Biomedical peptides, proteins & nucleic acids : structure, synthesis & biological activity最新文献

Dianthin 30, a ribosome inactivating protein (RIP) from Dianthus caryophyllus, has been expressed in Escherichia coli. Heterologous expression of a deletion mutant dianthin 30 delta 255-270 resulted in the production of a protein identical to carnation mature dianthin 30, including the absence at the carboxy-terminal of a putative 16 amino acid long pro-signal peptide. The production of a form of dianthin 30, which includes the pro-signal, is described as well. Both dianthin 30 delta 255-270 and dianthin 30 expressed in E. coli are mainly localized (90%) in the soluble fraction. Dianthin 30 delta 255-270 and dianthin 30 have been purified to homogeneity and were shown to inhibit protein synthesis in vitro with an IC50 of 8 and of 11 ng/ml, respectively. Secondary structure analysis, carried out by circular dichroism spectroscopy, indicated that the naturally occurring and the recombinant forms of dianthin 30 and dianthin 30 delta 255-270 possess the same secondary structure composition, accounting for an alpha + beta type architecture. RIPs as immunotoxins in clinical trial and as mitotoxins in experimental models have been extremely efficacious. In addition, growing evidence indicates their effective use as antiviral agents, including in HIV-1 infection. These data indicate the ability to produce either chemically linked or recombinant fusion proteins with dianthin 30 and cell-binding ligands for production of new reagents for clinical and experimental use.

Activating mutations of the p21 ras proto-oncogene are involved in the development of many common malignancies. Because activating mutations are limited in number, occur within otherwise completely conserved regions, and can be expressed by premalignant lesions, ras is an attractive target for subunit peptide vaccine approaches. Several studies in transplantable tumor models support the possibility that protection against tumors bearing activated ras can be achieved using peptide-based immunogens. We have identified an autochthonous tumor model, A/J mouse lung, which parallels human tumors in the progression of proliferative lesions from premalignant to malignant and which is a very sensitive in vivo system for the detection of activated ras. Although T-cells recognizing a number of activating substitutions can be elicited in this model, peptide immunogens corresponding to the most commonly observed activating mutations are weakly immunogenic. We have engineered a chimeric immunogen incorporating a promiscuous T-cell epitope to enhance the immunogenicity of an oligopeptide corresponding to a weakly immunogenic substitution. These and other challenges associated with developing subunit peptide vaccines to prevent tumors bearing activated ras are discussed.

Syntheses of peptides containing contiguous repeats of glutamine or asparagine, related to sequences derived from proteins associated with neurodegenerative diseases, are described. Such homo-polymeric peptides are notoriously difficult to prepare due to the formation of aggregated structures during their solid phase synthesis. A novel approach using a randomized array of side chain protected and unprotected residues proved to be an effective strategy for preventing intermolecular peptide-chain association.

A method to quantitatively predict peptide binding to HLA DRB1*0401, B1*0101, and B1*1501 has been developed using a dataset of the relative contributions of each of the naturally occurring amino acids in the context of a simplified peptide backbone. The prediction assumed that the relative role of each of the peptide sidechains could be treated independently and could be measured by assaying each of the twenty naturally occurring amino acids at the central eleven positions of a 13 residue peptide previously shown to contain the minimal requirements for high affinity binding to HLA DR proteins. Three separate databases were generated. They were shown to have predictive value when tested on a set of 13 unrelated peptides known to bind the DR proteins with a wide range of apparent affinity. The DRB1*0401 database was tested further by analyzing myelin basic protein. All 13 amino acid peptides containing a hydrophobic amino acid at the third position were synthesized and assayed for binding purified DRB1*0401. In every case, the measured affinity correlated with the predictive values within the experimental error of the assays. Finally, the ability to predict peptide binding to MHC class II molecules was shown to help in identifying T cell determinants. The specificity of DRB1*0401 restricted T cell hybridomas against human serum albumin corresponded to two peptides, predicted, and shown to bind the class II protein with high affinity.

