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Acceleration of amyloid fibril formation by carboxyl-terminal truncation of human serum amyloid A
Archives of Biochemistry and Biophysics 639 (2018) 9–15
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Acceleration of amyloid fibril formation by carboxyl-terminal truncation of human serum amyloid A
T
Masafumi Tanakaa,∗, Toru Kawakamib, Nozomi Okinoa, Kaoru Sasakia, Kiwako Nakanishia, Hiroka Takasea, Toshiyuki Yamadac, Takahiro Mukaia a
Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan Laboratory of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Suita 565-0871, Japan c Department of Clinical Laboratory Medicine, Jichi Medical University, Shimotsuke 329-0498, Japan b
A R T I C L E I N F O
A B S T R A C T
Keywords: Serum amyloid A Amyloid fibril AA amyloidosis Carboxyl-terminal truncation Native chemical ligation
Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis. Although the full-length SAA is 104 amino acids long, the C-terminal-truncated SAA lacking mainly residues 77–104 is predominantly deposited in AA amyloidosis. Nevertheless, the amyloid fibril formation of such truncated forms of human SAA has never been investigated. In the present study, we examined the effect of C-terminal truncation on amyloid fibril formation of human SAA induced by heparan sulfate (HS). Circular dichroism (CD) measurements demonstrated that the C-terminal truncation induces a reduced α-helical structure of the SAA molecule. HS-induced increases in thioflavin T fluorescence for SAA (1–76) peptide and less significant increases for full-length SAA were observed. CD spectral changes of SAA (1–76) peptide but not full-length SAA were observed when incubated with HS, although the spectrum was not typical for a β-structure. Fourier transform infrared experiments clearly revealed that SAA (1–76) peptide forms a β-sheet structure. Transmission electron microscopy revealed that short fibrillar aggregates of SAA (1–76) peptides, which became longer with increasing peptide concentrations, were observed under conditions in which full-length SAA scarcely formed fibrillar aggregates. These results suggested that the C-terminal truncation of human SAA accelerates amyloid fibril formation.
1. Introduction Amyloid A (AA) amyloidosis, one of the most common forms of lifethreatening systemic amyloidoses, is a complication of chronic inflammatory diseases such as rheumatoid arthritis [1]. Human serum amyloid A (SAA), an acute-phase protein whose concentration is markedly increased during inflammation, is a precursor protein detected in the amyloid deposits of patients with AA amyloidosis. The deposition of amyloid fibrils derived from SAA damages tissue structure and function, especially in the kidney [2]. In humans, there are four SAA genes (SAA1–4), of which SAA1 and SAA2 encode acute-phase proteins, SAA3 is a pseudogene that is not transcribed, and SAA4 encodes a constitutively expressed protein [3]. SAA1 is the main protein component found in AA amyloidosis. It is a 104-amino-acid protein in which the C-terminal region (mainly residues 77–104) is cleaved when deposited as amyloid fibrils. Although the underlying mechanism for this enzymatic cleavage remains to be elucidated, the truncation of the C-terminal region may influence the
amyloidogenic properties of the SAA molecule. However, since the Cterminal-truncated human SAA (namely, amyloid A protein) has not been produced by recombinant expression systems, we have utilized its shorter fragment peptide and full-length protein for the evaluation of amyloidogenic properties [4–6]. In the present study, we planned to obtain chemically the peptide corresponding to residues 1–76 of the SAA molecule. The peptide ligation method, which is achieved by the coupling of two or more peptide segments, was originally developed for the chemical synthesis of longer polypeptides possessing more than 50 amino acid residues [7]. In addition, synthetic yields of SAA (1–76) peptide were predicted to be markedly lower as the peptide elongates by conventional solid-phase peptide synthesis since the N-terminal region of the SAA molecule has a high propensity to aggregate. Thus, we decided to synthesize SAA (1–76) peptide by the peptide ligation method. Heparan sulfate (HS), a glycosaminoglycan (GAG) present in the extracellular matrix, is frequently detected in amyloid deposits, suggestive of its facilitating role in fibril formation [8]. In fact, GAG
Abbreviations: ATR-FTIR, Attenuated total reflection-fourier transform infrared; CD, Circular dichroism; HS, Heparan sulfate; SAA, Serum amyloid A; TEM, Transmission electron microscopy; ThT, Thioflavin T ∗ Corresponding author. Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. E-mail address: [email protected] (M. Tanaka). https://doi.org/10.1016/j.abb.2017.12.016 Received 16 November 2017; Received in revised form 12 December 2017; Accepted 21 December 2017 Available online 26 December 2017 0003-9861/ © 2017 Elsevier Inc. All rights reserved.
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For fibril formation experiments, HS was added to protein or peptide samples, followed by incubation at 37 °C without agitation.
promoted the rapid fibrillization of a peptide even with low intrinsic amyloidogenicity in vitro [9]. The precise role of GAG in the promotion of fibril formation is unclear, but it probably contributes to the initiation of amyloid formation as a scaffold [10], which is supported by the in vivo observation that over-expression of heparanase prevented amyloid deposition in mice [11]. The C-terminal region of the SAA molecule is considered to possess an HS-binding site [12,13]. Thus, the fibril formation of SAA induced by HS is supposed to be affected when the C-terminal region is truncated. SAA1 has three major isoforms, SAA1.1, SAA1.3, and SAA1.5, which vary in their central region at positions 52 and 57 [14]. Among the three SAA1 isoforms, SAA1.3 is associated with the highest risk of AA amyloidosis in the Japanese population [15]. In contrast, SAA1.1 is associated with the highest risk of AA amyloidosis in Caucasians [16]. Although the reasons for these discrepancies are still unknown, they are probably due to the overwhelming majority of Caucasians having the SAA1.1 isoform, but the frequencies of the three isoforms being approximately equal in Japanese. In the present study, using SAA (1–76) peptide obtained by the ligation method, we examined the effect of Cterminal truncation of human SAA1.3 on the amyloidogenic properties induced by HS.
Far-ultraviolet CD measurements were performed with a Jasco J820 spectropolarimeter (Hachioji, Japan). The results were corrected by subtracting the baseline of an appropriate blank sample. The mean residual ellipticity ([θ]) was calculated using the equation [θ] = (MRW) θ/101c, where θ is the measured ellipticity in degrees, 1 is the cuvette path length (0.2 cm), c is the peptide or protein concentration in g/mL, and the mean residue weight (MRW) is obtained from the molecular mass and the number of amino acids.
2. Material and methods
2.6. Fourier transform infrared (FTIR) spectroscopy
2.1. Materials
Attenuated total reflection (ATR)-FTIR spectra were recorded at ambient temperature (∼25 °C) on a Jasco FT/IR-4200 Fourier transform infrared spectrometer (Jasco Co., Tokyo, Japan) purged with N2 and equipped with a Mercury-Cadmium-Telluride detector. Samples immediately after the CD measurements were dried on the surface of an ATR plate for the FTIR measurements. For each sample, 256 interferograms were averaged at a spectral resolution of 4 cm−1 and processed using one-point zero-filling and Hamming apodization.
2.4. Fluorescence spectroscopy All fluorescence measurements were performed on a Hitachi F-7000 spectrophotometer (Tokyo, Japan). ThT fluorescence spectra were recorded in a 4 × 4 mm cuvette from 450 to 600 nm at an excitation wavelength of 440 nm. The ThT and protein or peptide concentrations were 10 μM and 50 μg/mL, respectively. 2.5. Circular dichroism (CD) spectroscopy
Heparan sulfate (HS) and thioflavin T (ThT) were purchased from Iduron Ltd. (Manchester, UK) and Sigma-Aldrich (St. Louis, MO), respectively. Fmoc amino acid derivatives were obtained from the Peptide Institute, Inc. (Minoh, Japan). The recombinant human SAA1.3, hereafter designated as SAA1.3 m because of a single methionine residue at the N-terminus, was produced in Escherichia coli as previously described [17]. Unless otherwise noted, 20 mM Tris buffer (pH 7.4) or 10 mM acetate buffer (pH 4.0) was used.
