Separation and characterization of aggregated species of amyloid-beta peptides

We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Aβ) peptides from their aggregates. From solutions of Aβ_1–40 or Aβ_1–42 monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6–6.5. The formation of Aβ aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for Aβ_1–40 and Aβ_1–42, respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the Aβ_1–42 peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the Aβ-selective antibody mAb1C3, where a monomeric Aβ_1–16 peptide was added to the protofibril preparation. Aβ_1–16 is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.     

Peptide synthesis catalysed by papain immobilised on polymer supports: Effect of the macromolecular structure and reaction conditions on synthesis

Papain immobilised on different types of polymeric supports was used for the synthesis of peptides in aqueous-organic solvent mixtures. The effects of the nature of the polymer support, degree of crosslinking, nature and length of the spacer grouping between the polymer backbone and the point of attachment of the enzyme, and reaction conditions like pH, concentration of nucleophile and the immobilised enzyme content on the course of the synthesis were investigated. Divinylbenzene-crosslinked polystyrene, divinylbenzenecrosslinked polyacrylamide and N,N′-methylene-bis-acrylamide-crosslinked polyacrylamide systems immobilised with papain were used for these studies. An increase in the length of the spacer arm and an increase in hydrophilicity invariably resulted in an increase in the yield of the peptide synthesis. Papain immobilised on polystyrene-PEG supports and tetraethyleneglycol-crosslinked polystyrene supports was determined to be more efficient in effecting peptide synthesis when compared to other polystyrene-based supports.     

Investigation of the noncovalent interactions between anti-amyloid agents and amyloid β peptides by ESI-MS

This paper describes an efficient and reproducible screening method for identifying low molecular weight compounds that bind to amyloid β peptides (Aβ) peptides using electrospray ionization mass spectrometry (ESI-MS). Low molecular weight compounds capable of interacting with soluble Aβ may be able to modulate/inhibit the Aβ aggregation process and serve as potential disease-modifying agents for AD. The present approach was used to rank the binding affinity of a library of compounds to Aβ1-40 peptide. The results obtained show that low molecular weight compounds bind similarly to Aβ1-42, Aβ1-40, as well as Aβ1-28 peptides and they underline the critical role of Aβ peptide charge motif in binding at physiological pH. Finally, some elements of structure-activity relationship (SAR) involved in the binding affinity of homotaurine to soluble Aβ peptides are discussed.     

A comparison of rigid and flexible crosslinked polymer-supported peptide synthesis

To correlate the coupling difficulty and nature of the support in solid-phase peptide synthesis, a set of difficult peptides were built under identical conditions on rigid and hydrophobic divinyl benzene-crosslinked polystyrene (DVB-PS) and flexible and polar 1,4-butanediol dimethacrylate-crosslinked polystyrene (BDDMA-PS) resins of same functional capacity (∼2 mmol/g). The difficult coupling stages observed on each support were identified by monitoring the acylation time, coupling yield and deprotection efficiency. The yield and purity of the peptides prepared on BDDMA-PS were higher than when DVB-PS support was used.     

An enzyme-linked immunosorbent assay to compare the affinity of chemical compounds for β-amyloid peptide as a monomer

Aβ_1–42 is the proteolytic cleavage product of cleavage of the amyloid precursor protein by β- and γ-secretases. The aggregation of Aβ_1–42 plays a causative role in the development of Alzheimer’s disease. To lock Aβ_1–42 in a homogenous state, we embedded the Aβ_1–42 sequence in an unstructured region of Bcl-x_L. Both the N-terminus and the C-terminus of Aβ_1–42 were constrained in the disordered region, whereas the conjunction did not introduce any folding to Aβ_1–42 but maintained the sequence as a monomer in solution. With Bcl-x_L-Aβ₄₂, we developed an enzyme-linked immunosorbent assay to compare the affinity of compounds for monomeric Aβ_1–42. Bcl-x_L-Aβ₄₂ was coated on a microplate and this was followed by incubation with different concentrations of compounds. Compounds binding to Leu17-Val24 of Aβ_1–42 inhibited the interaction between Bcl-x_L-Aβ₄₂ and antibody 4G8. The method can not only reproduce the activities of the reported Aβ_1–42 inhibitors such as dopamine, tannin, and morin but can also differentiate decoy compounds that do not bind to Aβ_1–42. Remarkably, using this method, we discovered a new inhibitor that binds to monomeric Aβ_1–42 and inhibits Aβ_1–42 fibril formation. As the structure of Bcl-x_L-Aβ₄₂ monomer is stable in solution, the assay could be adapted for high-throughput screening with a series of antibodies that bind the different epitopes of Aβ_1–42. In addition, the monomeric form of the Aβ_1–42 sequence in Bcl-x_L-Aβ₄₂ would also facilitate the identification of Aβ_1–42 binding partners by coimmunoprecipitation, cocrystallization, surface plasmon resonance technology, or the assay as described here.     

