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NDT Advance Access originally published online on May 8, 2008
Nephrology Dialysis Transplantation 2008 23(10):3247-3255; doi:10.1093/ndt/gfn231
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Lysophospholipids induce the nucleation and extension of β2-microglobulin-related amyloid fibrils at a neutral pH

Tadakazu Ookoshi1, Kazuhiro Hasegawa1, Yumiko Ohhashi1, Hideki Kimura2, Naoki Takahashi2, Haruyoshi Yoshida2, Ryoichi Miyazaki3, Yuji Goto4,5 and Hironobu Naiki1,5

1 Department of Pathological Sciences, Division of Molecular Pathology, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193 2 Department of General Medicine, Division of Nephrology, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193 3 Fujita Memorial Hospital, Fukui 910-0004 4 Institute for Protein Research, Osaka University, Osaka 565-0871 5 CREST, Japan Science and Technology Agency, Tokyo 103-0027, Japan

Hironobu Naiki, Department of Pathological Sciences, Division of Molecular Pathology, Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan. Tel: +81-776-61-8320; Fax: +81-776-61-8123; E-mail: Naiki{at}u-fukui.ac.jp



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
Background. In β2-microglobulin-related (Aβ2M) amyloidosis, partial unfolding of β2-microglobulin (β2-m) is believed to be prerequisite to its assembly into Aβ2M amyloid fibrils in vivo. Low concentrations of sodium dodecyl sulfate induce partial unfolding of β2-m to an amyloidogenic conformer and subsequent amyloid fibril formation in vitro, but the biological molecules that induce them under near-physiological conditions have not been determined.

Methods. We investigated the effect of some lysophospholipids on the nucleation, extension and stabilization of Aβ2M amyloid fibrils at a neutral pH, using fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy and electron microscopy. We also measured plasma concentrations of lysophospholipids in 103 haemodialysis patients and 14 healthy subjects and examined the effect of uraemic and normal plasmas on the stabilization of Aβ2M amyloid fibrils at a neutral pH.

Results. Some lysophospholipids, especially lysophosphatidic acid (LPA), induced not only the extension of Aβ2M amyloid fibrils but also the formation of Aβ2M amyloid fibrils from the β2-m monomer at a neutral pH, by partially unfolding the compact structure of β2-m to an amyloidogenic conformer as well as stabilizing the extended fibrils. Haemodialysis patients had significantly higher plasma concentrations of LPA than healthy subjects. Furthermore, uraemic plasmas with the highest ranking LPA concentrations stabilized Aβ2M amyloid fibrils significantly more potently than normal plasmas. On the other hand, simple addition of LPA to normal plasma did not enhance the fibril stabilizing activity.

Conclusions. These results suggest a possible role of lysophospholipids in the development of Aβ2M amyloidosis.

Keywords: amyloidosis; β2-microglobulin; lysophospholipid; thioflavin T



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary data
 References
 
β2-microglobulin-related (Aβ2M) amyloidosis, or dialysis-related amyloidosis (DRA), is a common and serious complication in long-term haemodialysis patients [1]. Carpal tunnel syndrome and destructive arthropathy with cystic bone lesions ensue on the deposition of Aβ2M amyloid fibrils in the tissue [2,3]. Intact β2-microglobulin (β2-m) is a major structural component of amyloid fibrils deposited in the synovial membrane of the carpal tunnel [4–7], but the mechanism of the deposition of these amyloid fibrils is not fully understood. Although the retention of β2-m in the plasma appears to be prerequisite [8], other factors, such as the age of the patient, the duration of haemodialysis, and less confidently the type of dialysis membrane used, may be involved [9–11]. A proinflammatory state induced by dialyzer membranes and contaminated dialysate may also contribute to the pathogenesis of DRA [12].