2.7. Transmission electron microscopy (TEM) Samples containing SAA peptides were spread on Formvar filmcoated copper grids (400 mesh) and were negatively stained with 2% sodium phosphotungstate (pH 7.0). The grids were observed by a JEM1400Plus transmission electron microscope (JEOL, Akishima, Japan) with an acceleration voltage of 80 kV. Digital images were acquired with a CCD camera.
2.2. Peptide synthesis Peptide synthesis by the ligation method was performed essentially as described previously [18]. Two peptide segments, SAA1.3 (1–44)Cys-Pro-OCH2COTle-NH2 and Cys-SAA1.3 (46–76), were prepared by standard Fmoc chemistry using an automated peptide synthesizer, ACT440Ω (AAPPTec, Louisville, KY) or Liberty Blue (CEM Corporation, Matthews, NC). After cleavage of peptides from the resin, they were purified by reversed-phase high-performance liquid chromatography (YMC-Pack ProC18 or Cosmosil 5C18-AR-II, 10 × 250 mm). Ligation reactions were performed under the conditions of 50 mM 4-mercaptophenylacetic acid, 20 mM Tris (2-carboxyethyl)phosphine, and 6.0 M guanidine hydrochloride in sodium phosphate buffer (pH 8.0) at 37 °C overnight. Desulfurization reactions were performed under the conditions of 10 mM 2,2′-azobis [2-(2-imidazolin-2-yl)propane] dihydrochloride, 0.10 M sodium 2-mercaptoethanesulfonate, 0.15 M Tris (2carboxyethyl)phosphine, and 6.0 M guanidine hydrochloride in sodium phosphate buffer (pH 7.0) at 37 °C overnight. After the final purification, peptide structures were confirmed by electrospray ionization mass spectrometry (Thermo Finnigan LCQ Deca XP spectrometer) or matrixassisted laser desorption ionization-time of flight mass spectrometry (Bruker AutoFLEX spectrometer) and amino acid analysis (Hitachi L2000 amino acid analyzer).
3. Results 3.1. Peptide design and analytical validation The amino acid sequence of the SAA1.3 (1–76) peptide is shown in Fig. 1. Owing to the length and the propensity for aggregation, especially in the N-terminal side of the sequence, we decided to employ the ligation method. Although a Cys residue will always be inserted by native chemical ligation, it can be converted into an Ala residue by a desulfurization reaction [19]. Since there are no Cys residues in the SAA sequence, ligation at the Ala residue at position 45 was chosen to consider reducing the deleterious side reactions. Details of the reaction
2.3. Sample preparation Lyophilized protein or peptides were dissolved in 4 M urea and freshly dialyzed into buffer extensively, and were centrifuged to remove insoluble or aggregated matter before use. Samples were kept at 4 °C throughout the preparation procedure. Protein or peptide concentrations were determined by measuring the absorption at 280 nm (A280).
Fig. 1. Amino acid sequence of SAA1.3 (1–76) peptide. Ligation site is indicated by an arrow. Amino acid positions which differ in other two human SAA isoforms are shown in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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3.3. Determination of experimental conditions for amyloid fibril formation Previously, we examined fibril formation of SAA molecules induced by the addition of heparin under acidic conditions (pH4.0) because SAA did not form amyloid fibrils in phosphate buffer (pH7.4) and acidification was required to facilitate it [5]. However, further observations revealed that the composition of the buffer influences the fibril formation. Hence, we first examined the effect of pH on the amyloid fibril formation of SAA1.3 (1–76) peptide upon the addition of heparin by measuring the fluorescence of ThT, an amyloidophilic dye, commonly used for the detection of a cross-β structure [22]. ThT fluorescence was observed at pH7.4 (20 mM Tris buffer), although the intensities were reduced compared with those at pH4.0 (10 mM acetate buffer) (Fig. S1), implying that the fibril formation of SAA1.3 (1–76) peptide occurs even at neutral pH. A cross-β structure is one of the hallmark features of amyloid fibrils [23]. The CD spectrum of SAA1.3 (1–76) peptide in the presence of heparin at pH4.0 was typical for a β-structure, with a single minimum at approximately 220 nm (Fig. S2A). Nevertheless, atomic force microscopy (AFM) images exhibited spherical aggregates probably because of the strong interactions between heparin and peptides that dominate the intermolecular association of peptides to elongate fibrils (Fig. S3A). In our previous study, such spherical morphology was observed when SAA peptide was incubated with heparin, which turned to become typical short fibrils by incubating with HS instead of heparin under the same experimental conditions [24]. In contrast, at pH7.4, although heparin induced some conformational changes of the SAA1.3 (1–76) peptide, the CD spectrum was not typical for a β-structure (Fig. S2B), but AFM images exhibited worm-like protofibrils (Fig. S3B). Taking these findings together, in the present study, we decided to evaluate the amyloid fibril formation of SAA1.3 (1–76) peptide in the presence of HS at pH7.4.
Scheme 1. Synthetic scheme for the preparation of SAA1.3 (1–76) peptide.
mechanisms of ligation and desulfurization are shown in Scheme 1. After the final purification, the mass of 8548.7 ± 0.9 (deconvoluted value) as observed by electrospray ionization mass spectrometry was equivalent to the calculated mass of 8548.3 for (M + H)+ (average) and the amino acid composition of the purified peptide was Asp10.2Ser5.5Glu6.2Pro1.3Gly9.3Ala13Met1.3Ile2.9Leu1.1Tyr4.2Phe6.9Lys2.1His2.0Arg8.1 (Table S1).
3.2. Influence of C-terminal truncation on the secondary structure of SAA Secondary structures of full-length SAA1.3 m and SAA1.3 (1–76) peptide were analyzed by CD spectroscopy immediately after the sample preparation. As shown previously in 10 mM phosphate buffer (pH 7.4), full-length SAA1.3 m in 20 mM Tris buffer (pH 7.4) exhibited a CD spectrum with double minima at approximately 208 and 222 nm at 4 °C, while showing a single minimum of approximately 200 nm at 37 °C (Fig. 2A). In contrast, the CD spectra for SAA1.3 (1–76) peptide at 4 °C and 37 °C were entirely distinct from those for fulllength SAA1.3 m (Fig. 2B), suggesting that the C-terminal truncation induces conformational changes of the SAA molecule. CD spectral deconvolution analyses using BeStSel software revealed that full-length SAA1.3 m and SAA1.3 (1–76) peptide possess similar amounts of βsheet structure based on the number of amino acid residues (Table S2) [20]. In terms of the characteristic that distinguished them, the amount of helix structure was markedly decreased in SAA1.3 (1–76) peptide, signifying that the truncated C-terminal region contains a helical structure or induces it somewhere else in the molecule by intramolecular interactions. These results are consistent with the latest findings that the absence of the C-terminal tail of murine SAA1.1 causes a significant loss in α-helical structure [21], although the spectral shapes of truncated forms of human and murine SAA are considerably different.