Supramolecular complex formation of β-cyclodextrin polymer with substituted salicylic acid or 3-hydroxy-2-naphthoic acid and their electrorheological behaviors

According to the chemical design, electrorheological properties of supramolecular complex from β-cyclodextrin polymer (β -CDP) were discussed. Six supramolecular complexes of β-cyclodextrin polymer with substituted salicylic acid and 3-hydroxy-2-naphthoic acid were synthesized by the solid-phase self-assembly method, and their component and structure were characterized by NMR, FT-IR, UV-vis and the fluorescence analysis. Then the electrorheological properties of their suspensions in silicone oil were investigated under DC electric fields. It was found that the yield stresses of these supramolecular complex ER fluids were 7.3–9.8 kPa at 4 kV/mm in DC electric field, which were enhanced by 34%–72% compared with that of pure β-CDP. Among them, that of β-CDP/3-hydroxy-2-naphthoic acid ER fluid was the highest. It was also found that the ER effect of supramolecular complexes can be controlled by changing different guests. When the substituted group is at phenyl ring, ER behavior can be slightly adjusted by the different substituted groups, their number as well as their position at phenyl ring. This can be proved by the measurement of dielectric properties.     

Effect of amyloid beta peptides Aβ<Subscript>1–28</Subscript> and Aβ<Subscript>25–40</Subscript> on model lipid membranes

To investigate the molecular interaction of amyloid beta peptides Aβ_1–28 or Aβ_25–40 with model lipid membranes differential scanning calorimetry (DSC) and DPH and TMA DPH fluorescence anisotropy approaches were used. The main transition temperature (T _m) and enthalpy change (ΔH) of model lipid membranes composed of DMPC/DPPG on addition of Aβ_25–40 or Aβ_25–40 at 10:1 (w/w) phospholipid/peptide ratio either non-aggregated or previously aggregated were examined. The effect of Aβ_1–28 and Aβ_25–40 on the membrane fluidity of liposomes made of DMPC/DPPG (98:2 w/w) was determined by fluorescence anisotropy of incorporated DPH and TMA DPH. The results of this study provide information that Aβ_1–28 preferentially interacts with the hydrophilic part of the model membranes, while Aβ_25–40 rather locates itself in the hydrophobic core of the bilayer where it reduces the order of the phospholipids packing.     

Tetraethyleneglycol diacrylate (TTEGDA)-crosslinked polystyrene resin as an efficient solid support for gel phase peptide synthesis

Various applications of the newly developed tetraethyleneglycol diacrylate (TTEGDA)-crosslinked polystyrene resin are illustrated by the synthesis of model peptides, fully protected peptides, peptide amides and biologically important sequences. PS-TTEGDA resin was prepared by suspension polymerization of styrene and TTEGDA and functionalized with chloromethyl, 4-cholromethyl-3-nitro, aminomethyl, α-bromopropionyl, a-aminopropionyl, 4-bromomethyl 3-nitrobenzamido, 4-aminomethyl-3-nitrobenzamido groups. Peptide synthesis was carried out using these modified resins by standard solid phase methodology. Coupling and deprotection in this synthetic strategy went to near completion showing the positive role of hydrophilic and flexible crosslinking agent TTEGDA in facilitating gelphase reactions. The peptides were removed from the support by photolysis, trifluoroaceticacid (TFA) treatment,trans-esterification or ammonolysis in high purity and yield. The crude peptides were purified by column chromatography/FPLC and characterized by aminoacid analysis, sequencing or¹H-NMR.     

α-(Phenylacetamido)benzylpolystyrene (pab-resin) : A new polymeric support for peptide synthesis with improved acid stability

Solid phase peptide synthesis carried out on conventional chloromethyl polystyrene support has the disadvantage of the acidolysis of the peptide-resin linkage during the deprotection step. In addition, when trifluoroacetic acid is used as deprotecting agent, intrapolymeric trifluoroacetyl transfer may give rise to truncated trifluoroacetylated peptides. A new polymer support devised to minimize these problems, α - (4 - chloromethyl-phenylacetamido) benzylcopoly(styrene-1%-divinylbenzene) (ClCH₂-Pab-resin), has been synthesized. The acetamido bridge between the peptide and the resin increases the acid stability of the new support thus allowing the synthesis of purer peptides in higher yields. Boc-aminoacyl-OCH₂-Pab-resins can be readily prepared in a versatile way by reaction of Boc-amino acid caesium salts with chloromethyl-Pab-resin. This fact contrasts with the rather elaborate procedures required for previously described similar resins.The synthetic viability of the chloromethyl-Pab-resin is shown by the synthesis of Ac-His-Arg-Tyr-Arg-Pro-OH (fragment 39–43 of histone H3). The synthesis and subsequent purification are exhaustively compared with the parallel synthesis carried out on a standard chloromethyl polystyrene support. The presence of the acetamido linkage provokes, as expected, a decrease in the HF cleavage yield. Nevertheless, this disadvantage is balanced by the increase in global purification yields even for the synthesis of a pentapeptide for which only five deprotection steps are required.     

Synthesis of enkephalins by the method of polymeric activated esters based on 4-hydroxy-3-nitrobenzophenone

Leucine- and methionine-enkephalins have been synthesized by the successive growth of the peptide chain from the C-end by the method of polymeric activated esters based on 4-hydroxy-3-nitrobenzophenone with yields of 90 and 70%, respectively, calculated on the initial C-terminal amino acid. Polystyrene with 2% of divinylbenzene was used as the polymeric matrix. Using the synthesis of methionine-enkephalin as an example, the possibility has been shown of using polymeric activated esters for the synthesis of peptides with a free carboxy group. Leningrad State University. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 547–553, July–August, 1989.