Arnold Trzeciak

New coupling reagents in peptide chemistry

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) has been applied as coupling reagent to solid phase peptide synthesis. Furthermore, a general synthetic procedure for new derivatives of different N-hydroxy compounds has been developed. They either act as excellent activating reagents causing low racemization during condensation of peptide segments or are useful tools for the formation of active esters suitable for couplings in mixed aqueous / organic media, respectively.

Identification and immunohistochemical mapping of GABAA receptor subtypes containing the δ-subunit in rat brain

Synaptic inhibition in brain is mainly mediated via GABAA receptors which display a striking structural heterogeneity. A novel type of GABAA receptor subunit, the δ‐subunit, has recently been described based on molecular cloning of its cDNA [1]. To identify the prevalence and distrubution of GABAA receptors which contain the δ‐subunit protein in situ, polyclonal site‐directed antisera were developed against three synthetic peptides derived form the rat δ‐subunit cDNA‐sequence. All antisera specifically recognized a 54 kDa protein in GABAA receptor preparation. Nearly 30% of the GABAA receptors contained the δ‐subunit immunoreactivity and displayed high affinity GABA and high affinity benzodiazepine binding sites as shown by immunoprecipitation. Receptors which contain the δ‐subunit were immunohistochemically shown to be restricted to a few brain areas such as the cerebellum, thalamus and dentate gyrus of the hippocampal formation. Thus, those neurons which express GABAA receptors with a δ‐subunit have now been visualized and made accessible for a functional analysis of this GABAA receptors subtype in situ.

Synthesis of ‘head-to-tail’ cyclized peptides on solid support by Fmoc chemistry

Abstract Two cyclic peptides were synthesized directly on solid support by ‘head-to-tail’ cyclizations. Key features are side chain attachment of an Asp residue to an amide or a hydroxy linker and orthogonal protection of the α-carboxyl function of this amino acid as allyl ester. Cyclization was performed with TBTU as coupling reagent. Depending on the attachment the cyclic peptides contain either an Asp or an Asn residue. The method is also applicable to Glx-containing cyclic peptides.

GABAA-Receptors: Drug Binding Profile and Distribution of Receptors Containing the α2-Subunit in Situ

The highest structural diversity of GABAA-receptor subunits is observed among members of the alpha-subunit class. Using subunit-specific antisera, the receptors containing the alpha 2-subunit were characterized. Western blots revealed an apparent molecular size of 52 kDa for the alpha 2-subunit. Immunohistochemically, the alpha 2-subunit was most preponderant in areas which lack the alpha 1-subunit, e.g. striatum and olfactory bulb granule cell layer, suggesting that these two subunits represent largely distinct receptor subtypes. Pharmacologically, the receptor population which was immunoprecipitated by the alpha 2-subunit-specific antisera displayed a drug binding profile characterized by a low affinity for CL 218872, beta CCM and zolpidem. This is in striking contrast to the high affinities of these ligands displayed by receptors immunoprecipitated by the alpha 1-subunit-specific antiserum. Thus, the alpha 1- and the alpha 2-subunit characterize two GABAA-receptor populations which greatly differ in brain distribution and pharmacological profile.

Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase

The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.

Identification of a domain of ETA receptor required for ligand binding

Various chimeric ETA and ETB receptors were produced in CHO cells for the elucidation of a specific domain which influences the affinity of the receptor toward BQ‐123, a selective ETA antagonist. Replacement of the first extracellular loop domain (B‐loop) of the ETA receptor with the corresponding domain of the ETB receptor, reduced the inhibition by BQ‐123 drastically, while the replacements of other extracellular domains of ETA did not. By contrast, the introduction of the B‐loop of ETA in place of the corresponding domain of the ETB receptor endowed the ETB‐based chimeric receptor with a sensitivity to BQ‐123. These observations suggest that the B‐loop domain of the ETA receptor is involved in ligand binding.

Functional Domains of Human Endothelin Receptor

The ligand binding site to the ETA receptor was investigated by substitution of each 5-amino acid sequence located in the second extracellular (B) region of the ETA receptor with the cognate sequences of the beta 2-adrenergic receptor. A 5-amino acid sequence (140-KLLAG-144) in the B-loop region was implicated as the most important element required for ligand binding. In addition, both the third and the fourth extracellular regions (C- and D-loops), including the flanking transmembrane regions, were found to play an important role in ligand selection. As for the biological significance of the intracellular regions of the ETA receptor, we have found that the C-terminal 8-amino acid residues located in close proximity to the seventh transmembrane region and the C-terminal 16-amino acid residues in the third intracellular loop are important for the binding of ET-1. Therefore, the intracellular third loop and C-terminal domains seem to contribute to the three-dimensional structure of the ligand binding site located in the extracellular domains. The same lines of experiment showed that the ETA receptor requires > 13 amino acid residues at the proximal cytoplasmic tail and 10 amino acid residues in the C-terminal region of the third intracellular loop to induce an ET-1-dependent increase in [Ca2+]i. Both regions are possibly involved in the interaction with G-protein.

N-Acyl-(α,γ Diaminobutyric Acid)n Hydrazide as an Efficient Gene Transfer Vector in Mammalian Cells in Culture

AbstractPurpose. This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells. Methods. Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring β-galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling. Results. Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a β-galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why α, γ-diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding. Conclusions. Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems.

Transient Transfection in Mammalian Cells

The aim of the presented work was to develop a cost-effective and easily scaleable transient transfection system with mammalian cells grown in serum-free suspension culture. For this purpose the cationic polyethylenimine (PEI) and the novel hybrid molecule dioleoyl-melittin (DOM) were used. Both substances are highly efficient transfection reagents for mammalian cells and have been described recently. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were performed in fresh medium (1/10 culture volume) and then added to the cells (indirect method).

Synergistic Enhancement of Transient Expression by Dioleoyl-Melittin (DOM) and Polyethylenimine (PEI) in Mammalian Cells in Suspensionculture

Dioleoyl-melittin, which is a conjugate of dioleoyl-phosphatidyl-ethanolamine-N-3-(2-pyridyldithio) propionate with the amphipatic peptide melittin Gly-Cys 1 represents a new class of peptide-based reagent for efficient transfection of mammalian cells. In this work we investigated the transfection efficiency of dioleoyl-melittin (DOM) combined with polyethylenimine (PEI) using HEK293(EBNA) cells grown in serum-free suspension cultures. Gene expression was monitored using the luciferase reporter gene and the human soluble TNF receptor p55 gene (TNFRp55) inserted into different vectors. Our data clearly show that DOM together with PEI exhibited synergistic enhancement for gene expression in EBNA cells. At the optimal DNA-DOM-PEI weight ratio the efficiency of transfection increases significantly compared to corresponding PEI and DOM transfection at low DNA concentration. In summary, our data show that dioleoyl-melittin and polyethylenimine act synergistically in transfecting 293(EBNA) cells grown in serum-free suspension culture.