Functionalization of amyloid fibrils via the Bri2 BRICHOS domain

Amyloid fibrils are mechanically robust and partly resistant to proteolytic degradation, making them potential candidates for scaffold materials in cell culture, tissue engineering, drug delivery and other applications. Such applications of amyloids would benefit from the possibility to functionalize the fibrils, for example by adding growth factors or cell attachment sites. The BRICHOS domain is found in a family of human proteins that harbor particularly amyloid-prone regions and can reduce aggregation as well as toxicity of several different amyloidogenic peptides. Recombinant human (rh) BRICHOS domains have been shown to bind to the surface of amyloid-β (Aβ) fibrils by immune electron microscopy. Here we produce fusion proteins between mCherry and rh Bri2 BRICHOS and show that they can bind to different amyloid fibrils with retained fluorescence of mCherry in vitro as well as in cultured cells. This suggests a “generic” ability of the BRICHOS domain to bind fibrillar surfaces that can be used to synthesize amyloid decorated with different protein functionalities.


Results
We cloned the mCherry protein 24 downstream of human Bri2 BRICHOS, encompassing residues 113-231 in human Bri2 21 (Fig. 1A) and produced the fusion protein together with a His6 tag and the solubility tag NT* from flagelliform spider silk protein (FlSp) 25 linked upstream of Bri2 BRICHOS via a thrombin cleavage site. The His6-NT*-Bri2 BRICHOS-mCherry protein was produced in E. coli, purified by immobilized metal affinity chromatography (IMAC), and cleaved with thrombin to release recombinant human (rh) Bri2 BRICHOS-mCherry, which was isolated by a second IMAC step. The yield was approximately 100 mg per liter bacterial culture. The fluorescence properties of rh Bri2 BRICHOS-mCherry are very similar to those of mCherry alone (Fig. 1B), which shows that mCherry is functional in the fusion protein. Likewise, the abilities of rh Bri2 BRICHOS to inhibit amyloid fibril formation of Aβ 42 is retained in the rh Bri2 BRICHOS-mCherry fusion protein as shown by kinetics measured by thioflavin T (ThT) fluorescence (Fig. 1C), indicating that the BRICHOS function is not perturbed by linking it to mCherry. The dual functionalities of rh Bri2 BRICHOS-mCherry motivated us to test whether the fibril binding properties of rh Bri2 BRICHOS 18,21 can be used to decorate amyloid fibrils with fluorescent mCherry.
The N-terminal fragment of mutant huntingtin (N-mutHtt), which is responsible for the neurodegenerative disorder Huntington's disease 26 , contains a polyglutamine repeat expansion that renders the protein aggregation prone 27 . When expressed in human cells, N-mutHtt forms amyloid-like fibrils that precipitate in large intracellular inclusions, which can be readily detected by microscopy 28 . Because of this feature, we selected N-mutHtt as a model to explore the ability of Bri2 BRICHOS to interact with amyloidogenic proteins in the cytoplasm, which is a non-physiological intracellular localization for this domain that is part of the secretory protein Bri2(Ref. 29 ). Green fluorescent protein (GFP)-tagged N-mutHtt with a 109 amino acid-long repeat (N-mutHtt109Q-GFP) was co-expressed with mCherry or mCherry-Bri2 BRICHOS in human osteosarcoma U2OS cells and human cervix carcinoma HeLa cells and the localization of the proteins were examined by fluorescence microscopy. While mCherry was typically excluded from the GFP-N-mutHtt inclusions or, alternatively localized at the rim of these structures, mCherry-Bri2 BRICHOS was enriched in the inclusions in the U2OS and HeLa cells (Fig. 2). We conclude that the ability of Bri2 BRICHOS to interact with amyloid-like fibrils is an intrinsic feature that is independent of its natural environment.
For in vitro analysis of binding properties of rh Bri2 BRICHOS, we generated amyloid fibrils of Aβ 42 , IAPP and α-synuclein, which are implied in Alzheimer's disease, type II diabetes and Parkinson's disease, respectively, as well as the de novo-designed β17 protein, according to published protocols 18,25,30,31 . The fibrils were isolated  www.nature.com/scientificreports/ by centrifugation and subsequently incubated with rh Bri2 BRICHOS-mCherry at a concentration that corresponds to 20% of the monomeric amyloid polypeptide concentration, at 37 °C for one hour. After washing, the rh Bri2 BRICHOS-mCherry incubated fibrils were analyzed by fluorescence microscopy. Green fluorescence (corresponding to ThT fluorescence) was observed for all tested amyloid fibrils incubated with rh Bri2 BRICHOS-mCherry. The results confirm amyloid-like properties for the different types of fibrils used. Red fluorescence was observed for rh Bri2 BRICHOS-mCherry suggesting that fusion protein bound to fibrils (Fig. 3). As controls, we incubated the fibrils with mCherry alone. The results show that Aβ42, IAPP, α-synuclein and β17 fibrils all bound rh Bri2 BRICHOS-mCherry, which resulted in fluorescent fibrils, while incubation with mCherry alone only gave marginal staining of the fibrils ( Fig. 3 and Supplementary Figure 1). To further confirm that Bri2 BRICHOS-mCherry binds to amyloid fibrils, we tested Aβ 42 E22G (the so-called Arctic mutation related to familial Alzheimer's disease 32 ) and a part of the central core region R3 (V306-K317) of Tau (referred to as PHF6) implicated in Tauopathies (e.g. Alzheimer's disease and Parkinson's disease), which resulted in similar results (Supplementary Figure 2). www.nature.com/scientificreports/ Next, we examined the localization of rh Bri2 BRICHOS-mCherry on fibrils by immune electron microscopy (EM). To this end, we incubated the rh Bri2 BRICHOS-mCherry decorated fibrils with a primary antibody against mCherry and a secondary antibody bound to 5 nm gold particles and studied them with negative stain EM. The immuno EM data (Fig. 4) confirmed the results from fluorescence studies of the Aβ 42 , IAPP, α-synuclein and β17 fibrils. The gold particles localized on the fibrils for each of the amyloidogenic proteins. While the gold particles gave a strong labeling of the fibrils incubated with rh Bri2 BRICHOS-mCherry, labeling was only sporadically detected with fibrils incubated with mCherry alone.
To quantify binding of rh Bri2 BRICHOS-mCherry and mCherry, we immobilized Aβ 42 fibrils on a carboxymethylated dextran surface and investigated binding by surface plasmon resonance (SPR). The SPR sensorgrams (Supplementary Figure 3) were used for determining dissociation constants, K D as described previously 33 . Rh Bri2 BRICHOS-mCherry showed strong binding to the immobilized Aβ 42 fibrils (K D = 75 nM), while mCherry bound significanly weaker (K D = 7 μM). The dissociation constant for rh Br2 BRICHOS-mCherry is in the same range as previously determined for rh proSP-C BRICHOS binding to Aβ 42 fibrils 22 . www.nature.com/scientificreports/

