Simplified ß-amyloid peptides for safer Alzheimer's vaccines development.

Over the past few years, new ways of fighting Alzheimer's disease have emerged based on stimulating the immunitary defence system of the patients. To avoid toxicity and autoimmune response related to the Aß[1-42] peptide immunotherapy, in the last decade a large number of works aimed at identifying new classes of safe Aß derivatives by modifying the full length ß-amyloid form. In strict agreement with the purposes of the sequence-simplification technology, Aß[1-16], Aß[13-28] and Aß[25-42] fragments were selected in order to retain the major immunogenic sites of the Aß[1-42] peptide, and corresponding simplified forms were designed and synthesized. All glycinated Aß derivatives showed immunogenic and antigenic properties similar to the parent Aß[1-42] peptide, and raised antibodies were all able to cross-recognize both Aß[1-42] and Aß[1-40] synthetic structures. All Aß simplified forms showed reduced fibrillogenic and inflammatory properties. In particular, the Aß[13-28]+G form failed to induce IFN-γ production thus suggesting that this molecule could represent a good candidate for potentially safer AD vaccine therapy.

Efficacy of PTX3 in a rat model of invasive aspergillosis.

Pentraxin 3 (PTX3) is an acute-phase glycoprotein with a nonredundant function in the host resistance to Aspergillus fumigatus. PTX3 activity was evaluated against pulmonary aspergillosis in rats immunosuppressed with cortisone acetate. PTX3 enhanced the survival rate and reduced the lung fungal burden of infected rats in both therapeutic and prophylactic modalities. Thus, we extended the protective activity of PTX3 in pulmonary aspergillosis to corticosteroid-induced immunodeficiency, which is a relevant clinical condition in graft-versus-host disease and in solid organ transplant.

The angiogenic inhibitor long pentraxin PTX3 forms an asymmetric octamer with two binding sites for FGF2.

The inflammation-associated long pentraxin PTX3 plays key roles in innate immunity, female fertility, and vascular biology (e.g. it inhibits FGF2 (fibroblast growth factor 2)-mediated angiogenesis). PTX3 is composed of multiple protomers, each composed of distinct N- and C-terminal domains; however, it is not known how these are organized or contribute to its functional properties. Here, biophysical analyses reveal that PTX3 is composed of eight identical protomers, associated through disulfide bonds, forming an elongated and asymmetric, molecule with two differently sized domains interconnected by a stalk. The N-terminal region of the protomer provides the main structural determinant underlying this quaternary organization, supporting formation of a disulfide-linked tetramer and a dimer of dimers (a non-covalent tetramer), giving rise to the asymmetry of the molecule. Furthermore, the PTX3 octamer is shown to contain two FGF2 binding sites, where it is the tetramers that act as the functional units in ligand recognition. Thus, these studies provide a unifying model of the PTX3 oligomer, explaining both its quaternary organization and how this is required for its antiangiogenic function.

Biochemical and biological characterization of a new oxidized avidin with enhanced tissue binding properties.

Chicken avidin and bacterial streptavidin are widely employed in vitro for their capacity to bind biotin, but their pharmacokinetics and immunological properties are not always optimal, thereby limiting their use in medical treatments. Here we investigate the biochemical and biological properties of a new modified avidin, obtained by ligand-assisted sodium periodate oxidation of avidin. This method allows protection of biotin-binding sites of avidin from inactivation caused by the oxidation step and delay of avidin clearance from injected tissue by generation of aldehyde groups from avidin carbohydrate moieties. Oxidized avidin shows spectroscopic properties similar to that of native avidin, indicating that tryptophan residues are spared from oxidation damage. In strict agreement with these results, circular dichroism and isothermal titration calorimetry analyses confirm that the ligand-assisted oxidation preserves the avidin protein structure and its biotin binding capacity. In vitro cell binding and in vivo tissue residence experiments demonstrate that aldehyde groups provide oxidized avidin the property to bind cellular and interstitial protein amino groups through Schiff's base formation, resulting in a tissue half-life of 2 weeks, compared with 2 h of native avidin. In addition, the efficient uptake of the intravenously injected (111)In-BiotinDOTA (ST2210) in the site previously treated with modified avidin underlines that tissue-bound oxidized avidin retains its biotin binding capacity in vivo. The results presented here indicate that oxidized avidin could be employed to create a stable artificial receptor in diseased tissues for the targeting of biotinylated therapeutics.

