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Glycosaminoglycans Enhance the Trifluoroethanol-Induced Extension of ₂-Microglobulin–Related Amyloid Fibrils at a Neutral pH

Suguru Yamamoto^*,, Itaru Yamaguchi^*, Kazuhiro Hasegawa^*, Shinobu Tsutsumi^*, Yuji Goto^,, Fumitake Gejyo and Hironobu Naiki^*,

*Department of Pathology, Fukui Medical University, Fukui, Japan; Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Science, Niigata, Japan; Institute for Protein Research, Osaka University, Osaka, Japan; and CREST of Japan Science and Technology Corporation, Saitama, Japan

Correspondence to Dr. Hironobu Naiki, Department of Pathology, Fukui Medical University, Fukui 910-1193, Japan. Phone: +81-776-61-8320; Fax: +81-776-61-8123;

	Abstract

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ABSTRACT. ₂-Microglobulin–related (A2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis, and ₂-microglobulin is a major structural component of A2M amyloid fibrils. Several biologic molecules inhibiting the depolymerization of A2M amyloid fibrils at a neutral pH were found recently. The effect of trifluoroethanol and glycosaminoglycans (GAG) on the extension of the fibrils at a neutral pH was investigated with the use of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. Trifluoroethanol at concentrations of up to 20% (vol/vol) caused fibril extension of heparin-stabilized seeds, inducing a subtle change in the tertiary structure of ₂-microglobulin and stabilizing the fibrils at a neutral pH. This extension reaction followed a first-order kinetic model. In addition, some GAG, especially heparin, dose-dependently enhanced the fibril extension. These results suggest that some GAG, especially heparin, may bind to the fibrils and enhance their deposition in vivo. Thus, the experimental system described here should be useful to search for the factors that accelerate A2M amyloid deposition in vivo. In addition, the interference of the binding of GAG to A2M amyloid fibrils may be an attractive therapeutic modality. E-mail: naiki@fmsrsa.fukui-med.ac.jp

	Introduction

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₂-Microglobulin–related (A2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis (1). Carpal tunnel syndrome and destructive arthropathy associated with cystic bone lesions are the major clinical manifestations of A2M amyloidosis (2,3). Several biochemical and cell biologic studies have revealed that intact ₂-microglobulin (2-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 still incompletely understood. Although the retention of 2-m in the plasma seems to be prerequisite (8), other factors, such as the age of the patient, the duration of dialysis, and the type of dialysis membrane used, may also be involved (9–11).

We and other groups have proposed that a nucleation- dependent polymerization model could explain the general mechanism of amyloid fibril formation in vitro, in various types of amyloidosis (12–17). This model consists of two phases: a nucleation phase and an extension phase. Nucleus formation requires a series of association steps of monomers, which are thermodynamically unfavorable, representing the rate-limiting step in amyloid fibril formation in vitro. Once the nucleus (n-mer) has been formed, further addition of monomers becomes thermodynamically favorable, resulting in rapid extension of amyloid fibrils according to a first-order kinetic model (12,14,16).

The extension of A2M amyloid fibrils, as well as the formation of the fibrils from 2-m, is greatly dependent on the pH of the reaction mixture, with the optimum pH at approximately 2.0 to 3.0 (16,17). However, they readily depolymerize into monomeric 2-m at a neutral pH (18). At pH 2.5, where the extension of A2M amyloid fibrils is optimum, 2-m loses much of the secondary and tertiary structures observed at pH 7.5 (17,18). Once incorporated into A2M amyloid fibrils at pH 2.5, 2-m becomes highly rich in -sheet structure and obtains the secondary and tertiary structures strikingly different from monomeric 2-m at both pH 7.5 and 2.5 (19). Heegaard et al. (20) demonstrated that 2-m forms the amyloidogenic conformer in the presence of acetonitrile or 2,2,2-trifluoroethanol (TFE) at a neutral pH. Chiti et al. (21,22) also reported that a partially structured species of 2-m closely similar to that at an acidic pH is significantly populated under physiologic conditions and transiently involved in the extension reaction of fibrils extracted from patients. However, for analyzing the effect of various biologic molecules and organic compounds on A2M amyloid fibril formation in vitro, it is essential to develop an experimental system whereby the kinetics of fibril formation is constantly and stably observed at a neutral pH.