Aβ2M amyloid deposition takes place predominantly in the cartilaginous and tendinous tissues [13,14], suggesting that the specific interaction between β2-m and the extracellular matrix molecules in these tissues, such as type II collagen, glycosaminoglycans (GAGs) and proteoglycans (PGs), causes Aβ2M amyloid deposition. Previously, we reported that various types of GAGs and PGs stabilize the Aβ2M amyloid fibrils and inhibit their depolymerization at a neutral pH [15]. Furthermore, we reported that some GAGs, especially heparin, dose-dependently enhanced the 2,2,2-trifluoroethanol-induced fibril extension at a neutral pH [16]. In the mechanism of amyloidogenesis of natively folded proteins such as β2-m and transthyretin, partial unfolding is believed to be prerequisite to its assembly into amyloid fibrils both in vitro and in vivo [17–19]. However, the biological molecules that induce partial unfolding of β2-m and subsequent amyloid fibril formation under the near-physiological conditions in vitro remain to be determined.

Various lipid molecules have been reported to induce the conformational change of various amyloid precursor proteins, as well as to initiate the amyloid fibril formation in vitro [20–28]. Recently, we reported that low concentrations of sodium dodecyl sulfate (SDS), an anionic detergent around the critical micelle concentration (CMC), unfold the compact structure of β2-m to an {alpha}-helix-containing aggregation-prone amyloidogenic conformer and stabilize the fibrils, resulting in the extension of Aβ2M amyloid fibrils at a neutral pH [29]. Given the structural similarity to SDS and the possible contribution of a proinflammatory state to the pathogenesis of DRA [12], a lysophospholipid (LPL) could be a candidate of the biological molecules inducing Aβ2M amyloid fibril formation. LPL is a biologically active, proinflammatory molecule with a glycerol backbone to which one hydrophobic fatty acid chain and a hydrophilic phosphate or phosphorylated alcohol is attached [30,31].



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary data
 References
 
Materials
Recombinant human β2-m (r-β2-m) was expressed and purified using the Escherichia coli expression system as previously described [32]. All other reagents are described in Supplementary Material. All LPLs analysed in this study are listed in the Supplementary Table.

Preparation of Aβ2M amyloid fibrils and seeds
Aβ2M amyloid fibrils used for the standard extension reaction were prepared from the patient-derived Aβ2M amyloid fibrils by the repeated extension reaction at pH 7.5 with r-β2-m, as described elsewhere [33]. We first obtained F1 fibrils by the extension of S0 seeds. F1 and S0 mean fibrils of generation 1 and seeds of generation 0, respectively. By repeating the algorithmic protocol, F6 fibrils were obtained from S5 seeds. S6 seeds were prepared by the extensive sonication of F6 fibrils. For the depolymerization assay (Figure 4), F7 fibrils were prepared by the repeated extension reaction of the patient-derived Aβ2M amyloid fibrils at pH 2.5 [34].


Figure 4
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  		Fig. 4 Effect of MPPA on the depolymerization of Aβ2M amyloid fibrils. The reaction mixture contained 300 µg/ml F7 fibrils extended at pH 2.5, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0.5 mM SDS or 0–0.5 mM MPPA. The reaction mixture was incubated at 37°C for 4 h and analysed by fluorescence spectroscopy as described in the Materials and methods section. Each column represents the average of three independent experiments. The error bars indicate SD.

 
Extension assay of Aβ2M amyloid fibrils
The reaction mixture was prepared on ice and contained 0–60 µg/ml S6 seeds, 0–25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0.5 mM SDS or 0–1.0 mM LPLs. Prior to the addition of S6 seeds and r-β2-m, the desired amounts of LPLs were pipetted from the thawed stock solutions into the tubes, evaporated under vacuum, then dissolved in the buffer solution using an ultrasonic bath (Bioruptor UCD-200TM, Cosmo Bio Co. Ltd, Tokyo, Japan) at 37°C at max power with 15 intermittent pulses (1 min: on, 1 min: off). We prepared the stock solutions of LPLs as described in the Supplementary Material. After being mixed by brief vortexing of the mixture, 30 µl aliquots were put into oil-free PCR tubes (0.5 ml in size; Takara Shuzo Co. Ltd, Otsu, Japan) on ice, then incubated at 37°C without agitation. After incubation for 0–80 h, the reaction was stopped by placing the tubes on ice. From each reaction tube, three 5-µl aliquots were removed and then subjected to fluorescence spectroscopy with thioflavin T (ThT) [35]. In Figure 5, ThT fluorescence was measured using a FusionTM universal microplate analyzer (Packard Instrument Co. Inc., Wellesley, MA, USA). Briefly, three 1-µl aliquots were removed from each reaction tube and mixed with 100 µl of 5 µM ThT solution (pH 8.5) on 96-well tissue culture treated microplate, flat bottom, black/clear (Matrix Technologies Corp., Hudson, NH, USA). The fluorescence was measured with a top detection mode and excitation/emission wavelengths of 440/505 nm.