Biotin derivatives carrying two chelating DOTA units. Synthesis, in vitro evaluation of biotinidases resistance, avidin binding, and radiolabeling tests.

The synthesis of four biotin derivatives carrying two DOTA moieties for each ligand (BisDOTA set) is reported, for increasing radiation/dose ratio and improving efficiency in the pretargeted avidin-biotin radioimmunotherapy. The biotin-containing scaffold of two BisDOTA was similar to the mono-DOTA derivative previously described. Then the scaffold was elongated by trifunctionalized spacers of different length and conjugated with one of the COOH groups of two DOTA. Two others were prepared starting from a on-resin lysine residue. The lysine alpha-NH2 was bonded to biotin, and then spacers were appended to the epsilon-NH2 and conjugated with two DOTA molecules. One compound contained a p-aminobenzoic acid spacer, which ensured higher head-to-tail distance and increased rigidity of the chain. These last two compounds had a very high ability to bond avidin and were labeled with 90Y at high specific activity. All the compounds were resistant to the action of serum biotinidases.

Fibroblast growth factor 2-antagonist activity of a long-pentraxin 3-derived anti-angiogenic pentapeptide.

Fibroblast growth factor-2 (FGF2) plays a major role in angiogenesis. The pattern recognition receptor long-pentraxin 3 (PTX3) inhibits the angiogenic activity of FGF2. To identify novel FGF2-antagonistic peptide(s), four acetylated (Ac) synthetic peptides overlapping the FGF2-binding region PTX3-(97-110) were assessed for their FGF2-binding capacity. Among them, the shortest pentapeptide Ac-ARPCA-NH(2) (PTX3-[100-104]) inhibits the interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in endothelial cells, without affecting the activity of unrelated mitogens. Also, Ac-ARPCA-NH(2) inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-overexpressing murine endothelial cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to Chinese Hamster ovary cells overexpressing the tyrosine-kinase FGF receptor-1 (FGFR1) and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction. In all the assays the mutated Ac-ARPSA-NH(2) peptide was ineffective. In keeping with the observation that hydrophobic interactions dominate the interface between FGF2 and the FGF-binding domain of the Ig-like loop D2 of FGFR1, amino acid substitutions in Ac-ARPCA-NH(2) and saturation transfer difference-nuclear magnetic resonance analysis of its mode of interaction with FGF2 implicate the hydrophobic methyl groups of the pentapeptide in FGF2 binding. These results will provide the basis for the design of novel PTX3-derived anti-angiogenic FGF2 antagonists.

Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction.

This study investigates the applicability of D-PAM, the inverso form of the Protein A Mimetic synthetic peptide affinity ligand (PAM) obtained from the screening of a multimeric combinatorial peptide library, in monoclonal IgG isolation from ascitic fluids and cellular supernatants. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture monoclonal antibodies in a single chromatographic step, with a recovery yield and purity degree above 90% and full recovery of antibody activity. D-PAM/Emphaze resin showed a host cell protein (HCP) and DNA reduction similar to protein A sorbent. Indeed, column capacity, determined by applying a large excess of purified antibodies to 1 mL of column bed volume, was always higher than 50 mg/mL. D-PAM/IgG interaction was characterized by isothermal titration calorimetry (ITC) and an analysis of binding isotherms, obtained for titration of ST2146, ST1485 and 7H3 IgG monoclonal antibodies, suggested that two peptides bind simultaneously to the IgG molecule, with a K(A) (equilibrium association constant) of 3.4, 6.2 and 3.4 x 10(4) M(-1), and a DeltaH (change in enthalpy) of -1.3, -4.2 and -4.1 kcal mol(-1), respectively.