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Even in the absence of HS incubated at 37 °C for 24 h, faint ThT fluorescence was observed in both full-length SAA1.3 m and SAA1.3 (1–76) peptide at pH7.4 (Fig. 3A and B). At 24 h after the addition of HS at a concentration of 50 μg/mL, increases in the ThT fluorescence were observed (Fig. 3A and B), which was more prominent for SAA1.3 (1–76) peptide than for full-length SAA1.3 m. The effect of HS concentrations on the time-dependent changes in ThT fluorescence was also monitored (Fig. 3C and D). In SAA1.3 (1–76) peptide, ThT fluorescence increased without an observable lag time regardless of the HS concentration, at least in the range of 25–100 μg/mL, and almost plateaued within 24 h. In contrast, less significant increases and no enhancing effects of HS on ThT fluorescence for full-length SAA1.3 m were observed. These results suggest that the C-terminal truncation facilitates fibril formation of the SAA molecule upon the addition of HS.
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3.4. Amyloid fibril formation of SAA molecules monitored by ThT fluorescence
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Fig. 2. CD spectra of (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide at 4 °C (blue lines) and 37 °C (red lines) in 20 mM Tris buffer (pH 7.4). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Archives of Biochemistry and Biophysics 639 (2018) 9–15
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Fig. 3. Representative ThT fluorescence spectra for (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide in the absence (dotted lines) or presence (solid lines) of HS (50 μg/mL, incubated at 37 °C for 24 h) in 20 mM Tris buffer (pH 7.4). Concentration dependence of HS on the ThT fluorescence kinetics for (C) the full-length SAA1.3 m and (D) the SAA1.3 (1–76) peptide at the HS concentrations of 0 (white), 25 (green), 50 (black), and 100 μg/mL (cyan). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
sheet architecture. However, the bands were influenced neither by the addition of HS nor the C-terminal truncation. Since samples are dried on the surface of an ATR plate, the protein or peptide concentrations are markedly elevated during the drying process, which markedly facilitated the formation of a β-sheet structure. Overall, taking these findings together with the ThT fluorescence results, it is conceivable that SAA1.3 (1–76) peptide has the propensity to form amyloid fibrils, which is facilitated by the presence of HS.
3.5. Conformational changes of SAA molecules monitored by CD and FTIR During the incubation in the absence of HS at 37 °C for 24 h, almost no significant changes were observed in the CD spectra for both fulllength SAA1.3 m and SAA1.3 (1–76) peptide (Fig. 4 compared with Fig. 2). Upon incubation with HS, CD spectral changes of full-length SAA1.3 m were subtle, but the changes were pronounced in SAA1.3 (1–76) peptide (Fig. 4). However, despite the increase in the ThT fluorescence, especially in SAA1.3 (1–76) peptide incubated in the presence of HS, the CD spectrum was not typical for a β-structure, distinct from the case at pH4.0 (Fig. S2A). The secondary structures of full-length SAA1.3 m and SAA1.3 (1–76) peptide in the absence or presence of HS were further examined by ATR-FTIR spectroscopy, which can more sensitively detect the presence of a β-sheet structure [25]. The amide I band that appears around the wavenumbers of 1600–1700 cm−1 is widely used for characterizing the secondary structure components of proteins [26]. In contrast to the CD results, each FTIR spectrum for the amide I region showed an absorbance maximum around 1625–1630 cm−1 (Fig. 5), suggesting that full-length SAA1.3 m and SAA1.3 (1–76) peptide possess a common β-
3.6. Morphological characterization of SAA aggregates visualized by TEM To verify the amyloid fibril formation, TEM images of samples containing full-length SAA1.3 m or SAA1.3 (1–76) peptide incubated in the absence and presence of HS were obtained (Fig. 6). In the presence of 50 μg/mL HS, full-length SAA1.3 m scarcely exhibited fibrillar aggregates (Fig. 6A). In contrast, SAA1.3 (1–76) peptide in the presence of 50 μg/mL HS exhibited short fibrillar aggregates (Fig. 6B), which was also observed in the absence of HS (Fig. 6C). Although neither the ThT fluorescence nor the TEM results reflect the amounts of amyloid fibrils, these observations imply that the C-terminal truncation of human SAA
Fig. 4. CD spectra of (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide at 37 °C after 24 h of incubation in the absence (dotted lines) or presence (black lines) of HS (50 μg/ mL) in 20 mM Tris buffer (pH 7.4).
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Fig. 5. IR absorbance spectra of (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide in the absence (dotted lines) or presence (solid lines) of HS.
accelerates amyloid fibril formation. When the SAA1.3 (1–76) peptide concentrations were increased from 50 to 200 μg/mL without changing the HS concentration, the short aggregates became elongated fibrils (Fig. 6D). In the absence of HS, such elongated fibrils were sparse but infrequently observed in spots.
during the purification and preparation processes. In fact, both fulllength SAA1.3 m and SAA1.3 (1–76) peptide exhibited evidence of carbamylation after the experiments, as determined by mass spectrometry analyses, although the degree of carbamylation was unclear. Thus, the effect of the N-terminal methionine and carbamylation, if any, needs to be prudently taken into account when comparing the amyloid fibril formation of full-length SAA1.3 and SAA1.3 (1–76) peptide. HS polysaccharides are ubiquitously expressed on the cell surface and extracellular matrix of animal tissues as components of proteoglycans. HS is composed of glucuronic acid (GlcA) or iduronic acid (IdoA) alternating with glucosamine that is either N-acetylated (GlcNAc) or N-sulfated (GlcNS). The degree of sulfation affects the facilitating effect of amyloid formation [24,31]. Thus, HS can be a regulating factor for amyloidogenesis by modulating the degree of sulfation. Differences in HS structures among organs may explain why amyloid is deposited in specific organs. The N- and C-terminal regions of the SAA molecule are considered to be critical for the heparin-induced amyloid formation and for the heparin/HS-binding, respectively [4,12]. Consequently, when the full-length SAA molecules were incubated with HS, binding of the C-terminal region to HS can compete with and hamper the HS-induced amyloid formation mediated by the Nterminal region. Therefore, in the present study, the C-terminal truncation could conceivably facilitate the fibril formation of SAA molecule in the presence of HS. SAA in amyloid deposits is generally found as the C-terminal-truncated form, which raises questions as to whether the cleavage is a prerequisite for the amyloid fibril formation and where or when it happens. Although several proteolytic enzymes have been proposed to cleave SAA molecule [32], which specific one contributes to the generation of amyloidogenic fragments is also unknown. In some proteins, proteolytic cleavage evoked by protein misfolding exposes amyloidogenic regions, which facilitates the amyloid fibril formation. Our finding that the C-terminal truncation accelerates the amyloid fibril
4. Discussion It has been reported that the truncation of the final 13 amino acids from the C-terminus of murine SAA2.2 retards the rate of fibril formation [27]. In the present study, we showed for the first time that the amyloid fibril formation of human SAA1.3 is accelerated by the truncation of the C-terminal region, which seems inconsistent with the mouse results. Pairwise sequence alignment using the NeedlemanWunsch algorithm revealed that human SAA1.3 and murine SAA2.2 share approximately 80% similarity. Additionally, the theoretical isoelectric points (pI) of these molecules are also similar (pI = ∼5.8). However, unlike human SAA1.3, it is noteworthy that the murine SAA2.2 isoform produced by the CE/J mouse strain does not form amyloid fibrils in vivo [28]. Because of the slower fibrillation rate, it is plausible that the C-terminal-truncated murine SAA2.2 is degraded into smaller fragments, which may serve to prevent amyloid fibril formation. Subtle differences in the amino acid sequence or the modifications of SAA strikingly impact on the amyloidogenicity. For example, the addition of a single N-terminal methionine greatly enhances the propensity for fibrillation and modulates the fibrillation pathway of human SAA [29]. More recently, it has been pointed out that carbamylation at the N-terminus of murine SAA influences the amyloid formation in a cell culture model [30]. Bacterially expressed full-length SAA1.3 contains a single N-terminal methionine, whereas chemically synthesized SAA1.3 (1–76) peptide contains no N-terminal methionine. In addition, it is likely that the samples are carbamylated since we utilized urea
Fig. 6. TEM images of the aggregates of (A) the full-length SAA1.3 m and (B–D) the SAA1.3 (1–76) peptide. Fibril formation was conducted at peptide concentrations of (A–C) 50 μg/mL and (D) 200 μg/mL in the (C) absence or (A, B, D) presence of HS (50 μg/mL) in 20 mM Tris buffer (pH 7.4). Scale bars indicate 200 nm.