A2M amyloid deposits in patients contain many kinds of amyloid-associated molecules: glycosaminoglycans (GAG), proteoglycans (PG) (23,24), apolipoprotein E (25), serum amyloid P component (26), and plasma proteinase inhibitors (27). The earliest deposition of A2M amyloid fibrils is observed in the cartilage tissue highly rich in GAG and PG (28,29). GAG such as heparan sulfate (HS) and chondroitin sulfate (CS) are constantly increased in A2M amyloid deposits (23,24,30). The roles of PG and their constituent GAG for amyloid fibril formation in vitro have been studied extensively in various types of human as well as murine amyloidosis (31–33). Recently, we reported that GAG and PG accelerate A2M amyloid fibril formation at an acidic pH (34) and that apolipoprotein E, GAG, and PG inhibit the depolymerization of fibrils at a neutral pH (18,34). These amyloid-associated factors, especially GAG, may act as an enhancing factor for the deposition of A2M amyloid fibrils in vivo.

	Materials and Methods

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Materials
TFE was obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Urea, acetonitrile, DMSO, and ethanol were obtained from Nacalai Tesque, Inc. (Kyoto, Japan). Heparin from porcine intestinal mucosa (grade I-A), HS from bovine intestinal mucosa, chondroitin sulfate A (CSA) from bovine trachea (70% chondroitin-4-sulfate, 30% chondroitin-6-sulfate), chondroitin sulfate C (CSC) from shark cartilage (90% chondroitin-6-sulfate, 10% chondroitin-4-sulfate), dermatan sulfate (DS) from porcine intestinal mucosa, and keratan sulfate (KS) from bovine cornea were obtained from Sigma (St. Louis, MO). Hyaluronic acid (HA) from human umbilical cord was obtained from Nacalai Tesque Inc.

Preparation of A2M Amyloid Fibrils, Unmodified Seeds, and Heparin-Stabilized Seeds
Unmodified A2M amyloid fibrils composed solely of recombinant 2-m (r-2-m) were formed by the repeated extension reaction at pH 2.5 with r-2-m expressed in methyltrophic yeast Pichia pastoris (35) and the patient-derived S0 seeds as described previously (36). The algorithmic protocol was repeated six times, and F6 fibrils and S6 seeds were obtained from S5 seeds. The S6 seeds were designated the unmodified seeds. Heparin-stabilized seeds were formed by the depolymerization reaction of the F6 fibrils with heparin at pH 7.5 as described previously (34).

Extension Assay of A2M Amyloid Fibrils
The reaction mixture was prepared on ice and contained 0 to 90 µg/ml heparin-stabilized or unmodified seeds, 0 to 50 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and various concentrations of protein denaturants or organic solvents: 0 to 30% (vol/vol) TFE, 0 to 4 M urea, 0 to 60% (vol/vol) ethanol, 0 to 60% (vol/vol) acetonitrile, and 0 to 30% (vol/vol) DMSO. In some experiments, 10 to 200 µg/ml heparin or other GAG were added to the reaction mixture (as indicated in each figure). 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. The reaction tubes were then transferred into a DNA thermal cycler (PJ480; Perkin-Elmer Cetus, Emeryville, CA) and incubated at 37°C without agitation. After incubation for 0 to 72 h, the reaction was stopped by placing the tubes on ice. From each reaction tube, three 5-µl aliquots were removed, then subjected to fluorescence spectroscopy with thioflavin T (ThT) (16), and the mean of each triplicate was determined.

