhibitors of the scavenger receptor binding, including maleylated BSA, polyinosinic acid, dextran sulfate and fucoidin, had no effect on the binding of Ox-LDL to Asp-hemolysin. Surface plasmon resonance studies revealed that Ox-LDL binds with high affinity (K D=0.63 μg/ml) to Asp-hemolysin. We concluded that Asp-hemolysin is a specific binding protein with a high affinity for Ox-LDL, and its binding specificity is distinct from any receptor for Ox-LDL. The present studies suggest that Asp-hemolysin may bind to Ox-LDL using a mechanism different from the scavenger receptors.
Keywords: Asp-hemolysin; Oxidized low density lipoprotein; Binding specificity; Scavenger receptor ligand; Surface plasmon resonance; Kinetic analysis;
Pyridoxal 5′-phosphate inhibits DNA binding of HNF1 by T Oka; H Sugitatsu; H Nordin; M.K Thakur; M Aoyama; T Sasagawa; I Suzuki; H Tsuji (189-196).
An efficient Escherichia coli expression system was constructed for the production of a variant form of HNF1 protein having the additional five amino acid residues (Asp-Arg-Trp-Gly-Ser) at the NH2-terminal. The cDNA encoding HNF1 was ligated to 6×His tag and inserted into an inducible bacterial expression vector pRSET A. After expression in E. coli, the recombinant product was purified by Ni-NTA affinity column chromatography. The purified product showed expected NH2-terminal sequence and specific binding to the HNF1 site. The effect of pyridoxal 5′-phosphate and its analogues on the binding activity of recombinant HNF1 was examined and found that only pyridoxal 5′-phosphate effectively inhibited the DNA binding. The concentration of pyridoxal 5′-phosphate that inhibited 50% of DNA binding was around 100 μM. Furthermore, we identified Lys197 of HNF1 molecule as the essential residue of DNA binding. These observations suggest that pyridoxal 5′-phosphate directly interacts with tissue-specific transcription factor HNF1 and modulates the binding to DNA.
Keywords: Pyridoxal 5′-phosphate; Recombinant HNF1; E.coli; DNA;
The binding of lactoferrin to glycosaminoglycans on enterocyte-like HT29-18-C1 cells is mediated through basic residues located in the N-terminus by Ikram El Yazidi-Belkoura; Dominique Legrand; Jan Nuijens; Marie-Christine Slomianny; Patrick van Berkel; Geneviève Spik (197-204).
Although lactoferrins (Lfs) isolated from milk of various mammals exhibit a close structural relationship, they show species-specific binding to cells. To define the specificity of recognition of human (hLf), bovine (bLf) and murine (mLf) lactoferrin by human intestinal cells, we analysed the binding of the three proteins to a subclone derived from human carcinoma cell line HT29. We observed that hLf and bLf interact with two types of binding sites (K d: 63±22 nM; 0.7±0.2 μM) while mLf was recognized only by the lowest affinity binding sites with a lower number of binding sites. Using N-terminal deleted human Lf variants, we found that the sequence G1RRRR5 is mainly responsible for the interactions with HT29 cells. Lactoferrin-binding sites on the surface of HT29 cells were further identified as heparan sulphate and chondroitin sulphate glycosaminoglycans. We conclude that the presence of the sequence A1PRK4 in bLf and K1ATT4 in mLf provides an insight into why the interaction of bLf with cell membrane-associated glycosaminoglycans is similar to that of hLf and why binding of these lactoferrin species differs from that of murine Lf.
Keywords: Lactoferrin; Proteoglycan; Glycosaminoglycan; Heparan sulfate; Enterocyte; HT29-18-C1 cell line;
Exploring computational lead optimisation with affinity constants obtained by surface plasmon resonance for the interaction of PorA epitope peptides with antibody against Neisseria meningitidis by Marcel J.E. Fischer; Christiaan Kuipers; Rolf P. Hofkes; Lovina J.F. Hofmeyer; Ed E. Moret; Nico J. de Mol (205-215).
