We have demonstrated that the polyethylene glycol (PEG) corona of long-circulating polymeric nanoparticles (NPs) favors interaction with the amyloid-beta (Aβ(1-42)) peptide both in solution and in serum. The influence of PEGylation of poly(alkyl cyanoacrylate) and poly(lactic acid) NPs on the interaction with monomeric and soluble oligomeric forms of Aβ(1-42) peptide was demonstrated by capillary electrophoresis, surface plasmon resonance, thioflavin T assay, and confocal microscopy, where the binding affected peptide aggregation kinetics. The capture of peptide by NPs in serum was also evidenced by fluorescence spectroscopy and ELISA. Moreover, in silico and modeling experiments highlighted the mode of PEG interaction with the Aβ(1-42) peptide and its conformational changes at the nanoparticle surface. Finally, Aβ(1-42) peptide binding to NPs affected neither complement activation in serum nor apolipoprotein-E (Apo-E) adsorption from the serum. These observations have crucial implications in NP safety and clearance kinetics from the blood. Apo-E deposition is of prime importance since it can also interact with the Aβ(1-42) peptide and increase the affinity of NPs for the peptide in the blood. Collectively, our results suggest that these engineered long-circulating NPs may have the ability to capture the toxic forms of the Aβ(1-42) peptide from the systemic circulation and potentially improve Alzheimer's disease condition through the proposed "sink effect".

Glycosyltransferases are sugar-processing enzymes that require a specific metal ion cofactor for catalysis. In the presence of other ions the catalysis is often impaired. Here, for the first time, the enzymatic catalysis in the presence of various metal ions was modeled for a glycosyltransferase using a large enzymatic model. The catalytic mechanism of α-1,2-mannosyltransferase Kre2p/Mnt1p in the presence of Mn(2+) and other ions (Mg(2+), Zn(2+) and Ca(2+)) was modeled at the two hybrid DFT-QM/MM (M06-2X/OPLS2005 and B3LYP/OPLS2005) levels. Kinetic and structural parameters of transition states and intermediates, as well as kinetic isotope effects, were predicted and compared with available experimental and theoretical data. The catalysis in the presence of the metal ions is predicted as a stepwise SNi-like nucleophilic substitution reaction (DNint*AN(‡)DhAxh) via oxocarbenium ion intermediates. In the rate-determining step the leaving phosphate group of the donor substrate plays a role of the base catalyst. The predicted increased enzymatic reactivity (kcat: Zn(2+) ≈ Mg(2+) < Mn(2+) < Ca(2+)) correlated with the metal ion ability to polarize the Kre2p environment (Mg(2+) > Zn(2+) > Mn(2+) > Ca(2+)). The formation of the retained anomeric configuration in the product is controlled by a strict geometry of the active site of Kre2p. The 6-OH group of the attacking acceptor substrate may assist in protection of the anomeric carbon against unwanted hydrolysis by a through-space interaction with the electron deficient C1[double bond, length as m-dash]O5(+) moiety of the oxocarbenium-ion-like transition state.

Hybrid quantum mechanics/molecular mechanics calculations were used to study the catalytic mechanism of the retaining human α-(1,3)-galactosyltransferase (GTBWT) and its E303C mutant (GTBE303C). Both backside (via covalent glycosyl-enzyme intermediate, CGEI) and frontside SNi-like mechanisms (via oxocarbenium-ion intermediate, OCII) were investigated. The calculations suggest that both mechanisms are feasible in the enzymatic catalysis. The nucleophilic attack of the acceptor substrate to the anomeric carbon of OCII is the rate-determining step with an overall reaction barrier (ΔE(‡) = 19.5 kcal mol(-1)) in agreement with an experimental rate constant (kcat = 5.1 s(-1)). A calculated α-secondary kinetic isotope effect (α-KIE) of 1.27 (GTBWT) and 1.26 (GTBE303C) predicts dissociative character of the transition state in agreement with experimentally measured α-KIE of other retaining glycosyltransferases. Remarkably, stable CGEI in GTBE303C compared with its counterpart in GTBWT may explain why the CGEI has been detected by mass spectrometry only in GTBE303C ( Soya N, Fang Y, Palcic MM, Klassen JS. 2011. Trapping and characterization of covalent intermediates of mutant retaining glycosyltransferases. Glycobiology, 21: 547-552).