Structural characterization of PTX3 disulfide bond network and its multimeric status in cumulus matrix organization.

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys(317) and Cys(318) are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3(-/-) mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3(-/-) mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

High-level expression and efficient purification of recombinant human long pentraxin PTX3 in Chinese hamster ovary cells.

PTX3 is a secreted multimeric glycoprotein which plays a key role in innate immunity by activating the classical complement pathway through specific recognition of the C1q subunit. A method is described for the high level expression of the recombinant human PTX3 in Chinese hamster ovary cells (CHO), adapted to a suspension growth in spinner flasks containing a serum-free chemically defined medium and producing about 50 mg of PTX3/L of culture. A purification procedure to produce a homogeneous protein preparation from the supernatant, by means of anion exchange, hydroxyapatite and size exclusion chromatography, is also reported. This three-step protocol allows us to obtain PTX3 with a recovery yield close to 70%, a purity degree exceeding 95%, and a final host cell protein (HCP) content lower than 150 ppm. The recombinant purified PTX3 retains its biological activity, as demonstrated by C1q binding ELISA assay, and displays a complex quaternary structure characterized by a high secondary structure content quite different from human short pentraxin C-reactive protein (CRP) and serum amyloid P component (SAP), as determined by circular dichroism, fluorescence analysis, and native and SDS-PAGE experiments.

Structure/function of KRAB repression domains: structural properties of KRAB modules inferred from hydrodynamic, circular dichroism, and FTIR spectroscopic analyses.

The abundant zinc finger proteins (ZFPs) sharing the KRAB motif, a potent transcription repression domain, direct the assembly on templates of multiprotein repression complexes. A pivotal step in this pathway is the assembly of a KRAB domain-directed complex with a primary corepressor, KAP1/KRIP-1/TIF1beta. The structure/function dependence of KRAB/TIF1beta protein-protein interaction and properties of the complex, therefore, play pivotal roles in diverse cellular processes depending on KRAB-ZFPs regulation. KRAB domains are functionally bipartite. The 42 amino acid-long KRAB-A module, indeed, is necessary and sufficient for transcriptional repression and for the interaction with the tripartite RBCC region of TIF1beta, while the KRAB-B motif seems to potentiate the assembly of the complex. The structural properties of KRAB-A and KRAB-AB domains from the human ZNF2 protein have been investigated by characterizing highly purified lone (A) and composite (AB) modules. Hydrodynamic and spectroscopic features, investigated by means of gel filtration, circular dichroism, and infrared spectroscopy, provide evidence that both KRAB-A and KRAB-AB domains present low compactness, structural disorder, residual secondary structure content, flexibility, and tendency to molecular aggregation. Comparative analysis among KRAB-A and KRAB-AB modules suggests that the presence of the -B module may influence the properties of lone KRAB-A by affecting the structural flexibility and stability of the conformers. The combined experimental data and the intrinsic features of KRAB-A and KRAB-AB primary structures indicate a potential role of specific subregions within the modules in driving structural flexibility, which is proposed to be of importance for their function.

Expression, purification and partial characterization of the Krüppel-associated box (KRAB) from the human ZNF2 protein.

The Krüppel-associated box (KRAB) domain is a potent transcription repression bipartite domain, shared by over 400 zinc finger proteins in humans, involved in the regulation of many functions. KRAB domains are both physically and functionally bipartite (A and B modules). The lone KRAB-A and composite KRAB-AB domains from the human ZNF2 protein were over-expressed as recombinant proteins in E. coli, isolated and purified to homogeneity to investigate their structure to function relationship.