LUDI is a program used for de novo structure-based design of ligands and can predict binding of ligands quantitatively using a scoring function. Here we evaluate LUDI in a lead optimisation study with ligands for the antibody MN12H2, that has been raised against outer membrane protein PorA epitope P1.16 of Neisseria meningitidis. The ligands were synthetic peptides that are derived from the smallest binding epitope 182DTNNN186. LUDI’s fragment building rules are used for the proposal of new peptide-ligands for MN12H2 and were focused on replacements of Asp186 in the epitope. Accordingly, a series of peptides was synthesised with isosteric mutations. The interaction of the peptides with MN12H2 was analysed with a surface plasmon resonance competition assay yielding equilibrium binding constants in solution (K S). The binding affinity seems to be largely determined by entropy, and the side chain of Asn186 is sensitive for charge, inversion, hydrophobicity and size. Head-to-tail cyclisation of the peptide in a nine-amino-acid ring gives little reduction in affinity. It is concluded that the scoring function of LUDI does not help in optimisation of the peptide lead for MN12H2 binding. Other more elaborate molecular mechanics calculations show similar results. This implies that our current knowledge of molecular recognition is insufficient for explaining a case of peptide-protein binding, where the design process requires subtle changes in structure (from lead finding to lead optimisation).
Keywords: MN12H2; SPR; LUDI; Binding; De novo design; Neisseria meningitidis;
Proteolytic cleavage of inducible nitric oxide synthase (iNOS) by calpain I by Gaby Walker; Josef Pfeilschifter; Uwe Otten; Dieter Kunz (216-224).
Proteolytic degradation of inducible nitric oxide synthase (iNOS or NOS2; EC 188.8.131.52) is one of the key steps by which the synthetic glucocorticoid dexamethasone controls the amount of iNOS protein and thus the production of nitric oxide (NO) in interferon-γ-stimulated RAW 264.7 cells. In the present study we examined the role of the calmodulin (CaM)-binding site present within iNOS protein for the proteolytic degradation by the calcium-dependent neutral cysteine protease calpain I (EC 184.108.40.206). Using pulse chase experiments as well as cell-free degradation assays we show that the iNOS monomer is a direct substrate for cleavage by calpain I. Two structural determinants are involved in proteolytic cleavage, the canonical CaM-binding domain present at amino acids 501–532 and a conformational determinant located within iNOS. The access of the CaM-binding region appears to be critical for substrate cleavage as incubation of in vitro synthesized iNOS with purified CaM inhibits iNOS degradation by calpain I. Moreover, cytosolic CaM levels are decreased upon treatment of RAW 264.7 cells with dexamethasone as assessed by immunoprecipitation. The data shown herein provide novel insights into the underlying mechanisms involved in the anti-inflammatory actions of glucocorticoids.
Keywords: Calmodulin-binding site; Calpain I; Dexamethasone; Inducible nitric oxide synthase; Macrophage cell line RAW 264.7;
NMR investigations of protein–carbohydrate interactions: insights into the topology of the bound conformation of a lactose isomer and β-galactosyl xyloses to mistletoe lectin and galectin-1 by José Manuel Alonso-Plaza; Marı́a Angeles Canales; Marta Jiménez; José Luis Roldán; Alicia Garcı́a-Herrero; Laura Iturrino; Juan Luis Asensio; Francisco Javier Cañada; Antonio Romero; Hans-Christian Siebert; Sabine André; Dolores Solı́s; Hans-Joachim Gabius; Jesús Jiménez-Barbero (225-236).
A hallmark of oligosaccharides is their often limited spatial flexibility, allowing them to access a distinct set of conformers in solution. Viewing each individual or even the complete ensemble of conformations as potential binding partner(s) for lectins in protein–carbohydrate interactions, it is pertinent to address the question on the characteristics of bound state conformation(s) in solution. Also, it is possible that entering the lectin’s binding site distorts the low-energy topology of a glycosidic linkage. As a step to delineate the strategy of ligand selection for galactosides, a common physiological docking point, we have performed a NMR study on two non-homologous lectins showing identical monosaccharide specificity. Thus, the conformation of lactose analogues bound to bovine heart galectin-1 and to mistletoe lectin in solution has been determined by transferred nuclear Overhauser effect measurements. It is demonstrated that the lectins select the syn conformation of lactose and various structural analogues (Galβ(1→4)Xyl, Galβ(1→3)Xyl, Galβ(1→2)Xyl, and Galβ(1→3)Glc) from the ensemble of presented conformations. No evidence for conformational distortion was obtained. Docking of the analogues to the modeled binding sites furnishes explanations, in structural terms, for exclusive recognition of the syn conformer despite the non-homologous design of the binding sites.