Figure 5
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  		Fig. 5 Effect of various LPLs on the extension of Aβ2M amyloid fibrils at a neutral pH. The reaction mixture contained 30 µg/ml S6 seeds, 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0.25 mM LPLs or 0.5 mM SDS (positive control). The reaction mixture was incubated at 37°C for 72 h and analysed by fluorescence spectroscopy as described in the Materials and methods section. The types of LPLs are indicated with the net charge of the hydrophilic groups in parentheses. Fatty acid chains attached to a glycerol backbone are also indicated with the numbers of carbons and double bonds in the parentheses. Each column represents the average of three independent experiments. The average value in the case of SDS was regarded as 100% in each experiment. The error bars indicate SD. Statistical analysis was performed by a two-factor factorial analysis of variance between factor A (LPA, LPG) and factor B (myristoyl, palmitoyl, stearoyl, oleoyl): P < 0.001 (LPA versus LPG), P < 0.0001 (types of fatty acid chains). Additionally, P < 0.01 (LPA versus LPG), P < 0.01 (stearoyl versus oleoyl) between factor C (LPA, LPG) and factor D (stearoyl, oleoyl).

 
Far-UV circular dichroism (CD) spectra of r-β2-m in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3- phosphate (MPPA)
Far-UV CD spectra (190–250 nm) of r-β2-m were recorded on a Jasco 725 spectropolarimeter at 37°C as described previously [36]. The reaction mixture containing 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0–0.5 mM MPPA was measured immediately after the preparation and after the incubation at 37°C for 24 h. The results are expressed in terms of mean residue ellipticity.

Depolymerization assay of Aβ2M amyloid fibrils
F7 fibrils extended at pH 2.5 were centrifuged at 21 500 x g for 2 h at 4°C. The precipitate was washed and re-suspended in ice-cold 100 mM NaCl. The reaction mixture (100 µl) was prepared on ice and contained 300 µg/ml F7 fibrils, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, 0 or 0.5 mM SDS, 0 to 0.5 mM MPPA and 0 to 1/5 v/v of plasma aliquots. Before and 4-h after incubation at 37°C without agitation, three 5-µl aliquots from each reaction tube were subjected to fluorescence spectroscopy.

Amyloid fibril formation from r-β2-m monomer
The reaction mixture (500 µl) contained 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0 or 0.5 mM SDS or 0.25 mM MPPA or 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (MPPG). The reaction tubes were sealed in a humidified box and incubated for 0–60 days in an air incubator at 37°C without agitation.

Determination of plasma concentrations of lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) in haemodialysis patients and healthy subjects
Plasma concentrations of LPC and LPA were determined by an enzymatic assay as previously reported [37] and a recently established colorimetric assay using enzymatic cycling [38], respectively. We investigated a total of 103 haemodialysis patients at a dialysis centre in the Fukui prefecture and a total of 14 healthy subjects. Venous EDTA-treated blood was collected from haemodialysis patients immediately before haemodialysis 2 to 3 h after meals. As control samples, venous EDTA-treated blood was collected from healthy subjects 2 to 3 h after meals. Blood samples were placed at 4°C immediately after collection. Plasma was separated after the centrifugation of blood samples at 2500 x g for 10 min at 4°C and stored at –80°C until assayed. Written informed consent was obtained from each patient and each healthy subject. Continuous variables were expressed as means ± SD.

Other analytical procedures
See Supplementary Material.