Discussion
Amyloid-like fibrils are versatile materials that are used in nature for various purposes, and designed nanomaterials built up from regular β-sheets have many potential applications in materials science, biology and medicine.
Here we show a potentially "generic" way to decorate amyloid-like fibrils with functional proteins. Our approach is based on the observations from immuno-EM that recombinant BRICHOS binds to the surface of Aβ and IAPP amyloid fibrils 18,21,22 . We show that a recombinant fusion protein between human Bri2 BRICHOS and mCherry can be efficiently produced using the solubility tag NT* FlSp 25 derived from the N-terminal domain of spider silk proteins 34 . Rh Bri2 BRICHOS-mCherry shows the same fluorescence properties as mCherry alone, binds to the surface of Aβ 42 (as well as to fibrils of the Aβ 42 E22G mutant), IAPP, α-synuclein, PHF6 and β17 amyloid fibrils and makes them fluorescent. The binding of rh Bri2 BRICHOS-mCherry to amyloid fibrils in vitro is accomplished simply by co-incubating the fusion protein and the fibrils in aqueous buffer at 37 °C for one hour under quiescent conditions, and specific binding of mCherry tagged Bri2 BRICHOS to amyloid-like inclusions of mutant huntingtin is observed in the cytoplasm of two different cell lines. These conditions will likely be tolerable by other Bri2 BRICHOS fusion proteins and other amyloid-like fibrils and nanomaterials. The approach presented here thus holds potential to be useful for decorating various fibrillar materials. Artificial spider silk-like fibers, with amyloid-like properties, are thought to have attractive properties for generation of novel biomaterials 35,36 and can form fibers also when linked genetically to other proteins 37 . One drawback with fusing proteins to a fiber-forming protein is that the added protein needs to withstand conditions required for silk fiber formation, which can entail for example presence of organic co-solvents or non-physiological pH. Such obstacles might be circumvented by using Bri2 BRICHOS based fusion proteins to decorate premade fibers. Amyloid fibrils can self-propagate and potentially also induce other proteins to form amyloid structures 38 . Amyloid-like fibrils and/ or oligomers that can be generated from them are potentially cytotoxic 39,40 and the seeding and cross-seeding www.nature.com/scientificreports/ phenomena are thus potentially harmful if amyloid and β-sheet based materials are implanted in living organisms. It can be noted that Aβ 42 or IAPP amyloid fibrils decorated with recombinant BRICHOS are markedly less prone to seed fibril formation and generate less cytotoxic oligomers compared to the corresponding naïve fibrils 18,21,22,41 . Moreover, addition of recombinant BRICHOS to mouse hippocampal slice preparations in vitro has been shown not to elicit detectable toxic effects and transgenic overexpression of BRICHOS in Drosophila fruit flies has no side effects on longevity or locomotor behavior 23,42,43 . It is thus conceivable that fibrils that have been decorated with Bri2 BRICHOS based fusion proteins have attenuated capacity to be cytotoxic and therefore can be well tolerated by cells and organisms.