Keywords: Lectins; Molecular recognition; Transferred-NOESY; Modeling; Carbohydrates;
Regulation of glutamine synthetase by metal-catalyzed oxidative modification in the marine oxyphotobacterium Prochlorococcus by Guadalupe Gómez-Baena; Jesús Diez; José Manuel Garcı́a-Fernández; Sabah El Alaoui; Lourdes Humanes (237-244).
The inactivation of glutamine synthetase (GS; EC 220.127.116.11) by metal-catalyzed oxidation (MCO) systems was studied in several Prochlorococcus strains, including the axenic PCC 9511. GS was inactivated in the presence of various oxidative systems, either enzymatic (as NAD(P)H+NAD(P)H-oxidase+Fe3++O2) or non-enzymatic (as ascorbate+Fe3++O2). This process required the presence of oxygen and a metal cation, and is prevented under anaerobic conditions. Catalase and peroxidase, but not superoxide dismutase, effectively protected the enzyme against inactivation, suggesting that hydrogen peroxide mediates this mechanism, although it is not directly responsible for the reaction. Addition of azide (an inhibitor of both catalase and peroxidase) to the MCO systems enhanced the inactivation. Different thiols induced the inactivation of the enzyme, even in the absence of added metals. However, this inactivation could not be reverted by addition of strong oxidants, as hydrogen peroxide or oxidized glutathione. After studying the effect of addition of the physiological substrates and products of GS on the inactivation mechanism, we could detect a protective effect in the case of inorganic phosphate and glutamine. Immunochemical determinations showed that the concentration of GS protein significantly decreased by effect of the MCO systems, indicating that inactivation precedes the degradation of the enzyme.
Keywords: Metal-catalyzed oxidation; Glutamine synthetase; Cyanobacterium; Regulation; Prochlorococcus;
Reoxygenation after cold hypoxic storage of cultured precision-cut rat liver slices: effects on cellular metabolism and drug biotransformation by Elleni Rekka; Ekaterina Evdokimova; Stéphane Eeckhaudt; Pedro Buc Calderon (245-251).
Cultured rat precision-cut liver slices (PCLS) were used to study the influence of hypothermic preservation and reoxygenation at 37°C on cellular metabolism and drug biotransformation. Cold hypoxic storage caused a depressed metabolism in rat liver slices, but reoxygenation for 8 h at 37°C partially restored the levels of both ATP and GSH and totally restored the capacity to synthesize proteins. Metabolism of midazolam (CYP3A-dependent oxidation) by cold preserved liver slices was decreased by 30% but no further affected by reoxygenation, showing the same profile as freshly cut slices. Such a reoxygenation at 37°C is accompanied by a dramatic loss of CYP3A2 protein while CYP3A1 protein was unaffected. These results suggest that CYP3A2 did not play a major role in midazolam oxidation. Such results are not consistent with a putative reoxygenation injury but rather with cold hypoxic damage. Since cold preserved liver slices did not respond to bacterial endotoxin stimulation (lipopolysaccharides), a minor role of non-parenchymal cells is suggested as mediators for deleterious effects developed during the cold storage.
Keywords: Liver slice; Midazolam metabolism; Hypoxia-reoxygenation; CYP3A;
Nitric oxide binding to oxygenated hemoglobin under physiological conditions by Zhi Huang; Joseph G. Louderback; Mansi Goyal; Fouad Azizi; S.Bruce King; Daniel B. Kim-Shapiro (252-260).
We have added nitric oxide (NO) to hemoglobin in 0.1 M and 0.01 M phosphate buffers as well as to whole blood, all as a function of hemoglobin oxygen saturation. We found that in all these conditions, the amount of nitrosyl hemoglobin (HbNO) formed follows a model where the rates of HbNO formation and methemoglobin (metHb) formation (via hemoglobin oxidation) are independent of oxygen saturation. These results contradict those of an earlier report where, at least in 0.01 M phosphate, an elevated amount of HbNO was formed at high oxygen saturations. A radical rethink of the reaction of oxyhemoglobin with NO under physiological conditions was called for based on this previous proposition that the primary product is HbNO rather than metHb and nitrate. Our results indicate that no such radical rethink is called for.
Keywords: Hemoglobin; Nitric oxide; EPR spectroscopy; Absorption; Allostery;
Author Index (261-263).
Cumulative Contents (264-265).
Information for Contributors (267-272).