Efficacy and sequence specific behaviour of antisense oligonucleotides in biological systems are attenuated by enzymatic degradation, which is predominantly dependent on the oligonucleotide modification. Quantitative data relating to the kinetics and pattern of enzymatic digestion are thus valuable for the interpretation of biological tests with novel antisense oligonucleotides. To study the stability of modified oligonucleotides against nuclease attack, in vitro experiments of enzymatic degradation have been carried out using micellar electrokinetic capillary chromatography (MECC) as a quantitative control and electrospray mass spectrometry (ES-MS) for fragment identification. In contrast to gel electrophoresis, which is commonly applied, monitoring of enzymatic digestion by MECC can be carried out directly from the incubated sample without the need for labeled substrate. Furthermore, exact quantitative analysis becomes possible. Phosphodiester oligonucleotides terminally conjugated with hexaethylene glycol have been prepared to investigate the stability and degradation process of 3'- and 5'-protected oligomers with natural backbones in serum-containing medium. The results demonstrate that 3'-protection is much more effective than 5'-protection for nuclease stability, both in fetal calf serum and in human blood serum. To examine the influence of backbone modification on nuclease stability, the digestion of dodecanucleotides containing different numbers of phosphorothioate groups has been investigated by MECC and ES-MS. Degradation rates vary by a factor of approximately 50. Most fragments have been identified and the degradation patterns allow conclusions about the variations of nucleolytic activity with changing substrates.

Using a random combinatorial synthetic peptide library method based on a one-bead one-peptide concept for ligand identification (Lam et. al, Nature 1991, 354, 82-84.), idiotype specific peptides were retrieved and optimized for interaction with the cell surface immunoglobulins [IgM(kappa)] of two murine B lymphoma cell lines. Several of the identified peptides were characterized with respect to cell binding and signal transduction. These peptides were able to bind specifically to the surface immunoglobulins of these lymphoma cells. In addition to binding, when synthesized in tetrameric or multimeric forms, the peptides were able to trigger signal transduction resulting in an increase in protein tyrosine phosphorylation. Since D-amino acid peptide libraries were used in some of our efforts to identify binding ligands, several of the idiotype-specific peptides are composed of all D-amino acids (e.g. wGeyvmvnG). These findings may have important therapeutic implications for targeted-therapy of B-cell lymphoma as these D-amino acid ligands are more resistant to proteolysis resulting in a prolonged pharmacokinetic disposition in vivo.

The receptor with high affinity for immunoglobulin E (Fc epsilon RI) on mast cells and basophils plays an important role in mediating many of the pathophysiological phenomena associated with allergy. Fc epsilon RI is a tetrameric complex, alpha beta gamma2, of non-covalently attached subunits: one IgE-binding alpha-subunit with the binding site in the extracellular part of the chain, one beta-subunit and a dimer of disulphide linked gamma-subunits. In the present work, prediction of the three-dimensional structure of the four membrane-spanning segments of the beta-subunit has been achieved using rules of helix-helix packing arrangements and molecular dynamics calculations. It yielded a four-helix bundle with specific Van der Waals interactions between the helices. This four-helix bundle was used as a framework upon which to calculate the conformation of the beta-subunit excluding the C and N terminal cytoplasmic tails, but including the three chains that connect the four helices in the bundle. Separately, these synthetic 11, 17 and 29 residue bridge peptides were examined by circular dichroism (CD) spectroscopy and a degree of alpha-helical content in these bridge peptides was found. Additional molecular modelling of the bridge peptides indicate the central residues of these as the location of the helical moieties. Finally, in the model proposed for the beta-subunit, for each pair of consecutive transmembrane (TM) helices and its bridge peptide, a helix-loop-helix-loop-helix motif was found.