Experimental procedures
Plasmids. The synthetic gene coding for Bri2 BRICHOS with mCherry attached to the C-terminal and a hexa-glycine linker between was ordered from GenScript (GenScript Biotech, Netherlands). The pU57 plasmid was digested with EcoRI and HindIII restriction enzymes and the Bri2 BRICHOS-mCherry gene was isolated on a 2% agarose gel, extracted with QIAquick Gel Extraction Kit (QIAGEN, Venlo Netherlands) and ligated into pT7-H 6 NT* FlSp plasmid that has previously been digested with the same restriction enzymes. To obtain mCherry, HiFi HotStart DNA polymerase (Kapa Biosystems, USA) was used for PCR amplification with the Bri2 BRICHOS mCherry gene as template. The gene was amplified with forward primer 5′-CCG GAA TTC CCT GGT GCC ACG CGG TTC TGT GAG CAA G-3′ and reverse primer 5′-GGG AAG CTT ACT TGT ACA GCT CGT CCA TGC CGC CGG T-3′ at 65 °C as annealing temperature. The PCR product was cleaved and ligated into pT7H 6 NT* FlSp as described above. pET21a-alpha-synuclein (αSN) was a gift from Michael J Fox Foundation MJFF (Addgene plasmid #51486) and was used as template to obtain pT7H 6  Expression and purification protocols. Expression and purification of rh Bri2 BRICHOS-mCherry were done as previously described for rh Bri2 BRICHOS 21 . Aβ42 and β17 were expressed and purified as described previously 25,30 . Expression of NT* FlSp (TEV recognition site, TRS)-α-synuclein and NT* FlSp MetIAPP was performed as described previously 25,30,34 . Purification of αSN was performed essentially as described earlier for Aβ 25  www.nature.com/scientificreports/ Aβ 42 E22G was expressed and purified as previously described for Aβ 42 (Ref. 25 ). Synthetic PHF6 (Tau R3 306 VQIVYKPVDLSK 317 ) was purchased as lyophilized peptide from GenScript (GenScript Biotech, Netherlands) with N-and C-termini capped by acetylation and amidation, respectively.  Fluorescence microscopy. Transfected cells were imaged using a Zeiss LSM 880 microscope equipped with a plan-Apo 63X/1.40 Oil objective. Line scan analysis was performed using ImageJ 44 . The values of each line scan was normalized to its maximum value. For quantifying the percentage of cells that displayed co-localization of mCherry-Bri2 BRICHOS or mCherry at N-mutHttQ109-GFP inclusions, cells were scored manually from three independent experiments (n = 35-50). Four μl of resuspended pelleted amyloid fibrils of Aβ 42, α-synuclein, IAPP, Aβ 42 E22G, PHF6, or β17 were transferred to a microscope slide (Thermo Scientific, USA) containing Vectashield mounting medium for fluorescence and cover slip was carefully mounted. Fluorescence images were collected with an EVOS FL Auto 2 imaging system (Invitrogen, USA) using GFP-channel (ex:470/22 nm; em:510/42 nm) to visualize Thioflavin T fluorescence and TX Red-Channel (ex:585/29 nm; em:624/40 nm) to visualize mCherry fluorescence. Fluorescence intensities were measured with ImageJ software 44 .