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formation of SAA molecules prompted us to speculate that the removal of the C-terminal region occurs before fibril formation. However, fulllength SAA is not necessarily unable to form amyloid fibrils, but rather possesses amyloidogenic potential, especially when the concentration increases, as implied by the FTIR experiments. Similarly, the truncation of murine SAA1.1, the sole isoform found in amyloid deposits in mice, is no prerequisite for amyloid formation [21]. Thus, the idea that the Cterminal truncation is a secondary event after the fibril formation cannot be completely ruled out yet. In fact, the proteolytic cleavage of SAA after the incorporation into fibrils has been proposed, although the data in support of this were obtained with murine SAA [33]. The susceptibility to proteolysis can also be influenced by the existence of lipid, as discussed below. One of the major concerns in amyloid studies of SAA is the influences of lipid, since SAA usually exists in a form bound to lipid [34]. Although crystallographic analysis revealed that human SAA is highly α-helical [35], lipid-free SAA in solution is intrinsically disordered at physiological temperature [36]. Lipid binding of SAA molecule induces stabilization of α-helical conformation, which serves to inhibit fibril formation [6]. Conformational changes upon lipid binding may also affect the interaction with glycosaminoglycans and the susceptibility to proteolysis or vice versa. In fact, HS has been shown to dissociate SAA from high-density lipoproteins, thereby facilitating amyloid fibril formation [37]. Alternatively, we have shown that lipid binding rendered SAA molecules resistant to protein degradation [38]. Furthermore, the lipid-bound conformation of SAA might be modulated by the alterations in lipid composition, which has been revealed to vary under chronic inflammatory conditions [39]. To obtain a comprehensive understanding of the detailed pathogenic mechanisms underlying AA amyloidosis, the amyloidogenic pathway of SAA should be argued in conjunction with the lipid metabolism. Thanks to the availability of biopharmaceuticals to control the underlying diseases such as rheumatoid arthritis, the number of patients with AA amyloidosis is decreasing. However, it has been reported that obesity-enhanced SAA secretion can be a risk factor for the development of AA amyloidosis [40]. In addition, there are several cases in which no clear-cut history of a defined chronic inflammatory condition was present at diagnosis or in which the underlying disease ultimately remains unknown [41]. Among systemic amyloidoses, model animals with AA amyloidosis, which can easily be induced by inflammatory stimuli, are most widely employed. Knowledge of the mechanisms of formation and the structures of amyloid fibrils gained from AA amyloidosis can be expanded to apply to other types of systemic amyloidoses. Thus, AA amyloidosis still warrants further investigation in order to develop treatments common to systemic amyloidoses. Further studies are required to uncover the multilayered mechanisms involved in order to elucidate the pathogenesis of AA amyloidosis.
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Pepys, SAA1alleles as risk factors in reactive systemic AA amyloidosis, Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis 5 (2009) 262–265. [17] T. Yamada, B. Kluve-Beckerman, J.J. Liepnieks, M.D. Benson, Fibril formation from recombinant human serum amyloid A, Biochim. Biophys. Acta 1226 (1994) 323–329. [18] T. Kawakami, R. Yoshikawa, Y. Fujiyoshi, Y. Mishima, H. Hojo, S. Tajima, I. Suetake, Synthesis of histone proteins by CPE ligation using a recombinant peptide as the C-terminal building block, J. Biochem. 158 (2015) 403–411. [19] Q. Wan, S.J. Danishefsky, Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides, Angew. Chem. Int. Ed. Engl. 46 (2007) 9248–9252. [20] A. Micsonai, F. Wien, L. Kernya, Y.H. Lee, Y. Goto, M. Refregiers, J. Kardos, Accurate secondary structure prediction and fold recognition for circular dichroism spectroscopy, Proc. Natl. Acad. Sci. U.S.A. 112 (2015) E3095–E3103. [21] M. Rennegarbe, I. Lenter, A. Schierhorn, R. Sawilla, C. Haupt, Influence of Cterminal truncation of murine Serum amyloid A on fibril structure, Sci. Rep. 7 (2017) 6170. [22] H. Naiki, K. Higuchi, M. Hosokawa, T. Takeda, Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1, Anal. Biochem. 177 (1989) 244–249. [23] M. Lopez De La Paz, K. Goldie, J. Zurdo, E. Lacroix, C.M. Dobson, A. Hoenger, L. Serrano, De novo designed peptide-based amyloid fibrils, Proc. Natl. Acad. Sci. U.S.A 99 (2002) 16052–16057. [24] H. Takase, M. Tanaka, A. Yamamoto, S. Watanabe, S. Takahashi, S. Nadanaka, H. Kitagawa, T. Yamada, T. Mukai, Structural requirements of glycosaminoglycans for facilitating amyloid fibril formation of human serum amyloid A, Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis 23 (2016) 67–75. [25] R. Sarroukh, E. Goormaghtigh, J.M. Ruysschaert, V. Raussens, ATR-FTIR: a "rejuvenated" tool to investigate amyloid proteins, Biochim. Biophys. Acta 1828 (2013) 2328–2338. [26] H. Hiramatsu, T. Kitagawa, FT-IR approaches on amyloid fibril structure, Biochim. Biophys. Acta 1753 (2005) 100–107. [27] S. Patke, R. Maheshwari, J. Litt, S. Srinivasan, J.J. Aguilera, W. Colon, R.S. Kane, Influence of the carboxy terminus of serum amyloid A on protein oligomerization, misfolding, and fibril formation, Biochemistry 51 (2012) 3092–3099. [28] J.D. Sipe, I. Carreras, W.A. Gonnerman, E.S. Cathcart, M.C. de Beer, F.C. de Beer, Characterization of the inbred CE/J mouse strain as amyloid resistant, Am. J. Pathol. 143 (1993) 1480–1485. [29] S. Patke, S. Srinivasan, R. Maheshwari, S.K. Srivastava, J.J. Aguilera, W. Colon, R.S. Kane, Characterization of the oligomerization and aggregation of human Serum Amyloid A, PLos One 8 (2013) e64974. [30] B. Kluve-Beckerman, J.J. Liepnieks, M.D. Benson, X. Lai, G. Qi, M. Wang, Carbamylation of the amino-terminal residue (Gly1) of mouse serum amyloid A
Acknowledgements This work was performed in part under the Cooperative Research Program of the Institute for Protein Research, Osaka University, CR-1501. We thank Ms. Naho Onoe and Ms. Kaho Matsumoto for their technical assistance. Part of this research was supported by JSPS KAKENHI Grant Number 15K08436. This publication was also subsidized by JKA through its promotion funds from KEIRIN RACE. The authors would like to thank Enago for the English language review. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.abb.2017.12.016. References [1] G.T. Westermark, M. Fandrich, P. Westermark, AA amyloidosis: pathogenesis and