Depolymerization Assay of A2M Amyloid Fibrils
Fresh F6 fibrils extended at pH 2.5 were centrifuged at 18,500 x g for 2 h at 4°C. The precipitate was washed and resuspended in ice-cold 100 mM NaCl. The reaction mixture was prepared on ice and contained 300 µg/ml F6 fibrils, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and 0 or 20% (vol/vol) TFE. After being mixed by pipetting, 30-µl aliquots were put into PCR tubes. The reaction tubes were then transferred into a DNA thermal cycler and incubated at 37°C without agitation. After a 0- to 24-h incubation, the reaction was stopped by placing the tubes on ice. From each reaction tube, three 5-µl aliquots were subjected to fluorescence spectroscopy, and the mean of each triplicate was determined.

CD Spectra of r-2-m in the Presence of TFE and Heparin
CD spectra of r-2-m were recorded on a Jasco 725 spectropolarimeter (Jasco, Tokyo, Japan) at 25°C as described previously (18). The reaction mixture contained 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 0 to 30% (vol/vol) TFE, and 0 to 100 µg/ml heparin. For measurements in the near-UV region (250 to 350 nm), 100 mM NaCl was also added to the mixture. The results are expressed in terms of mean residue ellipticity.

Quantification of Heparin Bound to the A2M Amyloid Fibrils
A2M amyloid fibrils were extended with r-2-m in the presence of heparin at pH 7.5 for 48 h, then collected by centrifugation at 18,500 x g for 1 h at 4°C. The precipitate was washed and resuspended in 100 mM NaCl, and this procedure was repeated three times to cleanse monomeric r-2-m and unbound heparin. Then a quantitative dimethylmethylene blue dye–binding method (Blyscan Proteoglycan and GAG Assay System) was used to determine the concentration of heparin on the A2M amyloid fibrils according to the manufacturer’s instruction (Accurate Chemical and Scientific Corp., Westbury, NY) (37).

Other Analytical Procedures
Electron microscopy and Congo red staining of A2M amyloid fibrils were performed as described previously (16). Protein concentrations of r-2-m and A2M amyloid fibrils were determined by the method using bicinchoninic acid (38) and a commercial protein assay kit (code 23235; Pierce, Rockford, IL).

Stastical Analysis
One-way ANOVA with post hoc test by Dunnett and linear least squares fit were used for statistical analysis.

	Results

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Extension of A2M Amyloid Fibrils at a Neutral pH in the Presence of TFE
As shown in Figure 1, when the heparin-stabilized seeds were incubated with 25 µM r-2-m at pH 7.5 in the presence of 20% (vol/vol) TFE, the ThT fluorescence increased without a lag phase and proceeded to equilibrium, whereas the ThT fluorescence was almost unchanged in the absence of TFE, as described previously (34). However, when the unmodified seeds were incubated with 25 µM r-2-m at pH 7.5 in the presence of 20% (vol/vol) TFE, the fluorescence increase was smaller than in the case of the heparin-stabilized seeds. In addition, in the absence of TFE, the fluorescence of the unmodified seeds decreased immediately after the initiation of the reaction, as described previously (18). In the absence of seeds, TFE did not cause polymerization of r-2-m at pH 7.5 after a 21-d incubation (data not shown).

View larger version (32K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Extension of 2-microglobulin–related (A2M) amyloid fibrils at a neutral pH. The reaction mixture contained 15 µg/ml heparin-stabilized (, ) or unmodified (•, ) seeds, 25 µM recombinant 2-microglobulin (r-2-m), 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and 0 (, ) or 20% (, •) (vol/vol) trifluoroethanol (TFE). The reaction mixture was incubated at 37°C for 0 to 48 h. At each incubation time, the reaction mixture was analyzed by fluorescence spectroscopy as described in Materials and Methods. This is a representative pattern of three independent experiments.

View larger version (35K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Effect of TFE concentration on the extension of A2M amyloid fibrils at a neutral pH. The reaction mixture contained 30 µg/ml heparin-stabilized seeds, 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and 0 to 30% (vol/vol) TFE. The reaction mixture was incubated at 37°C for 48 h and analyzed by fluorescence spectroscopy as described in Materials and Methods. Each column represents the average of three independent experiments. The error bars indicate SD.

