Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.

Structure and activity of native and thiolated α-chymotrypsin adsorbed onto gold nanoparticles.

A detailed understanding of protein-nanoparticle interactions is critical to realize the full potential of bioconjugate-enabled technologies. Parameters that lead to conformational changes in protein structure upon adsorption must be identified and controlled to mitigate loss of biological function. We hypothesized that the installation of thiol functional groups on a protein will facilitate robust adsorption to gold nanoparticles (AuNPs) and prevent protein unfolding to achieve thermodynamic stability. Here we investigated the adsorption behavior of α-chymotrypsin (ChT) and a thiolated analog of α-chymotrypsin (T-ChT) with AuNPs. ChT, which does not present any free thiols, was modified with 2-iminothiolane (Traut's reagent) to synthesize T-ChT consisting of two free thiols. Protein adsorption to AuNPs was monitored with dynamic light scattering and UV-vis spectrophotometry, and fluorescence spectra were acquired to assess changes in protein structure induced by interaction with the AuNP. The biological function of ChT, T-ChT, and respective bioconjugates were compared using a colorimetric enzymatic assay. The thiolated analog exhibited a greater affinity for the AuNP than the unmodified ChT, as determined from adsorption isotherms. The ChT protein formed a soft protein corona in which the enzyme denatures with prolonged exposure to AuNPs and, subsequently, lost enzymatic function. Conversely, the T-ChT formed a robust hard corona on the AuNP and retained structure and function. These data support the hypothesis, provide further insight into protein-AuNP interactions, and identify a simple chemical approach to synthesize robust and functional conjugates.

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody-Gold Nanoparticle Conjugates.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Probing the Mechanism of Antibody-Triggered Aggregation of Gold Nanoparticles.

The unique physicochemical properties of gold nanoparticles (AuNPs) provide many opportunities to develop novel biomedical technologies. The surface chemistry of AuNPs can be engineered to perform a variety of functions, including targeted binding, cellular uptake, or stealthlike properties through the immobilization of biomolecules, such as proteins. It is well established that proteins can spontaneously adsorb onto AuNPs, to form a stable and functional bioconjugate; however, the protein-AuNP interaction may result in the formation of less desirable protein-AuNP aggregates. Therefore, it is imperative to investigate the protein-AuNP interaction and elucidate the mechanism by which protein triggers AuNP aggregation. Herein, we systematically investigated the interaction of immunoglobulin G (IgG) antibody with citrate-capped AuNPs as a function of solution pH. We found that the addition of antibody triggers the aggregation of AuNPs for pH < 7.5, whereas a monolayer of antibody adsorbs onto the AuNP to form a stable bioconjugate when the antibody is added to AuNPs at pH ≥ 7.5. Our data identifies electrostatic bridging between the antibody and the negatively charged AuNPs as the mechanism by which aggregation occurs and rules out protein unfolding and surface charge depletion as potential causes. Furthermore, we found that the electrostatic bridging of AuNPs is reversible within the first few hours of interaction, but the protein-AuNP interactions strengthen over 24 h, after which the protein-AuNP aggregate is irreversibly formed. From this data, we developed a straightforward approach to acrylate the basic residues on the antibody to prevent protein-induced aggregation of AuNP over a wide pH range. The results of this study provide additional insight into antibody-nanoparticle interactions and provide a pathway to control the interaction with the potential to enhance the conjugate function.