Ilona Laczkó

Fmoc-protected, glycosylated asparagines potentially useful as reagents in the solid-phase synthesis of N-glycopeptides

1-Amino 1-deoxy derivatives of unprotected O-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyrano se, 2-acetamido-2-deoxy-D-galactose, D-galactose, lactose, D-fucose, D-mannose, and 2-deoxy-D-arabino-hexose were prepared and acylated with N-fluorenylmethoxycarbonylaspartic acid alpha-tert-butyl ester. The anomeric configuration of the N-glycosyl bond (including that of the mannose derivative) in each of the purified compounds was shown to be beta. The probable stability of the N-glycosyl and glycosidic bonds during the conditions of solid-phase peptide synthesis was investigated by treatment of the glycosylated asparagine derivatives with different concentrations of trifluoroacetic acid. Based on their stability, we found that Fmoc-Asn(sugar)-OH derivatives are excellent candidates for automated synthesis of biologically active glycopeptides.

Controlled in situ preparation of Aβ(1–42) oligomers from the isopeptide “iso-Aβ(1–42)”, physicochemical and biological characterization

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimers disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(1-42) by O-->N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to A beta(1-42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic A beta(1-42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O-->N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

Effects of polyhydroxy compounds on the structure and activity of α-chymotrypsin

Abstract The effects of glycerol, polyethylene glycol, fructose, glucose, sorbitol, and saccharose on the conformation and catalytic activity of α-chymotrypsin were studied in 0.1 M sodium phosphate buffer and buffered aqueous 60% ethanol (pH 8.0). The enzyme activity was practically completely lost within 10 min in 60% ethanol, but in the presence of stabilizers the activity was retained. With the exception of polyethylene glycol, the stabilizing effect decreased with increase of the incubation time. The preservation of the catalytic activity was accompanied by changes in the secondary and tertiary structures of α-chymotrypsin.

Endomorphin-2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo

The underlying cause of Alzheimers disease (AD) is thought to be the β‐amyloid aggregates formed mainly by Aβ1–42 peptide. Protective pentapeptides [e.g., Leu‐Pro‐Phe‐Phe‐Asp (LPFFD)] have been shown to prevent neuronal toxicity of Aβ1–42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin‐2 (End‐2, amino acid sequence: YPFF), defends against Aβ 1–42 induced neuromodulatory effects at the cellular level. Although End‐2 does not interfere with the kinetics of Aβ fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Aβ1–42 during aggregation, as revealed by tritium‐labeled End‐2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Aβ1–42 in a dose‐dependent manner, as measured by 3‐(4,5‐dimethylthiazolyl‐2)‐2,‐5‐diphenyltetrazolium bromide (MIT) assay. In vitro and in vivo electrophysiological experiments show that End‐2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA‐evoked responseenhancing effect of Aβ1–42. Studies using [d‐Ala (2), N‐Me‐Phe (4), Gly (5)‐ol]‐enkephalin (DAMGO), a µ‐opioid receptor agonist, show that the protective effects of the tetrapeptide are not µ‐receptor modulated. The endogenous tetrapeptide End‐2 mayserve as a lead compound for the drug development in the treatment of AD.—Szegedi, V., Juhász, G., Rózsa, E., Juhász‐Vedres, G., Datki, Z., Fülöp, L., Bozsó, Z., Lakatos, A., Laczkó, I., Farkas, T., Kis, Z., Tóth, G., Soós, K., Zarándi, M., Budai, D., Toldi, J., Penke, B. Endomorphin‐2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo. FASEB J. 20, E324–E333 (2006)

Aggregation of Aβ(1–42) in the presence of short peptides: conformational studies

CD and infrared spectroscopic studies were performed on (i) the inhibitory effects of equimolar quantities of LPFFD‐OH and LPYFD‐NH2 on the time‐dependent aggregation of amyloid β‐protein (Aβ) (1–42) and (ii) the β‐sheet‐breaker effects of two‐fold molar excess of the pentapeptides on aggregated Aβ(1–42) aged 1 week. The data obtained from the time‐dependent studies demonstrated that LPFFD‐OH did not significantly influence, whereas LPYFD‐NH2 exerted some inhibitory effect on the aggregation of Aβ(1–42). When added to a solution of Aβ(1–42) aged 1 week, LPFFD‐OH accelerated, while LPYFD‐NH2 delayed, but did not prevent further fibrillogenesis. The difference in the effects of these two pentapeptides on the aggregational profile of Aβ(1–42) is probably due to the difference in their conformational preferences: LPFFD‐OH adopts a β‐turn and extended structures, while LPYFD‐NH2 adopts a prevailing β‐turn conformation. Copyright

