Binding of serum-derived amyloid-associated proteins to amyloid fibrils

Abstract Background Amyloid signature proteins such as serum amyloid P component, apolipoprotein E (ApoE), and ApoA-IV generally co-localise with amyloid, regardless of the types of amyloid precursor protein or the organs. Most of these proteins derive from serum and have reportedly been involved in amyloid fibril formation and stabilisation, as well as in excretion and degradation of amyloid precursor proteins. However, the processes and mechanisms by which these specific proteins deposit together with amyloid fibrils have not been clarified. Methods We analysed the binding of serum proteins to amyloid fibrils derived from amyloid β and insulin in vitro by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results Specific serum proteins including ApoA-I, ApoE, ApoA-IV, ApoC-III and vitronectin adhered to amyloid fibrils at high concentrations in vitro. In addition, the profile of these proteins commonly occurred in both amyloid β and insulin amyloid fibrils and was mostly consistent with the composition of amyloid signature proteins. We also showed that high concentrations of serum proteins can adhere to amyloid fibrils in a short time. Conclusions Our in vitro results suggest that amyloid signature proteins coexist with amyloid primarily dependent on the binding of each serum protein, in the extracellular fluid, to amyloid fibrils.


Introduction
Amyloidosis consists of a group of diseases characterised by extracellular deposition of amyloid fibrils in various tissues and organs. To date, 36 different precursor proteins including amyloid b (Ab), immunoglobulin light chain (AL), transthyretin (ATTR), and insulin have been determined to cause the different amyloidoses [1]. Amyloid fibrils are formed by self-assembly of misfolded precursor proteins into ordered fibrillar structures. Despite the large variety of amyloid precursor proteins, amyloid fibrils have common structural and biochemical features: an antiparallel b-sheet structure that forms insoluble rigid, nonbranching fibrils, 10 nm in diameter, when viewed with an electron microscope; and apple-green birefringence after Congo red staining and visualisation with polarised light [1].
Sites of amyloid deposition show the coexistence of many types of proteins rather than the occurrence of an amyloid precursor protein alone. Among these proteins, certain ones are universally present regardless of the types of precursor protein or the organs; these proteins are often referred to as amyloid signature proteins [1][2][3][4]. These proteins include serum amyloid P component [5,6], apolipoprotein E (ApoE) [7,8], ApoA-I [9,10], vitronectin [11,12] and clusterin [13,14]. Most amyloid signature proteins derive from serum proteins, and some have reportedly been involved in the formation and stabilisation of amyloid fibrils, as well as in excretion and degradation of amyloid precursor proteins [15][16][17][18]. The co-localisation of amyloid signature proteins in tissues with amyloid deposits has been detected by using immunohistochemistry and proteomic analyses with laser microdissection. However, the significance of these proteins in amyloidogenesis and whether they are essential components of amyloid deposits have been issues of controversy, and the processes and the mechanisms by which these specific proteins deposit together with amyloid fibrils have not been clarified [1].
In this study, we analysed the adhesion of serum proteins to amyloid fibrils, and we showed that a significant amount of amyloid signature proteins preferentially adhered to amyloid fibrils in a short time in vitro.

Formation of Ab and insulin amyloid fibrils
In this study, we used Ab and insulin, which form rigid amyloid fibrils in vitro and cause clinically important amyloidosis. Ab 1-42 peptide (Peptide Institute Inc., Osaka, Japan) was dissolved in phosphate-buffered saline at a concentration of 1.0 mg/mL and was incubated at 37 C with agitation for 48 h. Recombinant human insulin (Humulin R; Eli Lilly, Kobe, Japan) solution (3.6 mg/mL) was mixed with an equal volume of 1 M glycine-HCl buffer at pH 2.5 and was incubated at 55 C with agitation for 48 h. After incubation, each reaction solution was centrifuged (20,000 g, 10 min, 4 C), and the amyloid pellets were washed with saline. This procedure was repeated five times to remove soluble proteins and buffer. Amyloid fibril formation was confirmed by measuring thioflavin T fluorescence and by using electron microscopy as described previously [19].
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of serum proteins bound to amyloid fibrils Serum samples (100 lL) from healthy volunteers (n ¼ 5) were mixed with 100 lL amyloid fibrils solution (2.0 mg/mL, in saline) in 1.5 mL low-protein-binding polypropylene tubes (Eppendorf, Hamburg, Germany), and incubated for 1 h at 37 C without agitation. After the incubation, the solutions were centrifuged (20,000 g, 10 min, 4 C), and the pellets were washed with saline. This procedure was repeated five times to remove soluble proteins and buffer in the supernatant that had not adhered to amyloid fibrils. The pellets were dissolved in Zwittergent (Calbiochem, San Diego, CA) and were incubated at 95 C for 5 min, and then trypsin (Promega, Madison, WI) was added and incubation proceeded at 37 C overnight. After reduction treatment with 25 mM ammonium hydrogen carbonate and 100 mM dithiothreitol, 1.0 mg (protein weight) of samples were analysed via LC-MS/MS by using the Velos Pro Dual-Pressure Linear Ion Trap Mass Spectrometer (Thermo Fisher Scientific, Waltham, MA). MS/MS spectra were searched against the database of Homo sapiens entries (Swiss-Prot) and the ImMunoGeneTics Information System via SEQUEST. We calculated the relative abundances of identified proteins by using the normalised spectral abundance factor (NSAF) with peptide spectrum matches divided by the number of amino acid residues [20,21].