View larger version (81K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Electron micrographs of extended A2M amyloid fibrils. (A through C) The reaction mixture containing 30 µg/ml heparin-stabilized seeds, 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and 0 (A), 20 (B), or 30% (vol/vol) TFE (C) was incubated at 37°C for 48 h. (D) The reaction mixture containing 5 µg/ml unmodified seeds, 25 µM r-2-m, 50 mM citrate buffer (pH 2.5), and 100 mM NaCl was incubated at 37°C for 24 h. (E) The reaction mixture containing 30 µg/ml heparin-stabilized seeds, 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, 20% (vol/vol) TFE, and 100 µg/ml heparin was incubated at 37°C for 48 h. These samples were prepared for electron microscopy as described in Materials and Methods. The bars are 250 nm long.

Effect of TFE on the Conformation of r-2-m
CD spectra of r-2-m were measured at pH 7.5 in the presence of 0 to 30% (vol/vol) TFE (Figure 4). In the absence of TFE, the far-UV CD spectrum of r-2-m exhibited a positive peak at 206 nm and a negative peak at 221 nm, and the near-UV CD spectrum of r-2-m exhibited positive peaks at 260, 269, 282, 288, and 293 nm. These data indicate that r-2-m in the absence of TFE at pH 7.5 is rich in -sheet conformation and has a compact tertiary structure, as described previously (18). However, the far-UV CD spectrum of r-2-m at 30% (vol/vol) TFE exhibited a major negative peak at 206 nm and a minor negative peak at 230 nm. In the near-UV CD spectrum of r-2-m at 30% (vol/vol) TFE, the positive ellipticity observed at 0% (vol/vol) TFE was eliminated, and negative peaks were observed between 250 and 290 nm. These data indicate that at 30% (vol/vol) TFE, the secondary structure of r-2-m had changed into the mixture of random and helical structures, and the tertiary structure was also fairly loosened as compared with the structure at 0% (vol/vol) TFE. The transit of the near-UV spectra was observed between 15 and 25% (vol/vol) TFE, whereas that of the far-UV spectra was observed between 20 and 30% (vol/vol) TFE. These data suggest that a subtle change in the tertiary structure may take place at approximately 20% (vol/vol) TFE with no significant change in the secondary structure.

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Far- (A) and near-UV CD spectra (B) of r-2-m in the presence of TFE. In each spectrum, the reaction mixture contained 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), and 0 to 30% (vol/vol) TFE. For the measurements in the near-UV region, 100 mM NaCl was also added to the above mixture.

Effect of TFE on the Depolymerization of A2M Amyloid Fibrils at a Neutral pH

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Effect of TFE on the depolymerization of A2M amyloid fibrils at a neutral pH. The reaction mixture contained 300 µg/ml fresh F6 fibrils, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, and 0 () or 20% (vol/vol) (•) TFE. The reaction mixture was incubated at 37°C for 0 to 24 h and analyzed by fluorescence spectroscopy as described in Materials and Methods. This is a representative pattern of three independent experiments.

Effect of GAG on the Extension of A2M Amyloid Fibrils at a Neutral pH in the Presence of TFE

View larger version (34K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Effect of glycosaminoglycans (GAG) on the extension of A2M amyloid fibrils in the presence of TFE at a neutral pH. The reaction mixture contained 30 µg/ml heparin-stabilized seeds, 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, 20% (vol/vol) TFE, and 100 µg/ml of various GAG; was incubated at 37°C for 48 h; and was analyzed by fluorescence spectroscopy as described in Materials and Methods. Each column represents the average of three independent experiments. The error bars indicate SD. The average without GAG was regarded as 100%. *P < 0.05, **P < 0.01 versus without GAG (one-way ANOVA, post hoc test by Dunnett).