   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary data
 References
 
Extension of Aβ2M amyloid fibrils in the presence of MPPA at a neutral pH
S6 seeds incubated with 25 µM r-β2-m at pH 7.5 in the absence of MPPA did not show a significant increase in ThT fluorescence (Figure 1A). On the other hand, in the presence of 0.25 mM MPPA or 0.5 mM SDS, the fluorescence increased without a lag phase and proceeded to equilibrium after a 24- to 36-h incubation. The linear semilogarithmical plot (r = 0.966) shown in Figure 1B, and the linear correlations between the extension rate and the seed or r-β2-m concentrations (Supplementary Figure 1) indicate that the extension of Aβ2M amyloid fibrils can be explained by a first-order kinetic model: i.e. fibril extension proceeds via the consecutive association of r-β2-m onto the ends of existing fibrils [39]. The fluorescence signal increased with MPPA concentration, peaking at 0.5 mM before steadily decreasing above this level of MPPA (Figure 1C). We estimated the CMC of MPPA to be 15.8 µM (Supplementary Figure 2). An electron microscopic study revealed that the fibrils extended with some lateral aggregation in the presence of 0.25 mM MPPA (Figure 2).


Figure 1
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  		Fig. 1 Extension of Aβ2M amyloid fibrils at a neutral pH. (A) The reaction mixture contained 30 µg/ml S6 seeds, 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and 0 (Figure 1) or 0.5 mM (Figure 1) SDS or 0.25 mM MPPA ({square}). The reaction mixture was incubated at 37°C for 0–80 h. At each incubation time, the reaction mixture was analysed by fluorescence spectroscopy as described in the Materials and methods section. Each point represents the average of three independent experiments. The error bars indicate SD. (B) The semilogarithmical plot of the difference: F(infinity)–F(t) versus reaction time. F(t) represents the fluorescence in the presence of 0.25 mM MPPA as a function of time ({square} in panel A). F(infinity) was tentatively determined and shown as a broken line in panel A. Linear regression and correlation coefficient were calculated (r = 0.966). (C) Effect of MPPA concentration on the extension of Aβ2M amyloid fibrils at a neutral pH. The reaction mixture contained 30 µg/ml S6 seeds, 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0.5 mM SDS or 0–1.0 mM MPPA. The reaction mixture was incubated at 37°C for 72 h. Each column represents the average of three independent experiments. The error bars indicate SD.

 

Figure 2
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  		Fig. 2 Electron micrographs of extended Aβ2M amyloid fibrils. The reaction mixture containing 30 µg/ml S6 seeds, 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0 (A) or 0.25 mM (B) MPPA was incubated at 37°C for 72 h. These samples were negatively stained with 1% phosphotungstic acid and prepared for electron microscopy as described in the Materials and methods section. The bars are 250 nm long.

 
Effect of MPPA on the conformation of r-β2-m
In the presence of 0 and 0.025 mM MPPA, far-UV CD spectra of r-β2-m exhibited a positive peak at about 202 nm and a negative peak at about 220 nm immediately after the addition of MPPA at 37°C (Figure 3A). In the presence of 0.05–0.5 mM MPPA, which is over the CMC, the spectra exhibited a transition state with the ellipticities of the positive peaks decreasing dose-dependently (Figure 3A). These spectra are similar to those of r-β2-m in the presence of 0–0.75 mM SDS as previously reported [29]. After the incubation at 37°C for 24 h, the ellipticities of both the positive and the negative peaks decreased in the presence of 0.25–0.5 mM MPPA, as compared to those immediately after the addition of MPPA (Figure 3B). Thus, the data in Figures 1C, 3 and Supplementary Figure 2 may indicate that in vitro, a significant amount of an amyloidogenic conformer of r-β2-m may be formed considerably above the CMC of MPPA.


Figure 3
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  		Fig. 3 Far-UV CD spectra of r-β2-m in the presence of MPPA. The reaction mixture containing 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0–0.5 mM MPPA was incubated at 37°C for 0 (A) or 24 h (B) before measurement.

 
Effect of MPPA on the depolymerization of Aβ2M amyloid fibrils at a neutral pH
When Aβ2M amyloid fibrils extended at pH 2.5 (F7 fibrils) were incubated in the absence of MPPA at pH 7.5 for 4 h, the ThT fluorescence decreased to about 1% of the initial fluorescence (Figure 4). On the other hand, the fluorescence decrease was suppressed dose-dependently with the increase in MPPA concentration (0.05–0.5 mM).