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promotes amyloid formation in a cell culture model, FEBS Lett. 590 (2016) 4296–4307. J.J. Aguilera, F. Zhang, J.M. Beaudet, R.J. Linhardt, W. Colon, Divergent effect of glycosaminoglycans on the in vitro aggregation of serum amyloid A, Biochimie 104 (2014) 70–80. C. Rocken, R. Menard, F. Buhling, S. Vockler, J. Raynes, B. Stix, S. Kruger, A. Roessner, T. Kahne, Proteolysis of serum amyloid A and AA amyloid proteins by cysteine proteases: cathepsin B generates AA amyloid proteins and cathepsin L may prevent their formation, Ann. Rheum. Dis. 64 (2005) 808–815. R. Kisilevsky, S. Narindrasorasak, C. Tape, R. Tan, L. Boudreau, During AA amyloidogenesis is proteolytic attack on serum amyloid a a pre- or post- fibrillogenic event? Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis 1 (1994) 174–183. R. Kisilevsky, P.N. Manley, Acute-phase serum amyloid A: perspectives on its physiological and pathological roles, Amyloid Int. J. Exp. Clin. Investig. Off. J. Int. Soc. Amyloidosis 19 (2012) 5–14. J. Lu, Y. Yu, I. Zhu, Y. Cheng, P.D. Sun, Structural mechanism of serum amyloid Amediated inflammatory amyloidosis, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 5189–5194. N.M. Frame, S. Jayaraman, D.L. Gantz, O. Gursky, Serum amyloid A self-assembles with phospholipids to form stable protein-rich nanoparticles with a distinct
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structure: a hypothetical function of SAA as a "molecular mop" in immune response, J. Struct. Biol. 200 (2017) 293–302, http://dx.doi.org/10.1016/j.jsb.2017.06.007. F. Noborn, J.B. Ancsin, W. Ubhayasekera, R. Kisilevsky, J.P. Li, Heparan sulfate dissociates serum amyloid A (SAA) from acute-phase high-density lipoprotein, promoting SAA aggregation, J. Biol. Chem. 287 (2012) 25669–25677. H. Takase, H. Furuchi, M. Tanaka, T. Yamada, K. Matoba, K. Iwasaki, T. Kawakami, T. Mukai, Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A, Biochim. Biophys. Acta 1842 (2014) 1467–1474. L. Gomez Rosso, M. Lhomme, T. Merono, P. Sorroche, L. Catoggio, E. Soriano, C. Saucedo, V. Malah, C. Dauteuille, L. Boero, P. Lesnik, P. Robillard, M. John Chapman, F. Brites, A. Kontush, Altered lipidome and antioxidative activity of small, dense HDL in normolipidemic rheumatoid arthritis: relevance of inflammation, Atherosclerosis 237 (2014) 652–660. R.A. van der Heijden, J. Bijzet, W.C. Meijers, G.K. Yakala, R. Kleemann, T.Q. Nguyen, R.A. de Boer, C.G. Schalkwijk, B.P. Hazenberg, U.J. Tietge, P. Heeringa, Obesity-induced chronic inflammation in high fat diet challenged C57BL/6J mice is associated with acceleration of age-dependent renal amyloidosis, Sci. Rep. 5 (2015) 16474. L. Obici, G. Merlini, AA amyloidosis: basic knowledge, unmet needs and future treatments, Swiss Med. Wkly. 142 (2012) w13580.
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Acceleration of amyloid fibril formation by carboxyl-terminal truncation of human serum amyloid A
T
Masafumi Tanakaa,∗, Toru Kawakamib, Nozomi Okinoa, Kaoru Sasakia, Kiwako Nakanishia, Hiroka Takasea, Toshiyuki Yamadac, Takahiro Mukaia a
Department of Biophysical Chemistry, Kobe Pharmaceutical University, Kobe 658-8558, Japan Laboratory of Protein Organic Chemistry, Institute for Protein Research, Osaka University, Suita 565-0871, Japan c Department of Clinical Laboratory Medicine, Jichi Medical University, Shimotsuke 329-0498, Japan b
A R T I C L E I N F O
A B S T R A C T
Keywords: Serum amyloid A Amyloid fibril AA amyloidosis Carboxyl-terminal truncation Native chemical ligation
Human serum amyloid A (SAA) is a precursor protein of AA amyloidosis. Although the full-length SAA is 104 amino acids long, the C-terminal-truncated SAA lacking mainly residues 77–104 is predominantly deposited in AA amyloidosis. Nevertheless, the amyloid fibril formation of such truncated forms of human SAA has never been investigated. In the present study, we examined the effect of C-terminal truncation on amyloid fibril formation of human SAA induced by heparan sulfate (HS). Circular dichroism (CD) measurements demonstrated that the C-terminal truncation induces a reduced α-helical structure of the SAA molecule. HS-induced increases in thioflavin T fluorescence for SAA (1–76) peptide and less significant increases for full-length SAA were observed. CD spectral changes of SAA (1–76) peptide but not full-length SAA were observed when incubated with HS, although the spectrum was not typical for a β-structure. Fourier transform infrared experiments clearly revealed that SAA (1–76) peptide forms a β-sheet structure. Transmission electron microscopy revealed that short fibrillar aggregates of SAA (1–76) peptides, which became longer with increasing peptide concentrations, were observed under conditions in which full-length SAA scarcely formed fibrillar aggregates. These results suggested that the C-terminal truncation of human SAA accelerates amyloid fibril formation.
1. Introduction Amyloid A (AA) amyloidosis, one of the most common forms of lifethreatening systemic amyloidoses, is a complication of chronic inflammatory diseases such as rheumatoid arthritis [1]. Human serum amyloid A (SAA), an acute-phase protein whose concentration is markedly increased during inflammation, is a precursor protein detected in the amyloid deposits of patients with AA amyloidosis. The deposition of amyloid fibrils derived from SAA damages tissue structure and function, especially in the kidney [2]. In humans, there are four SAA genes (SAA1–4), of which SAA1 and SAA2 encode acute-phase proteins, SAA3 is a pseudogene that is not transcribed, and SAA4 encodes a constitutively expressed protein [3]. SAA1 is the main protein component found in AA amyloidosis. It is a 104-amino-acid protein in which the C-terminal region (mainly residues 77–104) is cleaved when deposited as amyloid fibrils. Although the underlying mechanism for this enzymatic cleavage remains to be elucidated, the truncation of the C-terminal region may influence the
amyloidogenic properties of the SAA molecule. However, since the Cterminal-truncated human SAA (namely, amyloid A protein) has not been produced by recombinant expression systems, we have utilized its shorter fragment peptide and full-length protein for the evaluation of amyloidogenic properties [4–6]. In the present study, we planned to obtain chemically the peptide corresponding to residues 1–76 of the SAA molecule. The peptide ligation method, which is achieved by the coupling of two or more peptide segments, was originally developed for the chemical synthesis of longer polypeptides possessing more than 50 amino acid residues [7]. In addition, synthetic yields of SAA (1–76) peptide were predicted to be markedly lower as the peptide elongates by conventional solid-phase peptide synthesis since the N-terminal region of the SAA molecule has a high propensity to aggregate. Thus, we decided to synthesize SAA (1–76) peptide by the peptide ligation method. Heparan sulfate (HS), a glycosaminoglycan (GAG) present in the extracellular matrix, is frequently detected in amyloid deposits, suggestive of its facilitating role in fibril formation [8]. In fact, GAG
Abbreviations: ATR-FTIR, Attenuated total reflection-fourier transform infrared; CD, Circular dichroism; HS, Heparan sulfate; SAA, Serum amyloid A; TEM, Transmission electron microscopy; ThT, Thioflavin T ∗ Corresponding author. Department of Biophysical Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan. E-mail address: [email protected] (M. Tanaka). https://doi.org/10.1016/j.abb.2017.12.016 Received 16 November 2017; Received in revised form 12 December 2017; Accepted 21 December 2017 Available online 26 December 2017 0003-9861/ © 2017 Elsevier Inc. All rights reserved.
