Generation and Characterization of Human Interferon-beta Neutralizing Humanized Antibody

Russian Journal of Bioorganic Chemistry - Humanization of antibodies for the development of novel therapeutic agents with low immunogenicity remains a topical problem in modern science. In the...     

Development of a Humanized Antibody 5D3Hu against the PRAME Tumor Antigen

Russian Journal of Bioorganic Chemistry - The PRAME antigen, which is a significant target for monoclonal antibodies, is a tumor-specific marker that is active at all stages of tumor cell...     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Molecular modeling of interactions of agitoxin 2 with Kv1.3 voltage-gated potassium channel

The Kv1.3 voltage-gated potassium channel is involved in a number of processes in excitable and nonexcitable cells: maintenance of resting membrane potential, signal transduction, apoptosis, regulation of cell volume, activation and proliferation of white blood cells. Blocking this channel is an effective approach for the treatment of autoimmune, oncological, chronic inflammatory, and metabolic diseases. The most prospective blockers of Kv1.3 are toxins isolated from the venom of scorpions. Knowledge of the molecular aspects of binding of peptide blockers with the channel is an important condition for the creation of highly effective and selective ligands. In the present work, a complex of hybrid channel KcsA-Kv1.3 with agitoxin 2 was built using homology modeling and molecular dynamics simulation. Analysis of formed contacts allowed us to reveal a complete pattern of interactions and to identify key residues that are responsible for the toxin binding affinity. Results of computational experiment are consistent with the experimental data and important for drug development.     

Construction of TNF-binding proteins by grafting hypervariable regions of F10 antibody on human fibronectin domain scaffold

Tumor necrosis factor (TNF) plays a key role in the pathogenesis of various diseases. To study the possibility of constructing TNF-binding proteins by grafting hypervariable regions of immunoglobulins (CDR), we have replaced amino acid sequences of loops from the tenth type III domain of human fibronectin (¹⁰Fn3) by amino acid sequences of CDR from the light and heavy chains of the anti-TNF antibody F10. The assessment of TNF-binding properties of the resulting proteins by ELISA has revealed the highest activity of Hd3 containing sequences CDR-H1 and CDR-H2 of the antibody F10 and of Hd2 containing sequences CDR-H1 and CDR-H3. The proteins constructed by us on the fibronectin domain scaffold specifically bound TNF during Western blotting and also weakened its cytotoxic effect on L929 line cells. The highest neutralizing activity was demonstrated by the proteins Hd2 and Hd3, which induced, respectively, 10- and 50-fold increase in the EC₅₀ of TNF.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Molecular modeling of the tetramerization domain of human potassium channel Kv10.2 in different oligomeric states

A voltage-gated potassium channel Kv10.2 is expressed in the nervous system, but its functions and involvement in the development of human disease remain poorly understood. Mutant forms of the Kv10.2 channel were found in patients with epileptic encephalopathy and autism. Molecular modeling of the channel spatial structure is an important tool for gaining knowledge about the molecular aspects of the channel functioning and mechanisms responsible for pathogenesis. In the present work, molecular modeling of the helical fragment of the human Kv10.2 (hEAG2) C-terminal domain in dimeric, trimeric, and tetrameric forms was performed. The stability of all forms was confirmed by molecular dynamics simulation. Contacts and interactions, stabilizing the structure, were identified.