Electrophoresis and Western blotting of serum proteins that adhered to amyloid fibrils
Incubation of amyloid fibrils and serum as well as washing procedures were performed using the same protocols as those described for LC-MS/MS analysis. Amyloid pellets after incubation with serum samples were separated by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gels were stained with Coomassie brilliant blue. Lanes 1-5 were loaded with 10 lL of 100-fold diluted control serum samples, and lanes 6-10 were loaded with serum proteins bound to 10 lg of amyloid fibrils. Western blotting was performed with antibodies against ApoE (Abcam, Cambridge, UK), ApoA-I (Abcam), ApoA-IV (Proteintech, Rosemont, IL), ApoC-III (GeneTex, Irvine, CAA), vitronectin (Proteintech), clusterin (Santa Cruz Biotechnology, Dallas, TX), prothrombin III (GeneTex) and platelet factor 4 (Proteintech).
Kinetics and quantification of serum proteins that adhered to amyloid fibrils The bicinchoninic acid protein quantification method was used to analyse the kinetics and amount of serum proteins that bound to a certain amount of Ab or insulin amyloid fibrils. After incubation of serum samples (1.0 mg/mL, n ¼ 5) with and without Ab or insulin amyloid fibrils (1.0 mg/mL) at 37 C, samples were centrifuged (20,000 g, 10 min, 4 C), and the concentrations of serum proteins in sample supernatants were measured.

Electron microscopy
Ab and insulin amyloid fibrils incubated with and without serum were washed five times with diluted water. Samples were spread on carbon-coated grids (Oken, Tokyo, Japan), stained with 0.2% uranyl acetate, and observed via a transmission electron microscope (HT7700; Hitachi, Tokyo, Japan).

Results
Profile of serum proteins that adhered to amyloid fibrils obtained by using LC-MS/MS analysis Comprehensive in vitro analyses of serum proteins with higher adhesion activity to amyloid fibrils were performed. After 1 h of incubation of serum samples (n ¼ 5) with amyloid fibrils derived from Ab 1-42 or human insulin, proteins that bound to amyloid fibrils were detected by LC-MS/MS. NSAF values of serum proteins arranged in descending order reflected the abundance of each protein in serum (blue bars in Figure 1): albumin, immunoglobulins, apolipoproteins and transferrin. In contrast, the proteins that bound to amyloid fibrils (red bars in Figure 1) were quite different from those in the original serum. Merged profiles of proteins in the original serum and proteins that bound to amyloid fibrils highlighted the proteins that showed considerable adhesion to amyloid fibrils, that is, ApoA-I, ApoA-II, ApoC-III, ApoC-I, ApoA-IV, ApoE, vitronectin, prothrombin III, clusterin and platelet factor 4. The profiles of the proteins that preferentially bound to Ab 1-42 amyloid fibrils (Figure 1(A)) and insulin amyloid fibrils (Figure 1(B)) had similar compositions.
Western blotting of serum proteins that bound to amyloid fibrils SDS-PAGE of proteins that bound to Ab 1-42 amyloid fibrils (Figure 2(A)) and human insulin amyloid fibrils (Figure 2(B)) revealed patterns that were quite different from those for the original serum (n ¼ 5), results that are consistent with those of the LC-MS/MS analyses. In samples of pellets that contained proteins preferentially bound to amyloid, the primary bands in the original serum samples were obscured, and new bands were clearly observed instead (asterisks in Figure 2(A,B)).
Western blotting of the pellets showed that high concentrations of ApoA-I, ApoA-IV, ApoC-III, vitronectin, clusterin and platelet factor 4 bound to insulin amyloid fibrils ( Figure 2). The concentrations of these proteins in the serum supernatants were significantly reduced after incubation with insulin amyloid fibrils (Supplementary Figure 1), findings that are consistent with the preferential binding of these proteins to amyloid.