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For fibril formation experiments, HS was added to protein or peptide samples, followed by incubation at 37 °C without agitation.
promoted the rapid fibrillization of a peptide even with low intrinsic amyloidogenicity in vitro [9]. The precise role of GAG in the promotion of fibril formation is unclear, but it probably contributes to the initiation of amyloid formation as a scaffold [10], which is supported by the in vivo observation that over-expression of heparanase prevented amyloid deposition in mice [11]. The C-terminal region of the SAA molecule is considered to possess an HS-binding site [12,13]. Thus, the fibril formation of SAA induced by HS is supposed to be affected when the C-terminal region is truncated. SAA1 has three major isoforms, SAA1.1, SAA1.3, and SAA1.5, which vary in their central region at positions 52 and 57 [14]. Among the three SAA1 isoforms, SAA1.3 is associated with the highest risk of AA amyloidosis in the Japanese population [15]. In contrast, SAA1.1 is associated with the highest risk of AA amyloidosis in Caucasians [16]. Although the reasons for these discrepancies are still unknown, they are probably due to the overwhelming majority of Caucasians having the SAA1.1 isoform, but the frequencies of the three isoforms being approximately equal in Japanese. In the present study, using SAA (1–76) peptide obtained by the ligation method, we examined the effect of Cterminal truncation of human SAA1.3 on the amyloidogenic properties induced by HS.
Far-ultraviolet CD measurements were performed with a Jasco J820 spectropolarimeter (Hachioji, Japan). The results were corrected by subtracting the baseline of an appropriate blank sample. The mean residual ellipticity ([θ]) was calculated using the equation [θ] = (MRW) θ/101c, where θ is the measured ellipticity in degrees, 1 is the cuvette path length (0.2 cm), c is the peptide or protein concentration in g/mL, and the mean residue weight (MRW) is obtained from the molecular mass and the number of amino acids.
2. Material and methods
2.6. Fourier transform infrared (FTIR) spectroscopy
2.1. Materials
Attenuated total reflection (ATR)-FTIR spectra were recorded at ambient temperature (∼25 °C) on a Jasco FT/IR-4200 Fourier transform infrared spectrometer (Jasco Co., Tokyo, Japan) purged with N2 and equipped with a Mercury-Cadmium-Telluride detector. Samples immediately after the CD measurements were dried on the surface of an ATR plate for the FTIR measurements. For each sample, 256 interferograms were averaged at a spectral resolution of 4 cm−1 and processed using one-point zero-filling and Hamming apodization.
2.4. Fluorescence spectroscopy All fluorescence measurements were performed on a Hitachi F-7000 spectrophotometer (Tokyo, Japan). ThT fluorescence spectra were recorded in a 4 × 4 mm cuvette from 450 to 600 nm at an excitation wavelength of 440 nm. The ThT and protein or peptide concentrations were 10 μM and 50 μg/mL, respectively. 2.5. Circular dichroism (CD) spectroscopy
Heparan sulfate (HS) and thioflavin T (ThT) were purchased from Iduron Ltd. (Manchester, UK) and Sigma-Aldrich (St. Louis, MO), respectively. Fmoc amino acid derivatives were obtained from the Peptide Institute, Inc. (Minoh, Japan). The recombinant human SAA1.3, hereafter designated as SAA1.3 m because of a single methionine residue at the N-terminus, was produced in Escherichia coli as previously described [17]. Unless otherwise noted, 20 mM Tris buffer (pH 7.4) or 10 mM acetate buffer (pH 4.0) was used.
2.7. Transmission electron microscopy (TEM) Samples containing SAA peptides were spread on Formvar filmcoated copper grids (400 mesh) and were negatively stained with 2% sodium phosphotungstate (pH 7.0). The grids were observed by a JEM1400Plus transmission electron microscope (JEOL, Akishima, Japan) with an acceleration voltage of 80 kV. Digital images were acquired with a CCD camera.
2.2. Peptide synthesis Peptide synthesis by the ligation method was performed essentially as described previously [18]. Two peptide segments, SAA1.3 (1–44)Cys-Pro-OCH2COTle-NH2 and Cys-SAA1.3 (46–76), were prepared by standard Fmoc chemistry using an automated peptide synthesizer, ACT440Ω (AAPPTec, Louisville, KY) or Liberty Blue (CEM Corporation, Matthews, NC). After cleavage of peptides from the resin, they were purified by reversed-phase high-performance liquid chromatography (YMC-Pack ProC18 or Cosmosil 5C18-AR-II, 10 × 250 mm). Ligation reactions were performed under the conditions of 50 mM 4-mercaptophenylacetic acid, 20 mM Tris (2-carboxyethyl)phosphine, and 6.0 M guanidine hydrochloride in sodium phosphate buffer (pH 8.0) at 37 °C overnight. Desulfurization reactions were performed under the conditions of 10 mM 2,2′-azobis [2-(2-imidazolin-2-yl)propane] dihydrochloride, 0.10 M sodium 2-mercaptoethanesulfonate, 0.15 M Tris (2carboxyethyl)phosphine, and 6.0 M guanidine hydrochloride in sodium phosphate buffer (pH 7.0) at 37 °C overnight. After the final purification, peptide structures were confirmed by electrospray ionization mass spectrometry (Thermo Finnigan LCQ Deca XP spectrometer) or matrixassisted laser desorption ionization-time of flight mass spectrometry (Bruker AutoFLEX spectrometer) and amino acid analysis (Hitachi L2000 amino acid analyzer).
3. Results 3.1. Peptide design and analytical validation The amino acid sequence of the SAA1.3 (1–76) peptide is shown in Fig. 1. Owing to the length and the propensity for aggregation, especially in the N-terminal side of the sequence, we decided to employ the ligation method. Although a Cys residue will always be inserted by native chemical ligation, it can be converted into an Ala residue by a desulfurization reaction [19]. Since there are no Cys residues in the SAA sequence, ligation at the Ala residue at position 45 was chosen to consider reducing the deleterious side reactions. Details of the reaction
2.3. Sample preparation Lyophilized protein or peptides were dissolved in 4 M urea and freshly dialyzed into buffer extensively, and were centrifuged to remove insoluble or aggregated matter before use. Samples were kept at 4 °C throughout the preparation procedure. Protein or peptide concentrations were determined by measuring the absorption at 280 nm (A280).