Effects of Ca2+ on catalytic activity and conformation of trypsin and α-chymotrypsin in aqueous ethanol

The effects of calcium ions on the conformation and catalytic activity of trypsin and alpha-chymotrypsin were studied in aqueous ethanol. The activity of alpha-chymotrypsin was practically lost within 10 min in the presence of 60% ethanol while trypsin preserved about 40% of its original activity even in 85% ethanol at pH 3. The catalytic activity of alpha-chymotrypsin did not decrease in the presence of 1.2M CaCl2 and 0.6M CaCl2 with trypsin in ethanolic solvent. In the latter case an activation of enzyme was observed. The stabilizing effects of calcium ions were accompanied by an increase in the helical content in both enzymes, as followed by circular dichroism measurements.

Synthesis and Studies on Cell-Penetrating Peptides

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Conformational consequences of coupling bullous pemphigoid antigenic peptides to glutathione-S-transferase and their diagnostic significance.

Recombinant epitopic peptides BP1 and BP2 representing the Bullous pemphigoid autoantigens of BP230 and BP180 bound to the fusion partner glutathione‐S‐transferase (pGEX‐4T‐2, Pharmacia) have been previously shown to increase the efficacy of diagnosis of the disease. Using glutathione‐S‐transferase‐bound monomer peptides, the sensitivity of the immunological reaction exceeded that of the free synthetic epitopes and was further increased with the number of epitopic blocks in the multimer fusion products. This has been explained by the avidity effect of the fusion partner dimer formation and the high ligand affinity due to the tandem repetitions of epitopic sequences. However, a beneficial conformation of the bound epitopic peptides might also contribute to the above phenomenon. Circular dichroism (CD) and Fourier transform infrared (FTIR) absorption spectroscopic studies revealed the importance of glutathione‐S‐transferase to induce and stabilize ordered secondary structures of the epitopic peptides. The free monomer and multimer peptides in aqueous buffer were present as a mixture of unordered and β‐sheet conformation, while binding them to the fusion partner the proportion of ordered secondary structures increased in parallel with the number of antigenic epitopes. The most prominent changes in the conformational state of the monomers in the fusion form were the increase of α‐helical and β‐sheet and the decrease of unordered conformation, while in the case of oligomeric peptides the adoption of a helical conformation was accompanied by the decrease of β‐sheet structure. An outstanding α‐helix content (46%) was detected in the case of the trimeric BP1 in its recombinant fusion form. Copyright

A two step model aimed at delivering antisense oligonucleotides in targeted cells

To be efficient in vivo antisense oligonucleotides must reach the targeted cells and then cross the cellular membrane. We propose a two step system where the oligonucleotide is first electrostatically bound to a peptide coupled to a ligand of a cellular receptor. A complex is formed which allows the oligonucleotide to be bound to the membrane of the targeted cells. These oligonucleotides are then delivered inside the cells by the subsequent use of a transfection agent. As a reductionist model of peptide coupled to a ligand we have used a lipopeptide and characterized by a filter elution assay the stoichiometry between the peptide and the oligonucleotide in the complexes. Using HeLa cultured cells we have shown that addition of these complexes to the cells triggers the oligonucleotide binding to the cell membrane. The subsequent addition of dendrimers allows these antisense oligonucleotides to inhibit a reporter gene inside the cells.

Comparison of Photosystem-I and Photosystem-II activities of spheroplasts from normal and photobleached Anabaena cylindrica

The analysis of photochemical activities of Photosystem I and Photosystem II in spheroplasts from normal and photobleached Anabaena cylindrica showed an increase in Photosystem II activity relative to Photosystem I in photobleached cells. We suggest that the reasons for this modification in photochemical activity are, (i) a rearrangement of pigments between the two photosystems, and (ii) improved functional condition of the photosynthetic units in Photosystem II.