Morphology of amyloid fibrils with and without serum proteins
The morphology of amyloid fibrils with and without adhesion of serum proteins was not significantly different for both Ab and insulin amyloid fibrils, as Figure 4 shows.

Discussion
In this study, we analysed the binding of serum proteins to amyloid fibrils in vitro. We demonstrated that specific proteins including ApoA-I, ApoC-III, ApoE, ApoA-IV, clusterin and vitronectin showed preferential adhesion to amyloid fibrils. Most of these proteins are known as amyloid signature proteins that universally co-localise with amyloid deposits in all types of amyloidoses such as AL amyloidosis, ATTR amyloidosis, and Alzheimer's disease [1][2][3][4].
Analysis of proteins that deposit with amyloid fibrils has been performed by extracting amyloid deposits from tissue specimens via laser microdissection or by determining colocalisation via immunohistochemistry [4]. Our in vitro study clearly showed that these proteins are not only close to or co-localised with amyloid but also adhere to the amyloid fibrils themselves.
The amyloid signature proteins are believed to derive via interstitial fluid that flows through the extracellular matrix in which amyloid fibrils form and deposit. Most proteins in the interstitial fluid originate from plasma, and the protein composition of the interstitial fluid is quite similar to that of plasma and serum [22], although a small number of proteins are secreted by cells in local tissues.
In our in vitro experiments, certain serum-derived proteins including ApoA-I, ApoA-IV, ApoC-III, ApoE, vitronectin and clusterin demonstrated marked adhesion to amyloid fibrils. This protein profile was surprisingly consistent with that of well-known amyloid signature proteins that have been identified in various tissues containing amyloid deposits. We also showed that amyloid fibrils have adhesion activity for a significant amount of the serum proteinsapproximately 20% of the serum protein weightand that the binding occurs in a very short period. In tissues with amyloid deposits in vivo, a mixture of misfolded amyloid precursor proteins, oligomers and mature amyloid fibrils are seen; still unknown is the point at which amyloid signature proteins involved in amyloid formation co-localise with amyloid. However, our experiments suggest that once amyloid fibrils form in tissues, a significant amount of serum proteins can immediately bind to amyloid fibrils, because a sufficient amount of serum proteins is supplied via the interstitial fluid. The composition of the amyloid signature proteins may be a function of binding activity to amyloid fibrils and the concentration of each protein in the interstitial fluid in tissues with amyloid deposits.
Various effects of amyloid signature proteins on amyloid fibril formation, proteolytic degradation of amyloid fibrils and oligomers, and transport and clearance of amyloid precursor proteins and oligomers have been studied, especially in Alzheimer's disease [23,24]. Genome-wide association studies have confirmed that the e4 allele of ApoE, among the amyloid signature proteins, is a major genetic risk for sporadic Alzheimer's disease. However, the precise effect of amyloid signature proteins in the pathogenesis of amyloidoses remains controversial [15].
Our in vitro study showed that significant amounts and types of amyloid signature proteins can bind to amyloid fibrils rather than just co-localise with the fibrils, so these proteins may be involved in the pathogenesis of amyloidoses in a cooperative or competitive complex manner.
Our study does have limitations: we obtained consistent results with amyloid fibrils derived from insulin and Ab, but we did not investigate other types of precursor proteins. In addition, we did not conduct in vivo studies that would provide information about the binding activity of amyloidassociated proteins other than serum-derived proteins.
In conclusion, we demonstrated that a significant amount of proteins in serum showed preferential binding to amyloid fibrils in vitro and that the protein profile was consistent with that of the well-known amyloid signature proteins.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
This work was supported by JSPS KAKENHI [grant number 19K07869].