View larger version (40K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Effect of heparin on the extension of A2M amyloid fibrils at a neutral pH in the presence of TFE. The reaction mixture contained 30 µg/ml heparin-stabilized seeds, 25 µM r-2-m, 50 mM phosphate buffer (pH 7.5), 100 mM NaCl, 0 or 20% (vol/vol) TFE, and 0 to 200 µg/ml heparin and was incubated at 37°C for 48 h. (A) Correlation of the fluorescence increase () and 2-m concentration remaining in the supernatant (•) with the initial heparin concentration. The fluorescence increase and 2-m concentration in the supernatant were determined as described in Materials and Methods. Boxes and circles represent the average of three independent procedures, and the error bars represent SD. (B) Correlation of heparin concentration bound to the fibrils with the initial heparin concentration. The heparin concentration on the fibrils was determined as described in Materials and Methods. Boxes represent the average of three independent procedures, and the error bars represent SD.

	Discussion

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Extension of A2M Amyloid Fibrils at a Neutral pH in the Presence of TFE
Among several protein denaturants and organic solvents examined in this study, TFE was the most effective in accelerating A2M amyloid fibril extension at a neutral pH. TFE is known as a cosolvent to weaken the hydrophobic interactions within a polypeptide chain and to strengthen the intramolecular hydrogen bonds between residues close to each other (39). As a result of these effects, TFE changes the conformation of various proteins, often producing the helical structure stabilized by intramolecular hydrogen bonds (40,41). However, it is also known that the formation of intermolecular hydrogen bonds in the presence of TFE results in the aggregates (40) or amyloid fibrils (42). These dual properties of TFE may well explain the effect of TFE to accelerate A2M amyloid fibril extension at a neutral pH. At 20% (vol/vol) TFE, where the extension of A2M amyloid fibrils is optimum, TFE would weaken the hydrophobic interactions within 2-m and make 2-m an amyloidogenic conformer by unfolding the tertiary structure (Figure 4). At the same time, TFE could strengthen the intermolecular hydrogen bonds between 2-m molecules in the fibrils and stabilize the extended fibrils (Figure 5). Fezoui and Teplow (43) recently reported that 20% (vol/vol) TFE converts predominantly unstructured amyloid -peptide (A) monomers into partially ordered, helix-containing quasi-stable conformers, resulting in a temporal decrease in the lag phase for fibril formation and a significant increase in the rate of fibril elongation. They suggested that a partially folded helix-containing conformer may be an intermediate in A fibril assembly, and factors that affect helix formation and stability will have significant effects on the kinetics of A fibril formation in vivo.

Chiti et al. (21,22) detected two partially unfolded species of 2-m (I₁ and I₂) and reported that the population of I₂, which is similar to the species at an acidic pH, is significantly increased under physiologic conditions. They also demonstrated that after the denaturation of 2-m by hydrochloric acid, the population of I₂ increased only transiently from 5 s to 1 to 2 min after the initiation of refolding at pH 7.3 and is involved in the extension of patient-derived amyloid fibrils during the refolding. As shown in this article, TFE at a low concentration (20% [vol/vol]) has enabled us for the first time to perform constant and stable kinetic analysis of fibril extension at a neutral pH, as well as to investigate the effect of GAG on the extension reaction.

Effect of GAG on the Extension of A2M Amyloid Fibrils at a Neutral pH in the Presence of TFE
In this section, we consider the possible mechanism by which heparin and other GAG accelerate the extension reaction. We propose the following extension reaction of the heparin-stabilized seeds in the presence of heparin

where M is 2-m monomer, H is heparin, P_n-H and P_n+1-H are the terminals of n- and n+1-meric heparin-stabilized seeds, respectively, and P_n+1 is the newly extended terminal not stabilized by heparin. By binding to the newly extended terminal (P_n+1), heparin could reduce the depolymerization rate by stabilizing the terminal, thus promoting the extension reaction by stabilizing the newly extended terminals (P_n+2, P_n+3, P_n+4hellip;) one after another. This explanation may be consistent with the finding that in the absence of heparin, approximately 11 µM r-2-m (44% of the initial concentration) was still in the soluble fraction after a 48-h incubation with the heparin-stabilized seeds, whereas in the presence of 200 µg/ml heparin, the concentration of r-2-m remaining in the soluble fraction reduced to approximately 4 µM (16% of the initial concentration; Figure 7A). We cannot rule out the possibility that heparin could enhance the extension reaction by binding to monomeric 2-m. However, this interaction may not significantly contribute to the extension reaction because heparin did not change the far- and near-UV CD spectra of r-2-m at pH 7.5 in both the presence and the absence of 20% (vol/vol) TFE (data not shown).