Fig. 1. Amino acid sequence of SAA1.3 (1–76) peptide. Ligation site is indicated by an arrow. Amino acid positions which differ in other two human SAA isoforms are shown in red. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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3.3. Determination of experimental conditions for amyloid fibril formation Previously, we examined fibril formation of SAA molecules induced by the addition of heparin under acidic conditions (pH4.0) because SAA did not form amyloid fibrils in phosphate buffer (pH7.4) and acidification was required to facilitate it [5]. However, further observations revealed that the composition of the buffer influences the fibril formation. Hence, we first examined the effect of pH on the amyloid fibril formation of SAA1.3 (1–76) peptide upon the addition of heparin by measuring the fluorescence of ThT, an amyloidophilic dye, commonly used for the detection of a cross-β structure [22]. ThT fluorescence was observed at pH7.4 (20 mM Tris buffer), although the intensities were reduced compared with those at pH4.0 (10 mM acetate buffer) (Fig. S1), implying that the fibril formation of SAA1.3 (1–76) peptide occurs even at neutral pH. A cross-β structure is one of the hallmark features of amyloid fibrils [23]. The CD spectrum of SAA1.3 (1–76) peptide in the presence of heparin at pH4.0 was typical for a β-structure, with a single minimum at approximately 220 nm (Fig. S2A). Nevertheless, atomic force microscopy (AFM) images exhibited spherical aggregates probably because of the strong interactions between heparin and peptides that dominate the intermolecular association of peptides to elongate fibrils (Fig. S3A). In our previous study, such spherical morphology was observed when SAA peptide was incubated with heparin, which turned to become typical short fibrils by incubating with HS instead of heparin under the same experimental conditions [24]. In contrast, at pH7.4, although heparin induced some conformational changes of the SAA1.3 (1–76) peptide, the CD spectrum was not typical for a β-structure (Fig. S2B), but AFM images exhibited worm-like protofibrils (Fig. S3B). Taking these findings together, in the present study, we decided to evaluate the amyloid fibril formation of SAA1.3 (1–76) peptide in the presence of HS at pH7.4.
Scheme 1. Synthetic scheme for the preparation of SAA1.3 (1–76) peptide.
mechanisms of ligation and desulfurization are shown in Scheme 1. After the final purification, the mass of 8548.7 ± 0.9 (deconvoluted value) as observed by electrospray ionization mass spectrometry was equivalent to the calculated mass of 8548.3 for (M + H)+ (average) and the amino acid composition of the purified peptide was Asp10.2Ser5.5Glu6.2Pro1.3Gly9.3Ala13Met1.3Ile2.9Leu1.1Tyr4.2Phe6.9Lys2.1His2.0Arg8.1 (Table S1).
3.2. Influence of C-terminal truncation on the secondary structure of SAA Secondary structures of full-length SAA1.3 m and SAA1.3 (1–76) peptide were analyzed by CD spectroscopy immediately after the sample preparation. As shown previously in 10 mM phosphate buffer (pH 7.4), full-length SAA1.3 m in 20 mM Tris buffer (pH 7.4) exhibited a CD spectrum with double minima at approximately 208 and 222 nm at 4 °C, while showing a single minimum of approximately 200 nm at 37 °C (Fig. 2A). In contrast, the CD spectra for SAA1.3 (1–76) peptide at 4 °C and 37 °C were entirely distinct from those for fulllength SAA1.3 m (Fig. 2B), suggesting that the C-terminal truncation induces conformational changes of the SAA molecule. CD spectral deconvolution analyses using BeStSel software revealed that full-length SAA1.3 m and SAA1.3 (1–76) peptide possess similar amounts of βsheet structure based on the number of amino acid residues (Table S2) [20]. In terms of the characteristic that distinguished them, the amount of helix structure was markedly decreased in SAA1.3 (1–76) peptide, signifying that the truncated C-terminal region contains a helical structure or induces it somewhere else in the molecule by intramolecular interactions. These results are consistent with the latest findings that the absence of the C-terminal tail of murine SAA1.1 causes a significant loss in α-helical structure [21], although the spectral shapes of truncated forms of human and murine SAA are considerably different.
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Even in the absence of HS incubated at 37 °C for 24 h, faint ThT fluorescence was observed in both full-length SAA1.3 m and SAA1.3 (1–76) peptide at pH7.4 (Fig. 3A and B). At 24 h after the addition of HS at a concentration of 50 μg/mL, increases in the ThT fluorescence were observed (Fig. 3A and B), which was more prominent for SAA1.3 (1–76) peptide than for full-length SAA1.3 m. The effect of HS concentrations on the time-dependent changes in ThT fluorescence was also monitored (Fig. 3C and D). In SAA1.3 (1–76) peptide, ThT fluorescence increased without an observable lag time regardless of the HS concentration, at least in the range of 25–100 μg/mL, and almost plateaued within 24 h. In contrast, less significant increases and no enhancing effects of HS on ThT fluorescence for full-length SAA1.3 m were observed. These results suggest that the C-terminal truncation facilitates fibril formation of the SAA molecule upon the addition of HS.
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3.4. Amyloid fibril formation of SAA molecules monitored by ThT fluorescence
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Fig. 2. CD spectra of (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide at 4 °C (blue lines) and 37 °C (red lines) in 20 mM Tris buffer (pH 7.4). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Fig. 3. Representative ThT fluorescence spectra for (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide in the absence (dotted lines) or presence (solid lines) of HS (50 μg/mL, incubated at 37 °C for 24 h) in 20 mM Tris buffer (pH 7.4). Concentration dependence of HS on the ThT fluorescence kinetics for (C) the full-length SAA1.3 m and (D) the SAA1.3 (1–76) peptide at the HS concentrations of 0 (white), 25 (green), 50 (black), and 100 μg/mL (cyan). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
sheet architecture. However, the bands were influenced neither by the addition of HS nor the C-terminal truncation. Since samples are dried on the surface of an ATR plate, the protein or peptide concentrations are markedly elevated during the drying process, which markedly facilitated the formation of a β-sheet structure. Overall, taking these findings together with the ThT fluorescence results, it is conceivable that SAA1.3 (1–76) peptide has the propensity to form amyloid fibrils, which is facilitated by the presence of HS.
3.5. Conformational changes of SAA molecules monitored by CD and FTIR During the incubation in the absence of HS at 37 °C for 24 h, almost no significant changes were observed in the CD spectra for both fulllength SAA1.3 m and SAA1.3 (1–76) peptide (Fig. 4 compared with Fig. 2). Upon incubation with HS, CD spectral changes of full-length SAA1.3 m were subtle, but the changes were pronounced in SAA1.3 (1–76) peptide (Fig. 4). However, despite the increase in the ThT fluorescence, especially in SAA1.3 (1–76) peptide incubated in the presence of HS, the CD spectrum was not typical for a β-structure, distinct from the case at pH4.0 (Fig. S2A). The secondary structures of full-length SAA1.3 m and SAA1.3 (1–76) peptide in the absence or presence of HS were further examined by ATR-FTIR spectroscopy, which can more sensitively detect the presence of a β-sheet structure [25]. The amide I band that appears around the wavenumbers of 1600–1700 cm−1 is widely used for characterizing the secondary structure components of proteins [26]. In contrast to the CD results, each FTIR spectrum for the amide I region showed an absorbance maximum around 1625–1630 cm−1 (Fig. 5), suggesting that full-length SAA1.3 m and SAA1.3 (1–76) peptide possess a common β-
3.6. Morphological characterization of SAA aggregates visualized by TEM To verify the amyloid fibril formation, TEM images of samples containing full-length SAA1.3 m or SAA1.3 (1–76) peptide incubated in the absence and presence of HS were obtained (Fig. 6). In the presence of 50 μg/mL HS, full-length SAA1.3 m scarcely exhibited fibrillar aggregates (Fig. 6A). In contrast, SAA1.3 (1–76) peptide in the presence of 50 μg/mL HS exhibited short fibrillar aggregates (Fig. 6B), which was also observed in the absence of HS (Fig. 6C). Although neither the ThT fluorescence nor the TEM results reflect the amounts of amyloid fibrils, these observations imply that the C-terminal truncation of human SAA
Fig. 4. CD spectra of (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide at 37 °C after 24 h of incubation in the absence (dotted lines) or presence (black lines) of HS (50 μg/ mL) in 20 mM Tris buffer (pH 7.4).