Effect of various LPLs on the extension of Aβ2M amyloid fibrils at a neutral pH
LPA and lysophosphatidylglycerol (LPG) with the negatively charged hydrophilic groups induced the extension of Aβ2M amyloid fibrils at a neutral pH (Figure 5). LPA induced the extension significantly more potently than LPG (P < 0.001, two-factor factorial analysis of variance). The extension-inducing activity was also dependent on the types of fatty acid chains (P < 0.0001, two-factor factorial analysis of variance). LPA and LPG with palmitate (16:0) or stearate (18:0) were generally more effective than those with myristate (14:0) or oleate (18:1). LPA with a short fatty acid chain (6:0) did not induce the fibril extension. LPA and LPG with a saturated fatty acid chain (18:0) induced the extension significantly more potently than those with a monounsaturated fatty acid chain (18:1) (P < 0.01, two-factor factorial analysis of variance). The activity of lysophosphatidylserine (LPS) was weak even if it has a negatively charged hydrophilic group. LPC and lysophosphatidylethanolamine (LPE) with the neutral hydrophilic groups exhibited negligible activities except for 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine (18:0).

Formation of Aβ2M amyloid fibrils from r-β2-m monomer
When 25 µM r-β2-m was incubated without seeds for 60 days at pH 7.5 in the presence of 0.5 mM SDS or 0.25 mM MPPA, ThT fluorescence increased slightly (Figure 6A). No fluorescence increase was observed when r-β2-m was incubated alone or in the presence of 0.25 mM MPPG. Electron microscopically, SDS induced the formation of thin fibrils with raft-like aggregation (Figure 6B), while MPPA induced the formation of thin fibrils with bundle-like aggregation (Figure 6C). Moreover, the fibrils in Figures 6B and C were stained positively with Congo red and showed orange-green birefringence under polarized light (insets).


Figure 6
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  		Fig. 6 Effect of MPPA and MPPG on Aβ2M amyloid fibril formation from r-β2-m monomer. (A) The reaction mixture contained 25 µM r-β2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0 or 0.5 mM SDS, 0.25 mM MPPA or 0.25 mM MPPG. The reaction mixture was incubated at 37°C for 60 days and analysed by fluorescence spectroscopy as described in the Materials and methods section. Each column represents the average of three independent experiments. The error bars indicate SD. (B, C) Electron micrographs of Aβ2M amyloid fibrils formed from r-β2-m monomer in the presence of 0.5 mM SDS (B) or 0.25 mM MPPA (C). Phosphotungstic acid staining. The bars are 250 nm long. Insets represent the orange-green birefringence of amyloid fibril aggregates under polarized light.

 
Comparison of plasma LPC and LPA concentrations between haemodialysis patients and healthy subjects
Haemodialysis patients had significantly lower plasma concentrations of LPC and significantly higher plasma concentrations of LPA than healthy subjects (148 ± 38 µM versus 180 ± 32 µM, P < 0.005, and 0.15 ± 0.05 µM versus 0.08 ± 0.02 µM, P < 0.001, respectively) (Table 1). When non-diabetic haemodialysis patients of 50 years old or below were selected for analysis (Table 2), the patient subgroup was age- and gender-matched with the healthy group, but still had significantly higher plasma concentrations of LPA than the healthy group (0.15 ± 0.04 µM versus 0.08 ± 0.02 µM, P < 0.001). Sasagawa et al. [40] reported a similar change in the plasma LPL concentration in haemodialysis patients.


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  		Table 1 Clinical characteristics and LPL profile of haemodialysis patients and healthy subjects

 

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  		Table 2 Clinical characteristics and LPL profile of age-matched non-diabetic haemodialysis patients and healthy subjects

 
Comparison of the effect of normal plasmas with that of uraemic plasmas on the depolymerization of Aβ2M amyloid fibrils at a neutral pH
Uraemic plasmas with the highest ranking LPA concentrations stabilized Aβ2M amyloid fibrils significantly more potently at 1/25 and 1/100 v/v than normal plasmas (Table 3, Figure 7A). In contrast, uraemic plasmas with the lowest ranking LPA concentrations stabilized Aβ2M amyloid fibrils significantly less effectively at 1/5 v/v than normal plasmas (Table 3, Figure 7B). On the other hand, simple addition of sub-µM MPPA to normal plasma did not enhance the fibril stabilizing activity (Figure 8).