As shown in Figure 6, heparin, HS, DS, CSA, and CSC significantly enhanced the extension reaction in this order. The numbers of sulfate and carboxyl groups per disaccharide unit are 3-1, 2-1, 1-1, 1-1, 1-1, 1-0, and 0-1 for heparin, HS, DS, CSA, CSC, KS, and HA, respectively. Because both sulfate and carboxyl groups are negatively charged at a neutral pH, the enhancing ability of these GAG may well be correlated with the degree of the negative charge. Recently, we reported that the ability of GAG to inhibit the depolymerization of A2M amyloid fibrils at a neutral pH may roughly be correlated with the degree of sulfation (34). Comparison of heparin with HA may clearly indicate that the heavy sulfation of the same disaccharide unit increases the enhancing ability. In addition, that the uronic acid in HA does not undergo epimerization might suggest that glucuronate-iduronate epimerization present in heparin may have some influence on the enhancing ability. Here, we report that heparin may uniformly bind to the surface of extended A2M amyloid fibrils (Figure 7B). Because GAG constitute an extended and rigid structure, one GAG molecule would bind to the surface of the fibrils over many 2-m molecules by the electrostatic interaction between the negative charges of GAG and the positive charges of the specific residues of 2-m molecules.

Molecular Environment of A2M Amyloid Deposition In Vivo
Various types of GAG and PG, especially HS and perlecan, have been identified in many types of human and murine amyloidosis and implicated in their development (23,24,30–33,44–47). Even though HS and perlecan are ubiquitously synthesized in vivo as a representative component of the basement membrane and other extracellular matrices, the predominant sites of amyloid deposition in vivo are different from each other: A2M amyloid to be deposited in the cartilaginous and tendinous tissues (2,3,28,29), and amyloid protein A amyloid to be deposited in the spleen and kidneys (32). Although the present study may partly explain why A2M amyloid deposition takes place predominantly in the cartilaginous and tendinous tissues, which both are rich in GAG and PG (28,29), there have to be more specific reasons for the predominant deposition in them. Furthermore, although a low concentration of TFE is needed to extend A2M amyloid fibrils at a neutral pH in vitro, some factors that affect the conformation of 2-m and the stability of the fibrils will have significant effects on the kinetics of A2M amyloid fibril formation in vivo.

Heparin is widely used as an anticoagulant in hemodialysis (48). Although no significant difference in the prevalence of A2M amyloidosis was found between patients on continuous ambulatory peritoneal dialysis and those on hemodialysis carefully matched for time on dialysis and age at the onset of dialysis (49), the present study suggests that heparin could exert a subtle effect for the development of A2M amyloidosis under some clinical conditions.

Finally, interference with the binding of PG and GAG to 2-m and/or A2M amyloid fibrils in vivo may be an attractive therapeutic objective. Kisilevsky et al. (50) demonstrated that low-molecular-weight anionic sulfonate or sulfate compounds substantially reduce murine splenic amyloid protein A amyloid progression and interfere with HS-stimulated A fibril aggregation in vitro. The experimental system described in this article should be useful for searching for the drugs that prevent the interaction of these molecules.

	Acknowledgments

 
This research was supported in part by Grants-in-Aid for Scientific Research on Priority Areas—Life of Proteins—from Ministry of Education, Culture, Sports, Science and Technology, Japan, and for Research on Specific Diseases from Ministry of Health, Labor and Welfare, Japan.

We thank H. Okada and N. Takimoto for excellent technical assistance.

	References

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