Transfection of cells. U2OS and HeLa cells were transiently co-transfected with
Immuno EM. The amyloid fibril was incubated with mCherry or rh Bri2 BRICHOS-mCherry for 60 min at 37 °C in the same conditions as above. It was then centrifuged at 12,000 rpm for 10 min to pellet down the fibrils. The supernatant was discarded, and the pellet was washed 3 times with sodium phosphate buffer, and it was diluted to 10 µM for EM sample preparation. 5 µl diluted solution was applied on 200 mesh formvar coated nickel grid and excess solution was removed using blotting paper after 10 min of incubation. It was then washed twice with 10 µl MQ water. The nickel grid surface was blocked with 1% bovine serum albumin (prepared in PBS) for 30 min. It was then washed thrice with MQ water. 5 µl primary anti-RFP antibody (RF5R monoclonal antibody, Thermo Fisher Scientific, USA, 1:200 dilution in PBST) was applied to the grid and it was incubated for 60 min at room temperature. Then, the grids were washed thrice with MQ water. Further, the nickel grids were incubated with anti-mouse IgG-gold (BBI Solutions, Crumlin, UK) (1:40 dilution in PBST) secondary antibody for 60 min. Extra solutions were removed and then it was washed thrice with MQ water and then stained with 1% uranyl formate for 5 min. Extra stained was blotted with blotting paper and it was air-dried. Transmission electron microscopy (FEI Tecnai 12 Spirit BioTWIN, operated at 100 kV) was performed for analysis of fibril morphology using 2 k × 2 k Veleta CCD camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany) 0.15-20 images were recorded for each sample randomly. Images were obtained at a magnification of × 43,000 and × 87,000. For the manuscript × 87,000 magnified images were used.

Scientific Reports
| (2020) 10:21765 | https://doi.org/10.1038/s41598-020-78732-1 www.nature.com/scientificreports/ Surface plasmon resonance. Binding of Bri2 BRICHOS-mCherry and mCherry to Aβ 42 fibrils was measured with a BIACORE 3000 instrument. Sonicated Aβ 42 fibrils were immobilized on a CM5 sensor chips (Cytiva) using standard amine-coupling chemistry. A fresh mixture of 0.05 M NHS and 0.2 M EDC was added to the sensor-chip surface for activation, followed by incubation with 5.6 μM Aβ 42 diluted in 10 mM sodium acetate pH 4.5 and finally deactivation by ethanolamine. Blank channels for negative controls were prepared by omitting protein in the coupling step. Each experiment involved five different protein concentrations of either 313 nM-40 μM mCherry or 39 nM-5 μM Bri2 BRICHOS-mCherry and subsequent buffer flow to monitor dissociation. The experiments were performed with HBS-E (10 mM HEPES, 150 mM NaCl, 0.2 mM EDTA, pH 7.4) as running buffer and a flow rate of 25 μL/min. The chip surface was regenerated between each sample by injection of 30 mM NaOH for 30 s. Analysis of the SPR data was done as previously described 33 .

Data availability
Data are available from the corresponding author upon request.