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  		Table 3 Clinical characteristics and plasma profile of haemodialysis patients and healthy subjects selected for the depolymerization experiment

 

Figure 7
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  		Fig. 7 Comparison of the effect of normal plasmas with that of uraemic plasmas on the depolymerization of Aβ2 amyloid fibrils. The reaction mixture containing 300 µg/ml F7 fibrils extended at pH 2.5, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 0 or 0.5 mM SDS or 1/5, 1/25 or 1/100 v/v of normal plasmas (A, B) or uraemic plasmas with the highest ranking (A) or lowest ranking LPA concentrations (B) was incubated for 4 h at 37°C without agitation. In 0 and 0.5 mM SDS, each column represents the average of three incubations (A). In normal and uraemic plasmas, each column represents the average of five plasma samples (A, B). The error bars indicate SD. Statistical analysis was performed by Student's unpaired t-test. Note that in (A) and (B), different preparations of F7 fibrils were analysed in different experiments.

 

Figure 8
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  		Fig. 8 Effect of MPPA on the stabilizing activity of normal plasma. The reaction mixture containing 300 µg/ml F7 fibrils, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, 1/25 or 1/5 v/v of normal plasma and 0, 0.00025, 0.0025 or 0.025 mM MPPA was incubated for 4 h at 37°C without agitation. Plasma was obtained from a healthy subject whose plasma LPA concentration was 0.06 µM. Each column represents the average of three independent experiments. The error bars indicate SD. Statistical analysis was performed by a two-factor factorial analysis of variance between factor A (plasma concentration) and factor B (MPPA concentration): P < 0.0001 (plasma concentration), NS (MPPA concentration).

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary data
 References
 
Extension of Aβ2M amyloid fibrils at a neutral pH in the presence of LPL
The present study revealed that at micellar concentrations (100 µM order), LPLs harbouring a negatively charged hydrophilic group and a fatty acid chain with appropriate length (C:14–18) effectively induce not only the extension of Aβ2M amyloid fibrils but also the formation of Aβ2M amyloid fibrils from β2-m monomer at a neutral pH. These LPLs (i.e. LPA and LPG) are structurally similar to SDS. We consider that the basic structure shared by SDS, LPA and LPG, i.e. one alkyl chain and one negatively charged hydrophilic group, may be essential to the amyloidogenic effect based on the following observations. First, liposomes of phospholipids with two fatty acid chains (i.e. phosphatidic acid and phosphatidylcholine) exhibited no amyloidogenic effect at a neutral pH (data not shown). Second, some free fatty acids composed of an alkyl chain with one negatively charged carboxyl group induced the extension of Aβ2M amyloid fibrils at a neutral pH (Hasegawa et al. manuscript in preparation).

SDS had no amyloidogenic effect at micellar concentrations (5–10 mM), because micellar SDS unfolded β2-m to a random structure and destabilized the fibrils due to strong detergent activity [29]. On the other hand, MPPA exhibited amyloidogenic activity at micellar concentrations (100 µM order) (Figure 1C and Supplementary Figure 2). This may indicate that micellar MPPA exerts activity possibly due to weak detergent activity.

Many biological molecules have been reported to induce the formation/extension of Aβ2M amyloid fibrils at a neutral pH. Myers et al. [41] reported that the physiologically relevant factors, such as heparin, serum amyloid P component, apolipoprotein E, uraemic serum or synovial fluid enhance fibrillogenesis by stabilizing fibril seeds, thereby allowing fibril extension by rare assembly competent species formed by local unfolding of native monomers. Relini et al. [42] indicated that fibrillar collagen plays a crucial role in β2-m amyloid deposition under physiopathological conditions and suggested an explanation to the strict specificity of DRA for the tissues of the skeletal system.

Clinical relevance of LPLs to Aβ2M amyloid deposition in vivo
Haemodialysis patients had a significantly higher plasma concentration of LPA than healthy subjects (Tables 1 and 2). Moreover, uraemic plasmas with the highest ranking LPA concentrations stabilized Aβ2M amyloid fibrils significantly more potently than normal plasmas (Table 3, Figure 7). Serum LPLs, such as LPC, are released from membrane phospholipids mainly by the action of group IIA secretory phospholipase A2 (PLA2), then these LPLs are converted to LPA by the action of plasma lysophospholipase D (lysoPLD) [43]. Dorsam et al. [44] reported that uraemic sera contained significantly more type II PLA2 than control sera (median = 1025 µg/L, range = 52–3320 µg/L versus median = 9.2 µg/L, range = 4.6–17.5 µg/L; P = 0.002). These data suggest that uraemic dyslipidaemia with increased LPA concentration may contribute significantly to the pathogenesis of DRA.