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Fig. 5. IR absorbance spectra of (A) the full-length SAA1.3 m and (B) the SAA1.3 (1–76) peptide in the absence (dotted lines) or presence (solid lines) of HS.
accelerates amyloid fibril formation. When the SAA1.3 (1–76) peptide concentrations were increased from 50 to 200 μg/mL without changing the HS concentration, the short aggregates became elongated fibrils (Fig. 6D). In the absence of HS, such elongated fibrils were sparse but infrequently observed in spots.
during the purification and preparation processes. In fact, both fulllength SAA1.3 m and SAA1.3 (1–76) peptide exhibited evidence of carbamylation after the experiments, as determined by mass spectrometry analyses, although the degree of carbamylation was unclear. Thus, the effect of the N-terminal methionine and carbamylation, if any, needs to be prudently taken into account when comparing the amyloid fibril formation of full-length SAA1.3 and SAA1.3 (1–76) peptide. HS polysaccharides are ubiquitously expressed on the cell surface and extracellular matrix of animal tissues as components of proteoglycans. HS is composed of glucuronic acid (GlcA) or iduronic acid (IdoA) alternating with glucosamine that is either N-acetylated (GlcNAc) or N-sulfated (GlcNS). The degree of sulfation affects the facilitating effect of amyloid formation [24,31]. Thus, HS can be a regulating factor for amyloidogenesis by modulating the degree of sulfation. Differences in HS structures among organs may explain why amyloid is deposited in specific organs. The N- and C-terminal regions of the SAA molecule are considered to be critical for the heparin-induced amyloid formation and for the heparin/HS-binding, respectively [4,12]. Consequently, when the full-length SAA molecules were incubated with HS, binding of the C-terminal region to HS can compete with and hamper the HS-induced amyloid formation mediated by the Nterminal region. Therefore, in the present study, the C-terminal truncation could conceivably facilitate the fibril formation of SAA molecule in the presence of HS. SAA in amyloid deposits is generally found as the C-terminal-truncated form, which raises questions as to whether the cleavage is a prerequisite for the amyloid fibril formation and where or when it happens. Although several proteolytic enzymes have been proposed to cleave SAA molecule [32], which specific one contributes to the generation of amyloidogenic fragments is also unknown. In some proteins, proteolytic cleavage evoked by protein misfolding exposes amyloidogenic regions, which facilitates the amyloid fibril formation. Our finding that the C-terminal truncation accelerates the amyloid fibril
4. Discussion It has been reported that the truncation of the final 13 amino acids from the C-terminus of murine SAA2.2 retards the rate of fibril formation [27]. In the present study, we showed for the first time that the amyloid fibril formation of human SAA1.3 is accelerated by the truncation of the C-terminal region, which seems inconsistent with the mouse results. Pairwise sequence alignment using the NeedlemanWunsch algorithm revealed that human SAA1.3 and murine SAA2.2 share approximately 80% similarity. Additionally, the theoretical isoelectric points (pI) of these molecules are also similar (pI = ∼5.8). However, unlike human SAA1.3, it is noteworthy that the murine SAA2.2 isoform produced by the CE/J mouse strain does not form amyloid fibrils in vivo [28]. Because of the slower fibrillation rate, it is plausible that the C-terminal-truncated murine SAA2.2 is degraded into smaller fragments, which may serve to prevent amyloid fibril formation. Subtle differences in the amino acid sequence or the modifications of SAA strikingly impact on the amyloidogenicity. For example, the addition of a single N-terminal methionine greatly enhances the propensity for fibrillation and modulates the fibrillation pathway of human SAA [29]. More recently, it has been pointed out that carbamylation at the N-terminus of murine SAA influences the amyloid formation in a cell culture model [30]. Bacterially expressed full-length SAA1.3 contains a single N-terminal methionine, whereas chemically synthesized SAA1.3 (1–76) peptide contains no N-terminal methionine. In addition, it is likely that the samples are carbamylated since we utilized urea
Fig. 6. TEM images of the aggregates of (A) the full-length SAA1.3 m and (B–D) the SAA1.3 (1–76) peptide. Fibril formation was conducted at peptide concentrations of (A–C) 50 μg/mL and (D) 200 μg/mL in the (C) absence or (A, B, D) presence of HS (50 μg/mL) in 20 mM Tris buffer (pH 7.4). Scale bars indicate 200 nm.
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formation of SAA molecules prompted us to speculate that the removal of the C-terminal region occurs before fibril formation. However, fulllength SAA is not necessarily unable to form amyloid fibrils, but rather possesses amyloidogenic potential, especially when the concentration increases, as implied by the FTIR experiments. Similarly, the truncation of murine SAA1.1, the sole isoform found in amyloid deposits in mice, is no prerequisite for amyloid formation [21]. Thus, the idea that the Cterminal truncation is a secondary event after the fibril formation cannot be completely ruled out yet. In fact, the proteolytic cleavage of SAA after the incorporation into fibrils has been proposed, although the data in support of this were obtained with murine SAA [33]. The susceptibility to proteolysis can also be influenced by the existence of lipid, as discussed below. One of the major concerns in amyloid studies of SAA is the influences of lipid, since SAA usually exists in a form bound to lipid [34]. Although crystallographic analysis revealed that human SAA is highly α-helical [35], lipid-free SAA in solution is intrinsically disordered at physiological temperature [36]. Lipid binding of SAA molecule induces stabilization of α-helical conformation, which serves to inhibit fibril formation [6]. Conformational changes upon lipid binding may also affect the interaction with glycosaminoglycans and the susceptibility to proteolysis or vice versa. In fact, HS has been shown to dissociate SAA from high-density lipoproteins, thereby facilitating amyloid fibril formation [37]. Alternatively, we have shown that lipid binding rendered SAA molecules resistant to protein degradation [38]. Furthermore, the lipid-bound conformation of SAA might be modulated by the alterations in lipid composition, which has been revealed to vary under chronic inflammatory conditions [39]. To obtain a comprehensive understanding of the detailed pathogenic mechanisms underlying AA amyloidosis, the amyloidogenic pathway of SAA should be argued in conjunction with the lipid metabolism. Thanks to the availability of biopharmaceuticals to control the underlying diseases such as rheumatoid arthritis, the number of patients with AA amyloidosis is decreasing. However, it has been reported that obesity-enhanced SAA secretion can be a risk factor for the development of AA amyloidosis [40]. In addition, there are several cases in which no clear-cut history of a defined chronic inflammatory condition was present at diagnosis or in which the underlying disease ultimately remains unknown [41]. Among systemic amyloidoses, model animals with AA amyloidosis, which can easily be induced by inflammatory stimuli, are most widely employed. Knowledge of the mechanisms of formation and the structures of amyloid fibrils gained from AA amyloidosis can be expanded to apply to other types of systemic amyloidoses. Thus, AA amyloidosis still warrants further investigation in order to develop treatments common to systemic amyloidoses. Further studies are required to uncover the multilayered mechanisms involved in order to elucidate the pathogenesis of AA amyloidosis.
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Acknowledgements This work was performed in part under the Cooperative Research Program of the Institute for Protein Research, Osaka University, CR-1501. We thank Ms. Naho Onoe and Ms. Kaho Matsumoto for their technical assistance. Part of this research was supported by JSPS KAKENHI Grant Number 15K08436. This publication was also subsidized by JKA through its promotion funds from KEIRIN RACE. The authors would like to thank Enago for the English language review. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.abb.2017.12.016. References [1] G.T. Westermark, M. Fandrich, P. Westermark, AA amyloidosis: pathogenesis and
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