The improved chemical and biological properties of synthetic glycopeptides over peptides suggest their use as T cell agonists or antagonists. Recently, we prepared glycopeptide analogues of major T helper cell epitopic peptides corresponding to rabies virus proteins, and experimentally characterized their ability to bind to MHC class II proteins and stimulate T cell clones to rabies virus. In the current study, we investigated these MHC: peptide interactions by molecular modeling. We obtained structural support for our finding concerning the anomeric specificity of MHC with binding. While alpha-linked glycopeptides can bind to MHC without major alterations in the spatial arrangements and hydrogen bonding pattern of class II-peptide binding, the binding of beta-linked glycopeptides is considerably less favorable due to steric and columbic conflicts. Depending on where the saccharides are positioned along the peptide sequence, the MHC: glycopeptide complex may or may not produce the surface profile required for successful T cell receptor interaction. Application of this approach to other antigenic stimuli offers a good model to "dial in" the necessary sugar identity, length and anomeric configuration, as well as promising amino acid mutation sites, for successful design of T cell agonist or antagonist glycopeptides.

Peptide dendrimers such as Multiple Antigen Peptides (MAPs) are artificial proteins with branched architectures. They hold promise in biochemical and biomedical applications such as synthetic vaccines, serodiagnostics and intracellular delivery of peptides. We have shown that a new design of MAPs containing lipidated built-in adjuvant can be delivered by oral administration to elicit systemic and mucosal immunoglobulins as well as cytotoxic T-lymphocytes. For synthetic vaccines, it is desirable to obtain highly homogeneous preparations. To provide the precision and chemical unambiguity of this class of artificial proteins, we have devised several chemoselective approaches by thiol and carbonyl chemistries to facilitate their synthesis using unprotected peptide segments as building blocks and ligating them to the core matrix. This paper describes the methods of preparation, comparative studies of stability, and presents results of the preparation of antigens containing preformed multiple disulfides.

This study describes the development of passive and active vaccines directed at the Pseudomonas aeruginosa pilus adhesin. Passive immunization studies were carried out with P. aeruginosa strain K pilus-specific (PK3B, PK99H) and cross-reactive (PAK-13) monoclonal antibodies (MAbs). When A.BY/SnJ mice were passively immunized with a pilus-specific MAb (PK99H), which inhibited pilus-mediated adherence to respiratory epithelial cells, mice challenged with 5 x LD 50 of P. aeruginosa were completely protected while mice were not protected when animals were passively immunized with a pilus specific MAb (PK3B), which did not inhibit pilus adherence to epithilial cells. MAb PAK-13 was found to cross-react with the C-terminal portion of pili of different strains of P. aeruginosa. When mice were passively immunized with MAb PAK-13, subsequent challenge with KB7 (3 x LD50), PAO (8 x LD50) and PAK (3 x LD50) strains of P. aeruginosa resulted in a 70%, 60% and 90% protection of the mice, respectively. MAb PK99H has been previously shown to recognize a linear antigenic epitope consisting of the sequence DEQFIPK. This epitopic peptide was conjugated to protein carriers using different coupling strategies. Use of an appropriate adjuvant and the correct conjugation strategy were critical for raising high affinity antipeptide antisera. In a comparison of Freund's, alum, and Adjuvax, as adjuvants for a peptide-tetanus toxoid conjugate vaccine, highest titers for the synthetic peptide component of the conjugate were obtained with Adjuvax, while highest titers for the carrier protein components were obtained with Freund's. Of the four peptide-conjugates used in this study, only the C-terminal conjugated peptide failed to produce antibodies that bind to native antigen and did not protect mice in active immunization experiments (no survivors at 80 h in the mouse infection model). Conformationally restricted peptide conjugates in which the peptide was conjugated to the carrier at both ends provided better protection in mice challenged with lethal doses of P. aeruginosa than either N- or C-terminal linked peptide-conjugates. The pilus adhesin plays a critical role in P. aeruginosa pathogenesis and this is an excellent vaccine target for either active or passive immunization strategies.