The plasma concentration of LPA is about 103 times lower than the effective concentration in vitro (sub-µM versus 100 µM order). On the other hand, LPC exhibits small but significant amyloidogenic activity (Figure 5) and as shown in Tables 1 and 2, the plasma concentration of LPC is comparable to the effective concentration in vitro (100 µM order in both cases). Patients with the highest ranking LPA concentrations tended to have higher LPC concentration than patients with the lowest ranking LPA concentrations (Table 3). Moreover, our preliminary study indicated that MPPC significantly enhanced the activity of MPPA on the fibril extension in vitro. Thus, the fibrillogenic activity of LPA may be enhanced by LPC and other LPLs in vivo. Interestingly, Gellermann et al. [45] reported that amyloid fibrils derived from a subcutaneous node of a patient with Aβ2M amyloidosis are associated with LPC constituting about 10% of the associated lipid. On the other hand, simple addition of sub-µM MPPA to normal plasma did not enhance the fibril stabilizing activity (Figure 8). This suggests that LPA is not a single and major contributor to the fibril-stabilizing effect of uraemic plasmas in vitro (Figure 7). Uraemic plasmas contain a highly diverse group of uraemic toxins [46]. A proinflammatory state [12] and other factors in haemodialysis patients may increase the concentration of some uraemic toxins whose plasma concentrations more or less parallel those of LPLs. Together with LPLs, these uraemic toxins may stabilize Aβ2M amyloid fibrils and contribute to the development of DRA. Future studies are essential to reveal the exact roles of uraemic toxins for the development of DRA.

The interaction between β2-m and LPLs may take place not only in the blood circulation but also in the local tissue (e.g. joint and bone tissue). Interestingly, Jamal et al. [47] reported the increased expression of human type IIA secretory PLA2 antigen in arthritic synovium and Kehlen et al. [48] showed an increased lysoPLD mRNA expression in cultured fibroblast-like synoviocytes from patients with rheumatoid arthritis. In the tenosynovial tissue of DRA patients, LPLs produced locally with inflammation due to β2-m amyloid deposition may additively or synergistically enhance the fibrillogenic activity of extracellular matrix components, e.g. collagens, PGs. It is essential to examine whether the local concentration of LPLs rises significantly in the tenosynovial tissue of DRA patients.

The serum levels of β2-m in patients with end-stage renal failure can increase from ~0.1 µM to >5 µM [8]. Importantly, in the presence of 5 µM β2-m, Aβ2M amyloid fibrils became significantly extended (Supplementary Figure 1B). Thus, it may be reasonable to consider that the pathological interaction between β2-m and other molecules including LPLs will be significant only in haemodialysis patients, resulting in the manifestation of DRA after a long incubation period.

In conclusion, the present study shed some light on the essential role of the interaction between β2-m and lipid molecules in the manifestation of DRA. More extensive analysis of the overall profile of lipid metabolism in haemodialysis patients is needed to elucidate the molecular pathogenesis of DRA.



   Supplementary data
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data is available at NDT Journal online.



   Acknowledgments
 
This work was supported by Grants-in-Aid for Exploratory Research (T.O.), Scientific Research (B) (H.N.), Scientific Research on Priority Areas ‘Life of Proteins’ and ‘Water & Biomolecules’ (H.N.) and 21st Century COE Program (Medical Sciences) (H.N.) from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and for Research on Specific Diseases (H.N.) from the Ministry of Health, Labour and Welfare, Japan. The authors thank T. Kishimoto and N. Matsuyama (Alfresa Pharma Corporation, Osaka, Japan) for determination of plasma concentrations of LPLs and S. Yasuhara, R. Nomura, N. Takimoto and H. Okada for excellent technical assistance.

Conflict of interest statement. None declared.



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 

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Received for publication: 10. 2.08
Accepted in revised form: 3. 4.08


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